PSMA, a marker for prostate cancer: Molecular signalling and ......19th Meeting of German Veterinary...

Department of Physiological Chemistry

University of Veterinary Medicine Hannover

_____________________________________________________________

PSMA, a marker for prostate cancer:

Molecular signalling and transport mechanisms in humans and

their transferability to dogs

THESIS

Submitted in partial fulfilment of the requirements for the degree

DOCTOR OF PHILOSOPHY (PhD)

awarded by the University of Veterinary Medicine Hannover

by

Sonja Schmidt

(Hannover)

Hannover 2012

II

Supervisor: Prof. Dr. Hassan Y. Naim

Advisory Committee: Prof. Dr. Hassan Y. Naim

Prof. Dr. Rita Gerardy-Schahn

Prof. Dr. Wolfgang Bäumer

1st Evaluation: Prof. Dr. Hassan Y. Naim

Department of Physiological Chemistry,

University of Veterinary Medicine Hannover,

Foundation

Prof. Dr. Rita Gerardy-Schahn

Department of Cellular Chemistry,

Hannover Medical School

Prof. Dr. Wolfgang Bäumer

Department of Pharmacology, Toxicology and Pharmacy,

University of Veterinary Medicine Hannover,

Foundation

2nd Evaluation: Prof. Dr. Marco Colombatti

Department of Pathology and Diagnostics,

University of Verona, Italy

Date of oral exam: 05.11.2012

III

TABLE OF CONTENTS

TABLE OF CONTENTS.......................................................................................................III

LIST OF PUBLICATIONS AND PRESENTATIONS ....................................................... V

SCIENTIFIC PRESENTATIONS..................................................................................... V

MANUSCRIPTS............................................................................................................... VII

LIST OF OTHER PUBLICATIONS ............................................................................. VII

LIST OF ABBREVIATIONS................................................................................................. X

LIST OF FIGURES ............................................................................................................XIII

I. INTRODUCTION .............................................................................................................. 15

1.1. Prostate cancer ............................................................................................................ 17

1.1.1. Incidence and Epidemiology.................................................................................. 17

1.1.2. Screening for prostate cancer................................................................................ 19

1.1.3. Diagnosis................................................................................................................ 20

1.1.4. Therapy................................................................................................................... 20

1.1.5. Prostate cancer in dogs.......................................................................................... 21

1.2. Prostate-specific membrane antigen.......................................................................... 21

1.3. Lipid rafts..................................................................................................................... 24

1.3.1. Structure of lipid rafts............................................................................................ 25

1.3.2. Different types of lipid rafts................................................................................... 28

1.3.3. Size of lipid rafts..................................................................................................... 29

1.3.4. Function of lipid rafts............................................................................................ 30

1.4. Endocytosis .................................................................................................................. 31

1.5. Microtubules ................................................................................................................ 34

1.6. Aim of the study........................................................................................................... 36

II. DISCRIMINATORY ROLE OF DETERGENT-RESISTANT MEMB RANES IN

THE DIMERIZATION AND ENDOCYTOSIS OF PROSTATE-SPECIF IC

MEMBRANE ANTIGEN...................................................................................................... 39

III. CLONING AND CHARACTERISATION OF CANINE PROSTAT E-SPECIFIC

MEMBRANE ANTIGEN...................................................................................................... 63

IV

IV. DISCUSSION................................................................................................................... 87

4.1. Quaternary structure of PSMA ................................................................................. 89

4.2. Internalization of PSMA via microtubules ............................................................... 91

4.3. Association of PSMA with other proteins ................................................................. 96

4.4. Canine PSMA .............................................................................................................. 99

V. SUMMARY...................................................................................................................... 103

VI. ZUSAMMENFASSUNG............................................................................................... 107

VII. REFERENCES............................................................................................................. 111

VIII. APPENDIX.................................................................................................................. 137

8.1. Acknowledgements.................................................................................................... 139

V

LIST OF PUBLICATIONS AND PRESENTATIONS

Parts of the thesis have been published or communicated:

SCIENTIFIC PRESENTATIONS:

Poster presentations:

Activation and internalization of Prostate Specific Membrane Antigen result

in protein alterations in Lipid rafts

Sonja Schmidt, Martin Heine, Hassan Y. Naim

EMBO Meeting, 29. August – 01. September 2009, Amsterdam, Netherlands

Activation of prostate-specific membrane antigen results in protein alterations in detergent-

resistant membranes


19th Meeting of German Veterinary Society Division Physiology and Biochemistry, 15.-16.

February 2010, Hannover

Signalling pathway of prostate-specific membrane antigen implicates different types of

detergent-resistant membranes


Experimental Biology, 24.-28. April 2010, Anaheim, USA

Activation of prostate-specific membrane antigen implicates different types of detergent-

resistant membranes


1st International Symposium on „Protein Trafficking in Health and Disease“, 26.-28. May

2010, Hamburg

VI

Detergent-resistant membranes are essential along the signalling pathways of prostate-

specific membrane antigen

Sonja Schmidt and Hassan Y. Naim

Molecular Life Sciences 2011 - GBM, 25.-28. September 2011, Frankfurt

Detergent-resistant membranes are essential along the signalling pathways of prostate-

specific membrane antigen

Sonja Schmidt and Hassan Y. Naim

Annual Meeting of the American Society for Cell Biology, 03.-07. December 2011, Denver,

USA

Oral presentations:

Prostate-specific membrane antigen: Molecular and signalling aspects

Sonja Schmidt

Seminar in Biochemistry and Virology at the University of Veterinary Medicine,

8. December 2010, Hannover

PSMA, a marker for prostate cancer: Molecular signalling and transport mechanisms in

humans and their transferability to dogs

Sonja Schmidt

Seminar in Biochemistry and Virology at the University of Veterinary Medicine,

4. July 2012, Hannover

VII

MANUSCRIPTS:

Discriminatory role of detergent-resistant membranes in the dimerization and endocytosis

of prostate-specific membrane antigen

Sonja Schmidt1, Giulio Fracasso2, Dunia Ramarli2, Marco Colombatti2 and Hassan Y. Naim1

1Department of Physiological Chemistry, University of Veterinary Medicine Hannover,

Germany 2 Department of Pathology and Diagnostics, University of Verona, Italy

(submitted to Biochemical Journal)

Cloning and characterisation of canine prostate-specific membrane antigen

Sonja Schmidt1, Giulio Fracasso2, Marco Colombatti2 and Hassan Y. Naim1

1Department of Physiological Chemistry, University of Veterinary Medicine Hannover,

Germany 2 Department of Pathology and Diagnostics, University of Verona, Italy

(submitted to The Prostate)

LIST OF OTHER PUBLICATIONS:

The dual role of annexin II in targeting of brush border proteins and in intestinal cell

polarity (Differentiation, 2011)

Hein Z, Schmidt S, Zimmer KP, Naim HY

Abstract

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the

formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane

domains. Numerous transport pathways warrant the unique structural identity and

protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-

binding protein) is an important component of one of the apical protein transport machineries,

which involves detergent-resistant membranes and the actin cytoskeleton. Here, we

VIII

investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport

of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in

Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane

resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This

reduction is accompanied by a redistribution of these proteins to intracellular compartments

and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are

characterized by an almost flat apical membrane with sparse and short microvilli.

Concomitant with this alteration is the redistribution of the intermediate filament protein

keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19

interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in

lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and

polarization of intestinal cells.

Impairment of protein trafficking by direct interaction of gliadin peptides with actin (Exp

Cell Res, 2011)

Reinke Y, Behrendt M, Schmidt S, Zimmer KP, Naim HY

Abstract

Intestinal celiac disease (CD) is triggered by peptic-tryptic digest of gluten, known as Frazer's

Fraction (FF), in genetically predisposed individuals. Here, we investigate the immediate

effects of FF on the actin cytoskeleton and the subsequent trafficking of actin-dependent and

actin-independent proteins in COS-1 cells. Morphological alterations in the actin filaments

were revealed concomitant with a drastic reduction in immunoprecipitated actin from cells

incubated with FF. These alterations elicit impaired protein trafficking of intestinal sucrase-

isomaltase, a glycoprotein that follows an actin-dependent vesicular transport to the cell

surface. However, the actin-independent transport of intestinal lactase phlorizin hydrolase

remains unaffected. Moreover, the morphological alteration in actin is induced by direct

interaction of this protein with gliadin peptides carrying the QQQPFP epitope revealed by co-

immunoprecipitation utilizing a monoclonal anti-gliadin antibody. Finally, stimulation of cells

with FF directly influences the binding of actin to Arp2. Altogether, our data demonstrate that

FF directly interacts with actin and alters the integrity of the actin cytoskeleton thus leading to

IX

an impaired trafficking of intestinal proteins that depend on an intact actin network. This

direct interaction could be related to the endocytic segregation of gliadin peptides as well as

the delayed endocytic vesicle trafficking and maturation in gliadin-positive intestinal

epithelial cells and opens new insights into the pathogenesis of CD.

X

LIST OF ABBREVIATIONS

µg microgram

µl microliter

µm micrometer

67LR 67 kDa laminin receptor 1

Act activated

ARF 6 adenosine diphosphate-ribosylation factor 6

C celsius

CAS Crk-associated substrates

CCL5 chemokine (C-C motif) ligand 5

CD cytoplasmic domain

cDNA complementary deoxyribonucleic acid

cPSMA canine prostate-specific membrane antigen

DEAE diethylaminoethyl

DMEM Dulbecco’s modified eagle’s medium

DNA deoxyribonucleic acid

DRMs detergent-resistant membranes

DTT dithiothreitol

E. coli escherichia coli

e.g. for example

ECL enhanced chemiluminescence

ED extracellular domain

EEA1 early endosomal antigen-1

Endo F endoglycosidase F

Endo H endoglycosidase H

ER endoplasmatic reticulum

ERK extracellular-signal-regulated kinases

Fig. figure

g gram

GFP green fluorescent protein

XI

GPI glycosylphosphatidylinositol

GTP guanosine triphosphate

h hour

HIV human immunodeficiency virus

hPSMA human prostate-specific membrane antigen

IgE immunoglobulin E

IL-6 interleukin-6

kDa kilo Dalton

L liter

Lck lymphocyte-specific protein tyrosine kinase

LPH lactase phlorizin hydrolase

LPS lipopolysaccharides

M molar

mAb monoclonal antibody

MAPK mitogen-activated protein kinase

mg milligram

min minute

ml milliliter

mM millimolar

MOG myelin oligodendrocyte glycoprotein

nAct non activated

NF-kB nuclear factor kappa-light-chain-enhancer of activated B-cells

nm nanometer

P pellet

PBS phosphate buffered saline

PCR polymerase chain reaction

PED/PEA-15 phosphoprotein enriched in diabetes/phosphoprotein enriched in

astrocytes

PSA prostate-specific antigen

PSMA prostate-specific membrane antigen

PSMAC complex glycosylated form of PSMA

XII

PSMAM mannose-rich form of PSMA

PVDF polyvinylidene fluoride

RNA ribonucleic acid

rpm rounds per minute

RT-PCR real-time-polymerase chain reaction

S supernatant

SDS sodiumdodecylsulfate

SDS-PAGE sodiumdodecylsulfate-polyacrylamide gel electrophoresis

TCR T-cell receptor

TEMED tetramethylethylenediamine

TfR transferrin receptor

TGN trans-Golgi Network

TLR4 toll-like receptor 4

TM transmembrane region

TNM Tumour/Nodes/Metastasis

U units

uPAR urokinase-type plasminogen activator receptor

v volume

w weight

wt wild type

YFP yellow fluorescence protein

XIII

LIST OF FIGURES

Figure 1-1 Schematic diagram of prostate-specific membrane antigen (PSMA) 22

Figure 1-2 Model of lipid raft structure in biological membranes 27

Figure 1-3 Different routes of endocytosis 33

Figure 2-1 PSMA associates with Lubrol WX-DRMs as a dimeric protein 55

Figure 2-2 PSMA associates with Triton X-100-DRMs upon activation 56

Figure 2-3 Time course of association of PSMA with Triton X-100-DRMs 58

Figure 2-4 Internalization of PSMA is followed by its association with

Triton X-100 DRMs 59

Figure 2-5 Identification of proteins potentially interacting with PSMA 60

Figure 2-6 Scheme of DRM-associated internalization of PSMA 62

Figure 3-1 Alignment of canine PSMA and human PSMA show 91% amino acid

homology with the biggest difference in a longer cytoplasmic tail of

canine PSMA 81

Figure 3-2 GFP-cPSMA is transported to the cell surface and has a molecular

weight of approx. 140 kDa 82

Figure 3-3 Expression and co-localization of pCMV-3T1-cPSMA and

YFP-hPSMA in COS-1 cells 83

Figure 3-4 Glycosylation and trafficking pattern of GFP-canine PSMA are

similar to human PSMA 84

Figure 3-5 GFP-cPSMA is partially associated with Tween 20- and

Lubrol WX- DRMs, but not with Triton X-100-DRMs 85

Figure 3-6 GFP-cPSMA forms dimers 86

Figure 4-1 PSMA co-localizes and co-immunoprecipitates with 67LR 98

CHAPTER I:CHAPTER I:CHAPTER I:CHAPTER I:

IntroductionIntroductionIntroductionIntroduction

INTRODUCTION

17

I. INTRODUCTION

1.1. Prostate cancer Nowadays prostate cancer is recognized as one of the most important medical problems

facing the male population. Most prostate cancers are slow growing, however, there are cases

of aggressive prostate cancer. In such cases cancer cells can metastasize from the prostate to

other parts of the body, particularly bones, lymph nodes and lungs, and they may invade

rectum, bladder and lower ureters after local progression. Early prostate cancer usually causes

no symptoms. However, advanced prostate cancer is associated with urinary dysfunction as

the prostate gland surrounds the prostatic urethra. Changes within the gland, therefore,

directly affect urinary function including frequent urination, nocturia, and difficulty starting

as well as maintaining a steady stream of urine, hematuria and dysuria. Metastasis of

advanced prostate cancer can also cause symptoms in other parts of the body. The most

common symptom is bone pain, often in the vertebrae, pelvis or ribs.

1.1.1. Incidence and Epidemiology

In Germany, prostate cancer is the most common solid neoplasm among men, with more than

60100 newly diagnosed cases per year (DEUTSCHES KREBSFORSCHUNGSZENTRUM

2010). Furthermore, prostate cancer is currently the second most common cause of cancer

death in men (JEMAL et al. 2008). Since 1985 there has been a slight increase in most

countries in the number of deaths from prostate cancer, even in countries or regions where

prostate cancer is not common (QUINN and BABB 2002).

The factors that determine the risk of developing clinical prostate cancer are not well known;

nevertheless a couple of factors have been identified. At the moment there are three well-

established risk factors for prostate cancer:

• increasing age,

• heredity and

• ethnical origin (HEIDENREICH et al. 2010).

INTRODUCTION

18

Cancer of the prostate is very unusual in men younger than 45, but becomes more common

with increasing age. The median age at diagnosis of prostate cancer is 67 years and the

median age at death is 81 years (ALTEKRUSE and KRAPCHO 2010). Therefore prostate

cancer is a bigger health concern in developed countries with their greater proportion of

elderly people. Accordingly, in developed countries about 15% of male cancers are prostate

cancer, compared to 4% of male cancers in undeveloped countries (PARKIN et al. 2001).

Autopsy series suggest that 30% of men older than 50 years of age and 70% of those older

than 70 years of age have occult prostate cancer (COLEY et al. 1997). But the great majority

of men with a diagnosis of prostate cancer die from other causes. The 10-year risk of death

from prostate cancer ranged from approximately 8% among men with well-differentiated

tumours to 26% among those with poorly differentiated types. In comparison the 10-year risk

of death from competing causes was consistently nearly 60%, regardless of the tumour grade

(HOFFMAN 2011).

The genetic background may also contribute to the risk of developing prostate cancer. If one

first-line relative suffers from prostate cancer, the risk to develop prostate cancer is at least

doubled. If two or more first-line relatives are affected, the risk increases 5- to 11-fold

(STEINBERG et al. 1990; GRONBERG et al. 1996).

Autopsy-detected prostate cancers are diagnosed with roughly the same frequency in different

parts of the world (BRESLOW et al. 1977). This diagnostic finding is in sharp contrast to the

incidence of clinical prostate cancer, which differs widely between distinct regions of the

world, being high in Northern Europe and the USA and low in Southeast Asia (QUINN and

BABB 2002). Nevertheless, if Japanese men move from Japan to Hawaii, their risk of

developing clinical prostate cancer increases; if they move to California, their risk increases

even more, to approximately that of American men (ZARIDZE et al. 1984). These findings

indicate that exogenous factors affect the risk of progression from latent prostate cancer to

clinical prostate cancer. Factors such as food consumption, pattern of sexual behaviour,

alcohol consumption, exposure to ultraviolet radiation and job-related exposure have all been

discussed as being of etiological importance (KOLONEL et al. 2004).

INTRODUCTION

19

In summary, increasing age and hereditary are important risk factors of developing clinical

prostate cancer, while exogenous factors may have an important impact on this risk. But at the

moment there is not enough evidence to recommend lifestyle changes (for example, lowered

intake of animal fat and increased intake of fruits, cereals and vegetables) in order to decrease

the risk of developing clinical prostate cancer.

1.1.2. Screening for prostate cancer

The common argument for screening is an extended lifetime based on an early detection and

treatment of asymptomatic forms of prostate cancer, as compared with treatment at the time of

clinical diagnosis.

For many years, the digital rectal examination was the primary screening test for prostate

cancer. However, this screening method offers significant interexaminer variability (SMITH

and CATALONA 1995), and the majority of cancers remain undetected until reaching an

advanced stage (CHODAK et al. 1989).

In the late 1980s, prostate-specific antigen (PSA) testing was rapidly and widely adopted for

screening. PSA is a kallikrein-like serine protease produced by the epithelial cells of the

prostate. It is organ-specific but not cancer-specific. Therefore, serum levels may be elevated

in the presence of prostate cancer as well as benign prostatic hypertrophy, prostatitis, perineal

trauma and other non-malignant conditions. Moreover, a normal PSA value does not rule out

prostate cancer (HOFFMAN 2011).

The introduction of PSA testing has nearly doubled the lifetime risk of receiving a diagnosis

of prostate cancer and a substantial proportion of PSA-detected cancers are considered over-

diagnosed, because they would not cause clinical problems during normal lifetime.

Early results from large, randomized trials of screening showed a modest decrease in prostate

cancer mortality in Europe, whereas a U.S. study showed no decrease in prostate cancer

mortality at all (SCHROEDER et al. 2009; LU-YAO et al. 2002). However, these results may

be explained due to an increasing lifetime in Europe as well as the U.S., which compensates

the potential positive effect of PSA testing.

INTRODUCTION

20

Currently different other prostate-specific proteins are analysed as potential cancer

biomarkers to discriminate either between tumour tissue compared to its benign counterpart or

between low and high Gleason score tumours. One of these potential biomarker is the

prostate-specific membrane antigen (PSMA) as well as 60 kDa heat shock protein, lamin A,

prostatic acid phosphatase and others (SKVORTSOV et al. 2011).

1.1.3. Diagnosis

The primary diagnostic tools to obtain evidence of prostate cancer include digital rectal

examination, serum concentration of PSA and transrectal ultrasonography. Its final diagnosis

depends on the presence of adenocarcinoma cells in prostate biopsy cores or operative

specimens. Histopathological examination also allows grading and determination of the extent

of the tumour.

The Gleason score is the most commonly used system for grading adenocarcinoma of the

prostate. Gleason scores are assigned to prostate cancers based on their microscopic

appearance. They can only be assessed using biopsy material. Results range between 2 and

10, with 2 being the least aggressive and 10 being the most aggressive form of prostate

cancer.

Another major part to evaluate prostate cancer is to determine the stage, or how far the cancer

has spread. Knowing the stage also helps to assess a prognosis and is useful when selecting

therapies. A common system is the four-stage TNM system (abbreviated from

Tumour/Nodes/Metastases). It describes the size of the tumour, the number of involved lymph

nodes and the presence of any other metastases (HEIDENREICH et al. 2010).

1.1.4. Therapy

An active surveillance is acceptable for many men diagnosed with low-risk prostate cancer.

This idea implies careful observation of the tumour over time, with the intention of treatment

for cure, if there are signs of cancer progression. Aggressive prostate cancers can be treated

with surgery like radical prostatectomy, cryosurgery, radiation therapy, high-intensity focused

ultrasound, chemotherapy, oral chemotherapeutic drugs, hormonal therapy, or some

combination (BRAUN et al. 2009; PEYROMAURE et al. 2009).

INTRODUCTION

21

1.1.5. Prostate cancer in dogs

Besides humans, dogs are the only mammalian species developing prostate tumours

spontaneously. The incidence of tumours in dogs is lower than in humans, but the number of

affected dogs increases. Nearly all of these tumours are adenocarcinomas, metastasizing

mostly in lungs, lymph nodes and bones with high probability. As well as in men, dog

prostate cancer is most commonly found in elderly patients (WATERS et al. 1996). LAI et al.

(2008) could show that canine prostate cancer is very aggressive and of a less differentiated

type than most common human prostate cancers. Therefore it mostly resembles human,

androgen refractory, poorly differentiated prostate cancer with usually a bad prognosis (LAI

et al. 2008). In this respect the dog is of great importance as a model for this disease, filling a

gap between rodent model studies and human clinical trials.

1.2. Prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a type-II-transmembrane-glycoprotein with

folate hydrolase and carboxypeptidase activity (PINTO et al. 1996). It was initially found in

LNCaP cells by immunoprecipitation (HOROSZEWICZ et al. 1987). PSMA is expressed in

epithelial cells of the prostate and at low levels also in some other organs like kidney,

intestine and brain (ISRAELI et al. 1993; ISRAELI et al. 1994). Elevated levels of PSMA are

detected in prostate cancer cells including those that are metastatic (WRIGHT et al. 1996;

SILVER et al. 1997). Levels of PSMA are directly proportional to disease grade and stage

(ROSS et al. 2003). It has been shown that PSMA stimulates the development of prostate

cancer by increasing folate levels necessary for cell survival and growth (YAO et al. 2010).

Moreover, increased peptidase activity of PSMA is associated with aggressive and metastatic

prostate cancer (LAPIDUS et al. 2000). However, the consequence of this increased

enzymatic activity in the context of benign and malignant prostatic cells remains unclear.

Also in neovasculature of other nonprostatic tumours PSMA expression has been detected,

but it is not present in healthy vasculature (CHANG et al. 1999 a; CHANG et al. 1999 b).

As a consequence PSMA is a promising biomarker in the diagnosis and treatment of prostate

cancer. Antibodies conjugated to cytotoxic drugs are currently in clinical trials for use in

mAb-mediated immunotherapy (FRACASSO et al. 2002; BANDER et al. 2003; NANUS et

INTRODUCTION

22

al. 2003; BALLANGRUD et al. 2001; MC DEVITT et al. 2000). These mAbs combined with

cytotoxic drugs have shown the ability to induce apoptosis, especially in cells expressing high

levels of PSMA on their surface, like prostate cancer cells.

The extracellular domain of PSMA is highly glycosylated, with linked oligosaccharides

accounting for up to 25% of the molecular weight of the native protein (HOLMES et al.

1996). In particular, human PSMA has ten potential N-glycosylation sites. At least nine of

them, located in the PSMA ectodomain, are indeed glycosylated. Additionally PSMA is

heavily O-glycosylated (CASTELLETTI et al. 2006).

Figure 1-1. Schematic diagram of prostate- specific membrane antigen (PSMA). PSMA is a type-II-transmembrane protein with a short NH2- terminal cytoplasmic domain (CD), a hydrophobic transmembrane region (TM) and a large extracellular domain (ED). The CD contains an endocytic targeting motif and a filamin A binding site (A). The large ED is highly glycosylated. The ED contains two domains of unknown function that span amino acid residues 44–150 (B) and 151–274 (D), proline- and glycine-rich regions that span amino acid residues 145–172 and 249– 273, respectively (C and E), a catalytic domain that spans amino acid residues 274–587 (F), and a final domain of unknown function (amino acids 587–750) to which a helical dimerization domain (amino acids 601–750) is localized (G) (taken from RAJASEKARAN et al. 2005).

An interesting point in the biosynthetic features of PSMA is the presence of high proportions

of PSMA in an early immature mannose-rich glycosylated polypeptide throughout its life

cycle compatible with a slow transport rate between the ER and the Golgi (CASTELLETTI et

al. 2006).

INTRODUCTION

23

PSMA is transported along the secretory pathway via different detergent-resistant membranes

(DRMs). The mannose-rich form of PSMA associates predominantly with Tween 20-DRMs

in the ER, whereas the complex glycosylated form of later compartments is mainly insoluble

in Lubrol WX (CASTELLETTI et al. 2008). In contrast, in Triton X-100 PSMA is totally

soluble, meaning it does not associate with these kinds of DRMs. Triton X-100-DRMs are

enriched in sphingolipids and cholesterol, whereas Tween 20- as well as Lubrol WX-DRMs

show decreased amounts of these two lipids. In comparison to Triton X-100-DRMs,

phosphatidylethanolamine is increased approximately 6- and 8-fold in Tween 20- and Lubrol

WX-DRMs, respectively (CASTELLETTI et al. 2008).

In both the prostatic epithelium and transfected MDCK cells PSMA is localized primarily to

the apical plasma membrane (CHRISTIANSEN et al. 2003). Changes in the pattern of

glycosylation are described for several cancers, but glycan processing does not affect the

apical transport and folding of PSMA (CASTELLETTI et al. 2006).

PSMA shares homology with the transferrin receptor (TfR) at the levels of both amino acid

identity and domain organization (MAHADEVAN and SALDANHA 1999). As well as the

TfR also native human PSMA is expressed in a homodimeric fashion that constitutes the

enzymatically-active form of PSMA (SCHULKE et al. 2003). After complex glycosylation

homodimerization takes place in the Golgi (CASTELLETTI et al. 2008). The dimerization is

apparently mediated by epitopes within the extracellular domain, because truncated versions

of PSMA lacking the cytoplasmic as well as the transmembrane domain are still able to form

homodimers (SCHULKE et al. 2003).

PSMA is described to undergo constitutively internalization from the cell surface and binding

of antibodies or related antibody fragments to the extracellular domain increases the rate of

PSMA internalization (LIU et al. 1998). These antibodies may act like a natural ligand,

indicating that PSMA may have a receptor function involved in endocytosis of a putative

unknown ligand.

In LNCaP cells, PSMA undergoes internalization via clathrin-coated pits followed by

accumulation in endosomes (LIU et al. 1998). Furthermore PSMA associates with the actin

INTRODUCTION

24

cross linking protein filamin A and this association is involved in the localization of PSMA to

the recycling endosomal compartment (ANILKUMAR et al. 2003). In endothelial cells

internalization of PSMA is caveolae-dependent and an interaction with caveolin 1 could be

detected (ANILKUMAR et al. 2006). RAJASEKARAN et al. (2003) could demonstrate that

the cytoplasmic tail five N-terminal amino acids MXXXL are sufficient to mediate the

internalization of PSMA.

During the process of mAb-induced internalization of PSMA the small GTPases RAS and

RAC1 and the MAPKs p38 and ERK1/2 are activated. As following downstream effects a

strong induction of NF-kB activation together with an increased expression of IL-6 and CCL5

occur. IL-6 and CCL5 enhance the proliferative potential of LNCaP cells synergistically

(COLOMBATTI et al. 2009). The activation of signalling molecules as well as the peptidase

activity alludes to a role of PSMA in signal transduction.

Taken together, PSMA is a multifunctional protein and it has emerged as an important

biomarker for the management and therapy of prostate cancer in men, since its expression is

largely restricted to cells of the prostatic epithelium with protein levels proportional to tumour

grade.

1.3. Lipid rafts

The Singer-Nicolson fluid mosaic concept proposes that the lipid bilayer of biological

membranes functions as a neutral two-dimensional solvent, having little influence on

membrane protein function (SINGER and NICOLSON 1972). During the last 25 years this

model was replaced by the finding that lipids are distributed asymmetrically between the

outer and inner leaflets of the bilayer and this imposes a different organization of membrane

components on the lateral axis (VAN MEER 1998). Lipids exist in several phases in lipid

bilayers, including gel-like, liquid-ordered and liquid-disordered states, due to increasing

fluidity (BROWN and LONDON 1998). In the gel-like phase phospholipids are packed

tightly with cholesterol, but nevertheless remain mobile in the plane of the membrane. These

dynamic structures are called lipid rafts (or lipid microdomains, membrane microdomains, or

INTRODUCTION

25

detergent-resistant membranes (DRMs)). The lipids in these assemblies are enriched in

saturated hydrocarbon chains.

1.3.1. Structure of lipid rafts

Phospholipids with short and unsaturated acyl chains build up most biological membranes,

which are fluid and disordered (liquid-disordered phase). Whereas glycosphingolipids and

phospholipids with long and saturated acyl chains and therefore higher melting temperatures

can form highly ordered membrane microdomains (liquid-ordered phase) (SILVIUS et al.

1996; BROWN and LONDON 1998). Cholesterol supports the tight packing of lipid

microdomains by filling the gaps between bulky polar head groups of the lipids (VAN DER

GOOT and HARDER 2001).

In fact both proteins and lipids contribute to the generation of these membrane microdomains.

One important property of lipid rafts is their ability to include or exclude proteins to variable

extents. Proteins with raft affinity include glycosylphosphatidylinositol (GPI)-anchored

proteins, double acylated proteins, cholesterol-linked and palmitoylated proteins as well as

transmembrane proteins, particularly palmitoylated ones (SIMONS and TOOMRE 2000). It is

not yet clear why some proteins are included into rafts and others are not, but mutational

analysis revealed that amino acids in the transmembrane domains near the exoplasmic leaflet

are critical (SCHEIFFELE et al. 1997).

Recently it has been shown that proteins can modulate their transmembrane length and

composition with regard to the specific physical properties of the varying membranes in

which they reside (SHARPE et al. 2010) The membranes themselves have developed highly

specific lipid compositions. On the other hand membrane proteins alter their lipid

environment not only by binding specific lipids but also by influencing their surrounding lipid

environment (APAJALAHTI et al. 2010). In addition, proteins can stabilize lipid rafts by

restricting lateral diffusion through their association with the cytoskeleton (NAKADA et al.

2003).

For activation of numerous signalling proteins, many rafts have to cluster together, forming a

larger platform, in which functionally related proteins can interact as an obligatory first step

INTRODUCTION

26

towards participation in early signal transduction events (SIMONS and IKONEN 1997;

SIMONS and TOOMRE 2000; FRIEDRICHSON and KURZCHALIA 1998).

Such raft clustering occurs during the IgE signalling throughout the allergic immune response

(SHEETS et al. 1999; HOLOWKA and BAIRD 2001). Cross-linking of IgE receptors

(FcεRIs) leads to increased detergent resistance, due to a stronger association with lipid rafts.

Within these rafts, cross-linked FcεRI becomes phosphorylated. This leads to binding and

scaffolding of downstream signalling molecules and, finally, to the formation of a signalling

platform (SHEETS et al. 1999; HOLOWKA and BAIRD 2001).

The clustering of rafts can be triggered from the extracellular side, within the membrane or

from the intracellular side of a cell. Extracellularly ligands, antibodies or lectins can induce

the clustering of lipid rafts (SIMONS and EHEHALT 2002). Although occurring naturally via

multivalent ligands, similar responses of raft clustering have been observed using specific

antibodies directed against certain membrane receptors in vitro (SIMONS and TOOMRE

2000). Within the membrane raft clustering can be triggered by oligomerization of several

proteins and intracellularly it can be induced by cytosolic agents such as cytoskeletal

elements, adapters or scaffolds (SIMONS and EHEHALT 2002).

Proteins can move in and out of lipid rafts due to ligand binding or oligomerization

(HOLOWKA and BAIRD 2001; PALADINO et al. 2004). Increased raft affinity of a certain

protein and its activation within rafts (e.g. phosphorylation) can initiate a cascade of events,

leading to further increase of raft size by clustering (SIMONS and EHEHALT 2002).

Therefore lipid rafts are dynamic, nanoscale, sterol-sphingolipid-enriched, ordered assemblies

of proteins and lipids. These assemblies can coalesce into larger, more stable raft domains by

specific lipid-lipid, protein-lipid and protein-protein oligomerizing interactions (SIMONS and

GERL 2010). Such phenomena have been demonstrated in artificial membranes consisting of

two or three lipid species (AHMED et al. 1997; MILHIET et al. 2001; LAWRENCE et al.

2003). Cohesion within lipid rafts is assumed to be due to the packing of long, aliphatic tails

of sphingolipids or phospholipids against the smooth α-side of the sterol ring system thus

giving rise to condensed complexes (MC CONNELL and VRLJIC 2003).

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27

Transmembrane proteins

Figure 1-2. Model of lipid raft structure in biological membranes. Sphingolipids are enriched in the outer leaflet of the bilayer, whereas cholesterol and phospholipids are distributed in both leaflets. Lipids within the raft domain usually have long and saturated acyl chains, whereas lipids excluded from these domains have shorter and unsaturated acyl chains. GPI-anchored and dually acylated proteins are concentrated in raft domains. Prenylated proteins generally reside in non-raft regions, while some transmembrane proteins do associate with raft domains and others do not (taken from WAHEED and FREED 2009).

A universal physico-chemical feature of membrane microdomains is their ability to resist

extraction with non-ionic detergents at 4°C (BROWN and LONDON 1998). Upon

solubilization of cellular extracts with a non-ionic detergent (such as Triton X-100), DRMs

can be recovered either by sedimentation or in the floating fractions of sucrose density

gradients. This method has now become widely established for their isolation from biological

samples and has led to the term “detergent-resistant membranes, DRMs”.

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1.3.2. Different types of lipid rafts

It has been shown that the plasma membrane does not hold a monopoly on membrane

domains. For example, the existence of lipid rafts along the early secretory pathway has been

proposed by ALFALAH et al. (2005), where the detergent Tween 20 was found to completely

solubilize basolateral proteins, whereas apical proteins remained insoluble after extraction

with the same detergent. Also BROWMAN et al. (2006) identified the two ER-localized

proteins erlin-1 and erlin-2 which are highly enriched in Triton X-100-DRMs.

The ER contains relatively low levels of cholesterol and sphingolipids proposing a different

composition or size of lipid rafts in this compartment compared to those of the plasma

membrane or the TGN. Interestingly, utilization of different non-ionic detergents such as

CHAPS, Brij, Lubrol and Tween-20, which have different solubilizing powers to isolate

DRMs, have shown different lipid and protein compositions (DROBNIK et al. 2002;

SCHUCK et al. 2003; ALFALAH et al. 2005). They have been proven to be a successful tool

to discriminate biochemically between various biosynthetic forms of proteins based on their

solubility or insolubility in a particular detergent (CHAMBERLAIN 2004; SCHUCK et al.

2003). For example, Tween 20- or Brij 98-insoluble membranes contain predominantly the

mannose-rich forms of apical proteins and segregate them from a number of basolateral

proteins. In comparison, raft-associated Golgi-processed or mature proteins were mainly

found in Triton X-100- or Lubrol WX-specific DRMs (ALFALAH et al. 2005;

CASTELLETTI et al. 2008).

Different biosynthetic forms of PSMA, for example, can be differentiated from each other

through their capacity to be retained in various types of DRMs (ALFALAH et al. 2005;

CASTELLETTI et al. 2008). Mannose-rich PSMA of the ER is retained in Tween 20-DRMs,

whereas mature and complex glycosylated PSMA is associated with Lubrol WX-DRMs.

Therefore it could be hypothesized that a particular biosynthetic form associates preferentially

with a specific lipid environment and is transferred to another environment when its

maturation state is altered (CASTELLETTI et al. 2008; LINDNER and NAIM 2009).

Due to varying types of detergents not only different biosynthetic forms are isolated also

variants in the structural lipid components were detected. Triton X-100-DRMs show high

amounts of sphingolipids and cholesterol, whereas DRMs isolated with either Tween 20 or

Lubrol WX show decreased amounts of these two lipids; however they are enriched in other

INTRODUCTION

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lipids such as phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol

(CASTELLETTI et al. 2008). The lipid composition of Tween-20-DRMs is therefore in line

with lipids detected in the endoplasmic reticulum (low levels of cholesterol and

sphingolipids).

1.3.3. Size of lipid rafts

The evidence on the existence of lipid rafts in vivo has been put into question by several

aspects on their size and lifetime (HARDER et al. 1998; PRALLE et al. 2000; SHARMA et

al. 2004).

During the last few years improving techniques in electron microscopy alone or coupled with

spatial statistics, heterofluorescence and homofluorescence resonance energy transfer,

fluorescence quenching, fluorescence lifetime imaging microscopy, fluorescence recovery

after photobleaching, fluorescence correlation spectroscopy, raster scan image correlation

spectroscopy, and single particle tracking could reduce open questions (PIKE 2006). PIKE

(2006) summarized the question how small or how large a raft can be as followed:

“Complexes in the range of only a few nanometers, referred to as lipid shells or nanoclusters,

were thought to be too small and potentially similar to thermodynamic fluctuations occurring

near critical points in lipid phase diagrams. At the other end of the scale, large complexes

such as the immunological synapse, thought to be derived from the coalescence of multiple

smaller domains, were deemed too large and too complex to be considered as simple

membrane rafts.” Finally they determined membrane rafts as small (10–200nm),

heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that

compartmentalize cellular processes. Small rafts can coalesce to form larger platforms

through protein-protein and protein-lipid interactions. The upper limit of 200 nm was chosen

to include the surface area of caveolae, a member of the membrane raft family (PIKE 2006).

Size and composition of lipid rafts also vary according to the cell type (SCHUCK et al. 2003),

nutrition conditions (PERETTI et al. 2006) and the differentiation stage of the cells

(FITZNER et al. 2006).

On the other hand, Triton X-100-resistant membranes purified on flotation gradients turned

out to be 0.1–1 µm-sized vesicles (BROWN and ROSE 1992). Even if structures of similar

size and composition do not exist in living cells, these structures can be induced by cross-

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30

linking of putative lipid raft components (KUSUMI et al. 2004), by polarizing or activating

signals (GOMEZ-MOUTON et al. 2001), or by cell contact (GAUS et al. 2005).

1.3.4. Function of lipid rafts

Lipid rafts play an important role in many cellular processes, including membrane sorting and

trafficking, cell polarization, and signal transduction processes that have been best studied in

T-cells (JANES et al. 2000), B-cells (CHERUKURI et al. 2001), and the allergic response

(SHEETS et al. 1999; HOLOWKA and BAIRD 2001). Raft clustering is also involved in

ceramide/sphingomyelin signalling, which regulates several cellular functions, including cell

growth, survival, and death (KOLESNICK 2002). Several toxins, bacteria, prions, viruses and

parasites also use lipid rafts for their purposes (VAN DER GOOT and HARDER 2001). For

example, different viruses make use of lipid rafts to infect host cells. One of the best

characterized viruses is influenza virus. The two integral glycoproteins of the virus,

hemagglutinin and neuraminidase, are both raft-associated (ZHANG et al. 2000). Budding of

influenza virus from the apical membrane of epithelial cells takes place in lipid rafts

(SCHEIFFELE et al. 1999) and the virus preferentially includes raft lipids in its envelope

during budding. During this process polymerization of M proteins forms a layer facing the

cytosolic side of the nascent viral envelope and thus drives raft clustering (ZHANG et al.

2000). Also other viruses like HIV-1, Epstein-Barr virus, Filoviridae and Papillomaviridae

utilize lipid rafts to infect host cells (SIMONS and EHEHALT 2002). While entering host

cells bacteria avoid acidic lysosomes and consequent degradation by using raft-mediated

endocytic pathways. Other bacteria induce the activation of MAP-kinases as well as the

production of different cytokines by stimulating host cells via LPS and its GPI-anchored

receptor, which resides in lipid rafts (SOLOMON et al. 1998).

Apart from infections, lipid rafts also play an important role in different metabolic diseases

such as lysosomal storage diseases, diabetes, Alzheimer´s disease, Parkinson´s disease,

neuropathies and chronic inflammation. Moreover lipid rafts are involved in different

malignancies. An accumulation of cholesterol is detectable in various solid tumours,

including prostate cancer and oral cancer (FREEMAN and SOLOMON 2004;

KOLANJIAPPAN et al. 2003). Additionally, cholesterol metabolism is dysregulated in many

INTRODUCTION

31

other cancer types, such as myeloid leukemia, lung, and breast cancers (LI et al. 2003; EL-

SOHEMY and ARCHER 2000; BENNIS et al. 1993).

1.4. Endocytosis

The term “endocytosis” describes a process in which molecules gain entry into a cell without

passing through the cell membrane. According to DE DUVE (1963) endocytosis includes two

activities, known as phagocytosis and pinocytosis. The former is defined as the ingestion of

large particles (such as bacteria) in small vesicles. The latter describes the uptake of fluids or

macromolecules in vesicular structures (DE DUVE 1963).

In general, endocytosis provides many important cellular functions, including the uptake of

extracellular nutrients, regulation of cell-surface receptor expression, recycling of proteins

and lipids (SMYTHE and AYSCOUGH 2006), maintenance of cell polarity, and antigen

presentation (HARDING and GEUZE 1992). Endocytic pathways are also utilized by distinct

pathogens and symbiotic microorganisms to gain entry into cells (MUKHERJEE et al. 1997;

MELLMAN 1996).

The best-characterized form of endocytosis is receptor-mediated. This allows a selective

uptake of particular macromolecules. The molecules first bind to specific cell surface

receptors, which are concentrated in specialized regions of the plasma membrane, called

clathrin-coated pits. As a clathrin-coated pit invaginates, a GTP-binding protein, called

dynamin, forms a spiral around the neck of these pits. This results in the formation of clathrin-

coated vesicles containing the receptors and their bound molecules (ligands). Afterwards the

vesicles rapidly uncoat and fuse with early endosomes. These are vesicles with tubular

extensions located at the periphery of the cell. Fusion of endocytic vesicles with endosomes

requires interactions between complementary pairs of transmembrane proteins of the vesicles

and the target membranes (v-SNAREs and t-SNAREs) and Rab GTP-binding proteins. Early

endosomes act as molecular sorting stations and their contents are sorted for either transport

to lysosomes or recycling to the plasma membrane. This pathway is used by recycling

receptors, such as transferrin and low-density lipoprotein receptors (COOPER 2000).

INTRODUCTION

32

Another pathway of endocytosis, which is clathrin-independent, involves the uptake of

molecules in small invaginations of the plasma membrane, called caveolae. Caveolae are a

special type of lipid rafts, found in many eukaryotic cells, especially in endothelial cells and

adipocytes. Dynamin is also important for budding of caveolin-containing vesicles, known as

caveosomes, which can also fuse with early endosomes. Substances internalized by caveolin-

dependent endocytosis include toxins, viruses, bacteria and circulating proteins (MAYOR and

PAGANO 2007).

A third, distinct mechanism of internalization requires the action of the GTPase ARF6. It does

not use clathrin, caveolin or dynamin. This pathway is utilized by integrins, major

histocompatibility complex molecules and GPI-anchored proteins (MAYOR and PAGANO

2007).

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Figure 1-3. Different routes of endocytosis. Endocytosis can take place clathrin-dependent, caveolin-dependent or independent of both clathrin and caveolin, but requiring the action of the GTPase ARF6. Afterwards internalized vesicles fuse with early endosomes. Canonical early endosomes are characterized by the presence of the small GTPase RAB5, its adaptor molecule EEA1 and the lipid phosphatidylinositol-3-phosphate. These early endosomes serve as a sorting compartment, from which molecules are either recycled to the plasma membrane or transported to lysosomes for degradation (modified from GOULD and LIPPINCOTT-SCHWARTZ 2009).

During the process of endocytosis, the composition of distinct vesicles changes continuously

due to maturation or fusion with other compartments of the cell (COOPER 2000).

In polarized cells, internalized receptors can also be transferred across the cell to the opposite

domain of the plasma membrane - a process called transcytosis. For example, a receptor

INTRODUCTION

34

endocytosed from the basolateral domain of the plasma membrane can be sorted in early

endosomes for transport to the apical membrane.

As well as actin filaments, microtubules play an important role in endocytosis (BREITFELD

et al. 1990). During endocytosis the generation and movement of endocytic vesicles is

mediated by dynamic associations with the cytoskeleton. Furthermore, the maintenance of the

characteristic spatial distribution and morphology of endocytic organelles within the cell is

regulated by actin filaments and microtubules (NIELSEN et al. 1999). During this process,

RAB5 stimulates both association of early endosomes with microtubules and early-endosome

motility towards the minus ends of microtubules (NIELSEN et al. 1999). Several other studies

also show that the translocation and clustering of endosomes and lysosomes depends on

microtubules and that colchicine blocks the formation of endocytic invaginations, also

consistent with a block of endocytosis (MATTEONI and KREIS 1987; ELKJAER et al.

1995).

Furthermore, the microtubule motor proteins dynein and kinesin (VALE 1987; WOEHLKE

and SCHLIWA 2000) and the class I unconventional myosin motors are involved in different

steps of this process (MERMALL et al. 1998; TUXWORTH and TITUS 2000).

1.5. Microtubules

Like actin filaments, microtubules are a component of the cytoskeleton within the cytoplasm

of the cell. They are dynamic structures that undergo frequent assembly and disassembly.

Microtubules play an important role in a variety of cell movements, including some forms of

cell locomotion, the intracellular transport of organelles and the separation of chromosomes

during mitosis. In addition, they strongly influence the cell shape (COOPER 2000).

Microtubules are polymers of globular tubulin subunits and are shaped like a hollow cylinder.

The tubulin subunits are heterodimers of α- and β-tubulin, which are found in all eukaryotic

cells, and their sequences are highly conserved. A subunit rarely dissociates under normal

conditions, because the interactions holding α-tubulin and β-tubulin in a heterodimeric

complex are very strong.

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35

Multiple tubulin subunits are arranged in a cylindrical tube measuring 24 nm in diameter. The

length of these tubes can vary from a fraction of a micrometer to hundreds of micrometers,

and because of their particular tube-structure, microtubules are much stiffer than either

microfilaments or intermediate filaments. Longitudinal as well as lateral interactions between

the tubulin subunits are responsible for maintaining the tubular form. Neighbouring subunits

are connected into a linear protofilament due to longitudinal contacts between their heads and

their tails. These protofilaments are linked side by side due to lateral interactions and thus

form a sheet or cylinder (LODISH et al. 2000).

There are many proteins that bind to microtubules, including the microtubule-based motor

proteins (dynein, kinesin and related motors) during processes like endocytosis, and the

microtubule-associated proteins that play a more structural role.

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36

1.6. Aim of the study

Along the secretory pathway, PSMA is transported via different detergent-resistant

membranes (DRMs). After interacting with Tween 20-insoluble microdomains in the ER and

as soon as PSMA enters the Golgi, it associates with Lubrol WX-DRMs and this interaction is

either maintained or renewed once PSMA reaches the plasma membrane (CASTELLETTI et

al. 2008). However, so far nothing is known about the role of different microdomains during

activation and internalization of PSMA. Therefore it was my aim to investigate the

assessment of the modulatory effect of antibody binding on the association of PSMA with

distinct DRMs.

Furthermore these microdomains are described as platforms for different protein-protein,

protein-lipid or lipid-lipid interactions.

I investigated potential interaction partners of PSMA in these DRMs, which trigger the

signalling capacity of PSMA. For this purpose I utilized 2-dimensional gel electrophoresis to

compare the expression levels of particular proteins in DRMs before and after antibody-

induced activation of PSMA. Varying proteins were analysed by mass spectrometry and

checked for direct interactions with PSMA by confocal analysis and co-immunoprecipitation

experiments.

In addition to humans, dogs are the only mammalian species that develop prostate cancer

spontaneously. Also in dogs prostate cancer is most commonly found in elderly patients

(WATERS et al. 1996) and as well as in humans, almost all of these tumours are

adenocarcinomas with metastasis most commonly found in lungs, lymph nodes and bones.

LAI et al. (2008) could show that canine prostate cancer is very aggressive and of a less

differentiated type than most common human prostate cancers. Therefore it mostly resembles

human, androgen refractory, poorly differentiated prostate cancer with usually a bad

prognosis. In this respect, the dog is of great importance as a model for this disease, filling a

gap between rodent model studies and human clinical trials.

Recent studies using immunohistochemistry revealed controversial results regarding the

expression of PSMA in prostatic tissue of dogs, for example one group could only detect

PSMA in the prostate of castrated animals (LAI et al. 2008; AGGARWAL et al. 2006).

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37

However, results obtained by RT-PCR showed clear expression of PSMA transcripts in the

canine prostate that was enhanced fivefold in carcinomas (LAI et al. 2008).

Therefore another objective was to identify a homologous protein to human PSMA in prostate

tissue of dogs (cPSMA). This was followed by cloning and recombined expression of this

antigen in different cell lines such as COS-1 cells and MDCK cells. As initial steps for

developing diagnostic and therapeutical approaches against prostate cancer in dogs, further

experiments were done on the biosynthesis, the posttranslational processing, the sorting as

well as the association of cPSMA with different detergent-resistant membranes.

CHAPTER ICHAPTER ICHAPTER ICHAPTER II:I:I:I:

Discriminatory role of detergentDiscriminatory role of detergentDiscriminatory role of detergentDiscriminatory role of detergent----resistant resistant resistant resistant

membranes in the dimerizatiomembranes in the dimerizatiomembranes in the dimerizatiomembranes in the dimerization and n and n and n and

endendendendocytosis of pocytosis of pocytosis of pocytosis of prostaterostaterostaterostate----specific membrane specific membrane specific membrane specific membrane

antigenantigenantigenantigen

Running title:

Dimerization and endocytosis of PSMA

DIMERIZATION AND ENDOCYTOSIS OF PSMA

41

Discriminatory role of detergent-resistant membranes in the dimerization and endocytosis of prostate-specific membrane antigen Sonja SCHMIDT*, Giulio FRACASSO†, Dunia RAMARLI†, Marco COLOMBATTI† and Hassan Y. NAIM*

*Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany and †Department of Pathology and Diagnostics, University of Verona, Policlinico G.B. Rossi, P. le L.A. Scuro n.10, 37134 Italy Running title: Dimerization and endocytosis of PSMA ___________________________________________________________________________ Prostate-specific membrane antigen (PSMA) is a surface protein overexpressed in prostate cancer and neovasculature of further tumours. It is therefore one of the most promising biomarkers in diagnosis and treatment of prostate cancer. PSMA is associated with detergent-resistant membranes (DRMs). The mature form of PSMA is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. Recently we could demonstrate that antibody-induced cross-linking of cell surface PSMA activates different signalling cascades like the MAPK-pathway. Here we show that internalization of PSMA is increased after antibody binding and this leads to redistribution of PSMA to Triton X-100-DRMs in a time-dependent manner and to alterations in the expression levels of several proteins in these DRMs. One of the proteins also clustering in Triton X-100-DRMs is α-tubulin and we could show that PSMA is internalized via microtubules. Our results propose an essential role for different DRM types in the signalling pathways involving PSMA. Keywords: Prostate cancer, PSMA, lipid rafts, dimerization, endocytosis, microtubules ___________________________________________________________________________ Abbrevations used: PSMA, prostate-specific membrane antigen; PSMAC, complex glycosylated form of PSMA; PSMAM, mannose-rich form of PSMA; DRM, detergent-resistant membrane; mAb, monoclonal antibody; ER, endoplasmic reticulum; LPH, lactase phlorizin hydrolase 1 To whom correspondence should be addressed: Hassan Y. Naim, PhD, Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany, Tel.: 0049511 / 953-8780; Fax: 0049511 / 953-8585; Email: [email protected]


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INTRODUCTION Adenocarcinomas of the prostate are one of the most common malignancies in men in developed countries. Conventional treatment like prostatectomy or radiation can only be sufficient if prostate cancer is diagnosed at an early stage. Prostate-specific membrane antigen (PSMA) is a type-II-transmembrane-glycoprotein with folate hydrolase and carboxypeptidase activity (1), found initially in LNCaP cells by immunoprecipitation (2). PSMA is expressed in epithelial cells of the prostate and at low levels also in some other organs like kidney, intestine and brain (3,4). Elevated levels of PSMA are detected in prostate cancer cells including those that are metastatic (5,6). Levels of PSMA are directly proportional to disease grade and stage (7). Also in neovasculature of other non prostatic tumours PSMA expression has been detected, but it is not present in healthy vasculature (8,9). In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in endosomes (10). Furthermore PSMA associates with the actin cross linking protein filamin A and this association is involved in the localization of PSMA to the recycling endosomal compartment (11). In endothelial cells internalization of PSMA is caveolae-dependent and an interaction with caveolin 1 could be detected (12). Rajasekaran et al. could demonstrate that the cytoplasmic tail five N-terminal amino acids MXXXL are sufficient to mediate the internalization of PSMA (13). As a consequence PSMA is one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. Antibodies conjugated to cytotoxic drugs are currently in clinical trials for use in mAb mediated immunotherapy (14-18). Different specific mAbs conjugated to cytotoxic drugs have shown the ability to induce apoptosis, especially in cells expressing high levels of PSMA on their surface, like prostate cancer cells. However the function of PSMA, the direct correlation between its expression and increasing tumour aggressiveness in prostate cancer and details about internalization still remain unclear. To further understand the mechanism of PSMA internalization we investigated PSMA during internalization in lipid rafts or detergent-resistant membranes (DRMs). Lipid rafts are described as dynamic, nanoscale, sterol-sphingolipid-enriched, ordered assemblies of proteins and lipids, in which the metastable raft resting state can be stimulated to coalesce into larger, more stable raft domains by specific lipid-lipid, protein-lipid and protein-protein oligomerizing interactions (19). These rafts are involved in signalling processes, trafficking and endocytosis. Extraction with distinct detergents allows isolation of DRMs with different composition (20,21). Triton X-100-DRMs are enriched in sphingolipids and cholesterol, whereas Tween 20-DRMs as well as Lubrol WX-DRMs show decreased amounts of these two lipids. In contrast phosphatidylethanolamine is increased approx. 6- and 8-fold in Tween 20- and Lubrol WX-DRMs respectively. Along the secretory pathway PSMA is transported via different microdomains. After interacting with Tween 20-insoluble microdomains and as soon as PSMA enters the Golgi, it associates with Lubrol WX-DRMs and this interaction is either maintained or renewed once it reaches the plasma membrane (21). In this study we are interested in the association of PSMA with these different DRMs during internalization. Therefore we induce internalization by antibody cross-linking of PSMA. We investigated already that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during this process of activation. As downstream effects of the activation we observed a strong induction of NF-kB activation associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically (22).


43

Due to these facts we hypothesize a fundamental role of DRMs during activation and internalization of PSMA as platforms for signalling, trafficking and endocytosis. In this study we demonstrate for the first time that [1] homodimers of PSMA are associated with Lubrol WX-DRMs, [2] antibody-induced cross-linking of these PSMA molecules results in a partitioning of PSMA and α-tubulin into Triton X-100-DRMs and [3] concomitantly internalization of PSMA occurs along tubulin filaments. Along these lines an essential role for different types of DRM in the signalling pathways implicating PSMA can be proposed. EXPERIMENTAL Reagents and antibodies Tissue culture material was purchased from Sarstedt, RPMI-Medium and Dulbecco´s Modified Eagle´s Medium and supplemented reagents (penicillin-streptomycin, fetal calf serum) were from PAA Laboratories. Triton X-100, proteinase inhibitors, protein A-sepharose and bovine serum albumin were from Sigma. Acrylamide, TEMED, SDS, Tris, sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium di-hydrogen phosphate, sodium deoxycholate, paraformaldehyde, saponin, chaps, glutathione, urea, thiourea, DTT and PVDF membranes were purchased from Carl Roth. The ECL plus Western Blotting Detection System and Biotin were from Thermo Scientific. Lubrol WX was from MP Biomedicals, ammonium chloride from Merck and coomassie blue G-250 from Serva. The anti-PSMA mAb D2B recognizing a luminal epitope of PSMA was produced in our laboratory (22). 7e11c antibody recognizing a cytosolic epitope of PSMA was purified from an ATCC hybridoma. Horseradish peroxidase-coupled goat anti-mouse antibody was from Dako. The anti-tubulin antibodies were from Sigma and secondary antibodies coupled to Alexa Flour dyes were purchased from Invitrogen. Cell culture LNCaP cells and CHO cells were cultured in humidified atmosphere containing 5% CO2 in air at 37°C in RPMI-Medium containing 2 g/L glucose in the presence of fetal calf serum (10% v/v) and penicillin-streptomycin (100 U/ml and 0.1 mg/ml respectively). COS-1 cells and MDCK cells were cultured under equal conditions in Dulbecco´s Modified Eagle´s Medium (DMEM) containing 1 g/L glucose, fetal calf serum and penicillin-streptomycin. Activation of PSMA by antibody-induced cross-linking Activation of PSMA by antibody-induced cross-linking was performed according to the protocol described in (22). Briefly LNCaP cells that reach 70% confluence were incubated with 5 µg/ml of the appropriate mAb for 45 minutes at room temperature, washed and placed at 37°C for 15 minutes with 10 µg/ml goat-anti-mouse antibody to induce the clustering of PSMA molecules. DRM extraction LNCaP cells that reach 70% confluence were washed twice in ice-cold PBS and solubilized in PBS containing 1% (w/v) detergent (Lubrol WX or Triton X-100 respectively) supplemented with a mixture of proteinase inhibitors. Cells were homogenized with a Luer-


44

21 Gauge needle 20times/ml and then maintained on ice for 2 hours. Afterwards, samples were pre-centrifuged 15 minutes at 8.000 x g and cell debris was discarded followed by a centrifugation at 100.000 x g, 4°C for 90 minutes (Beckman Optima LE-80, SW 55Ti-rotor). The supernatant and pellet obtained, corresponding to the soluble and insoluble fractions respectively, were separately analysed. The pellet fractions were lysed over night in a lysis-buffer containing 25 mM Tris, 50 mM sodium chloride, 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate supplemented with a mixture of proteinase inhibitors. On the next day the protein amounts of all fractions were determined using Bradford protein assay (Bio-Rad) and 50 µg protein of each fraction was analysed by SDS/PAGE and western blotting. Immunoassays PSMA was immunoprecipitated from supernatant and pellet fractions after DRM extraction from LNCaP cells. Immunoprecipitation was performed by using D2B mAb for 1 hour at 4°C. Antigen-antibody complexes were then recovered with protein A-sepharose over night, denatured and loaded on SDS/PAGE followed by western blot analysis. Specific bands on PVDF membranes detected by the appropriate antibodies were visualized by a ChemiDoc XRS Molecular Imager (Bio-Rad) device. Digital images obtained were quantified by using image processing and analysis software ImageJ. For indirect immunofluorescence, cells were fixed in 4% paraformaldehyde in PBS. Quenching was performed twice for 10 minutes in 50 mM ammonium chloride in PBS. Blocking and addition of the antibodies were performed in PBS containing 1% bovine serum albumin and 0.5% saponin. Preparations were visualized using a Leica TCS SP5 confocal laser microscope with an x 63 oil planapochromat lens (Leica Microsystems, Wetzlar). Analysis of the quaternary structure Monomers and dimers of PSMA were separated by sucrose density gradients under native conditions. LNCaP cells were lysed for 2h in PBS containing 1% of Triton X-100 supplemented with a mixture of proteinase inhibitors. After a centrifugation step at 8.000 x g to remove cell debris, cell lysates were loaded on top of 5-25% (w/v) continuous sucrose gradients which were centrifuged at 100.000 x g for 18h at 4°C (Beckman Optima LE-80, SW 40Ti-rotor). 24 fractions of 500µl each were collected from bottom to top. Half of each fraction was precipitated using ethanol and PSMA was detected by western blot analysis. For analysis of the quaternary structure of PSMA in Lubrol WX-DRMs we prepend a step of DRM extraction followed by lysis of the pellet for 2h in PBS containing 1% of Triton X-100 supplemented with a mixture of proteinase inhibitors. Afterwards the pellet and supernatant containing Lubrol WX-DRMs and soluble proteins respectively were loaded on top of continuous sucrose density gradients. Biotinylation of surface proteins 1 µg/ml Biotin-NHS in PBS was added to PBS-washed LNCaP cells and left on ice for 30 minutes. The cells were then washed with ice-cold PBS, quenched twice with 0.1% bovine serum albumin in PBS for 10 minutes and washed again twice with PBS. Afterwards, internalization of PSMA was induced by activation via antibody cross-linking. Remaining biotin on surface proteins was then cleaved by glutathione (1.5 mg/ml). Cells were washed again twice with PBS, solubilized and DRMs were extracted as described above. Immunoprecipitation with anti-PSMA antibody (D2B) was performed and immunoprecipitates were subjected to SDS/PAGE and western blot analysis. The


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chemiluminescent detection of biotinylated PSMA was carried out using streptavidin horseradish peroxidase (Amersham). 2-dimensional gel electrophoresis After activation of PSMA and DRM extraction protein concentrations of pellet fractions were determined using Bradford protein assay. Then equal amounts of protein (400 µg) were precipitated with ethanol over night at -20°C. After centrifugation (10.000 x g, 20min, 4°C) pellets were air-dried and resuspended in rehydration buffer containing 7 M urea, 4% (v/v) chaps, 2 M thiourea and 18 mM DTT for 2 hours at room temperature followed by isoelectric focussing on ReadyStrip IPG Strips (Bio-Rad). Afterwards strips were used for SDS/PAGE as second dimension. Gels were stained with colloidal coomassie G-250 and quantified by using ImageJ. Statistical analysis Statistical analysis of results was performed using GraphPad Prism5 applying the parametric paired or unpaired t test (depending on the experimental setup). Significance was accepted when p<0.05. RESULTS Dimers of PSMA are associated with Lubrol WX-DRMs LNCaP cells were solubilized with two different detergents, Lubrol WX and Triton X-100, and DRMs were extracted. As previously reported (21) mature PSMA is completely soluble in Triton X-100 and is partially insoluble in Lubrol WX (Fig. 1A). Castelletti et al. (21) could also show that as soon as PSMA enters the Golgi, it associates with Lubrol WX-DRMs and this interaction is either maintained or renewed once it reaches the plasma membrane. Homodimerization is also taking place in the Golgi (21), but so far nothing is known about the quaternary structure of PSMA associated with these special types of Lubrol WX-DRMs. To determine the quaternary structure of PSMA associated with Lubrol WX-DRMs, we performed continuous sucrose gradients (5 – 25%) based on the ability of dimers to migrate to fractions with higher sucrose density than monomers. As shown in Fig. 1B (also previously shown in ref. (21)) the monomeric form of PSMA peaks in fractions 16-19 where the mannose-rich glycosylated form (PSMAM) can be distinguished from its complex glycosylated counterpart (PSMAC). Dimers of PSMA are located in fractions 13-15 that exclusively contains complex glycosylated PSMA (PSMAC). To analyse the quaternary structure of PSMA in Lubrol WX-DRMs, cells were first solubilized in Lubrol-WX and DRMs were extracted by ultracentrifugation. After extraction of the insoluble (P) Lubrol WX-DRMs and the soluble (S) material, these were individually loaded on top of a 5-25% continuous sucrose gradient. PSMA in Lubrol WX-DRMs migrates like the dimeric form of PSMA, peaking in fractions 14 and 15. Detergent-soluble PSMA was distributed in the later fractions (16 and 17), which contains both the mannose-rich and the complex glycosylated forms in their monomeric state. Taken together, it can be concluded that homodimerization of PSMA is required for its association with Lubrol WX-DRMs.


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Appearance of PSMA in Triton X-100-DRMs upon activation Upon ligand or antibody cross-linking, some plasma membrane receptors undergo enhanced partitioning into sphingolipid-cholesterol membrane microdomains as an obligatory first step toward participation in early signal transduction events (23-25). Experimentally, these microdomains can be isolated based on their insolubility in cold, non ionic detergents (26). Upon solubilization of cellular extracts with a detergent, detergent-resistant membranes (DRMs) can be recovered by ultracentrifugation or in the floating fractions of sucrose-gradients. For PSMA both procedures revealed comparable results, so that we utilized here the ultracentrifugation approach through out. Here, LNCaP cells were activated by antibody-induced cross-linking and then Lubrol WX- as well as Triton X-100-DRMs were extracted. As previously reported (21) non activated mature PSMA is mainly insoluble in Lubrol WX and does not associate with Triton X-100-DRMs. Strikingly, activation of PSMA by antibody-induced cross-linking results in redistribution of PSMA to Triton X-100-DRMs. To underline the specificity of PSMA clustering in DRMs upon activation we followed three approaches. In the first we used LNCaP cells in conjunction with an antibody that is directed against the cytoplasmic tail of PSMA (7e11c), i.e. do not bind the extracellular luminal part of the protein. The second approach employed mAb D2B in MDCK cells stably expressing canine PSMA. This antibody does not recognize the canine PSMA (data not shown). Finally we used CHO cells that stably express intestinal lactase phlorizin hydrolase (LPH) that does not undergo internalization and utilized a mAb anti-LPH antibody that is directed against an epitope on the luminal domain of this protein. In all these control experiments neither clustering nor association of PSMA, canine PSMA and LPH with Triton X-100 DRMs could be detected. The clustering and subsequent association of PSMA with Triton X-100 DRMs occurs in a time-dependent manner as shown in Fig. 3. In fact, increased levels of PSMA are recovered in DRMs with increasing time points and reach a plateau between 45 and 60 min. To examine a possible correlation between clustering into Triton X-100-DRMs and internalization of PSMA after antibody cross-linking we performed the activation assay at 4°C where internalization is blocked. Redistribution of PSMA to Triton X-100-DRMs occurs also after activation at 4°C. Therefore distribution of PSMA into Triton X-100-DRMs is likely to be independent of internalization. In conclusion, antibody-induced cross-linking of PSMA at the surface results in a major change in its detergent solubility properties and addressed in what follows the role of DRMs during activation of PSMA. Internalization of PSMA is mediated by its association with DRMs The clustering of PSMA occurs already at the cell surface. We wanted to determine whether this event is required for internalization. Fig. 4A shows that PSMA is internalized and appears in vesicular structures after activation with antibody cross-linking at the cell surface. To confirm these data at the protein level the plasma membrane of LNCaP cells was biotinylated and internalization of PSMA was induced by antibody cross-linking. Fig. 4B (upper panel) demonstrates a strong protein band corresponding to internalized PSMA in activated cells. The glutathione-treated and non-treated activated cells revealed almost similar band intensity of mature PSMA indicating that the internalization of PSMA is almost complete. On the other hand the non activated cells revealed faint bands corresponding to complex (PSMAC) as well as mannose-rich glycosylated PSMA (PSMAM). This is likely due to (i) slight diffusion of biotin through the membrane labelling thus both intracellular forms of PSMA or (ii) due to incomplete action of glutathione. Nevertheless this does not alter the


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significance of this result, which clearly demonstrates that PSMA undergoes substantial internalization upon antibody cross-linking. To investigate the role of Triton X-100-DRMs during internalization DRM extraction was performed. The data (Fig. 4B, lower panel) clearly show that biotinylated PSMA in the antibody-activated samples was localized in Triton X-100-DRMs in contrast to PSMA in the non-activated cells. Again the supernatant of non activated cells shows a faint band of lower molecular weight likely corresponding to the high-mannose glycosylated form of PSMA (PSMAM) which is presumably due to slight diffusion of biotin through the membrane. Identification of candidate proteins potentially interacting with PSMA in DRMs Antibody-induced internalization of PSMA is involved in signalling processes (22) and interactions of PSMA with different proteins like the actin cross-linking protein filamin A (11) and clathrin as well as adaptor protein complex-2 (27) have been shown to participate in internalization of PSMA. To identify additional proteins potentially interacting with PSMA the Triton X-100-DRMs of non activated cells were compared with those of activated cells by 2D-gel electrophoresis. Mean relative spot volumes and differences in expression were calculated using ImageJ and the differing spots were analysed by mass spectrometry followed by Swiss-Prot database search with MASCOT to assign identities. As shown in Fig. 5A the expression levels of a number of proteins varied in Triton X-100-DRMs due to antibody-induced activation of PSMA. One of these proteins is tubulin that increased substantially in Triton X-100-DRMs isolated from activated cells. To confirm this finding by western blot analysis LNCaP cells were activated or non-activated by antibody-induced cross-linking followed by Triton X-100-DRM extraction. Fig. 5B shows a significant clustering of α-tubulin in Triton X-100-DRMs upon activation. Also the levels of β-tubulin increase in these DRMs, yet not to the same extent as α-tubulin (Fig. 5B). These findings strongly suggest that microtubules are involved in internalization of PSMA. The colocalization of PSMA before and after cross-linking and internalization with α-tubulin was examined in COS-1 cells that transiently express the YFP-tagged form of PSMA. Fig. 5D revealed YFP-PSMA in the Golgi apparatus and at the cell surface in non-activated COS-1 cells. α-tubulin that was detected by indirect immunofluorescence did not colocalize with YFP-PSMA in these cells. Upon antibody-induced cross-linking of PSMA internalization occurred and several endosomes containing YFP-PSMA can be observed to colocalize with α-tubulin along or in close vicinity to the cell surface (Fig 5E). These observations together suggest that YFP-PSMA is transported along the tubulin filaments. DISCUSSION PSMA is a potential target for immunotherapy of prostate cancer (10,28), as well as for solid tumours due to its de novo expression in tumour neovasculature (8,9). In this manuscript we addressed the role of lipid rafts in two major physiological events that are of pivotal importance in the life cycle of PSMA, homodimerization and internalization. Homodimerization of PSMA is required for its enzymatic activity of PSMA (29) and occurs late along the secretory pathway (21). Our results demonstrate that the transition from monomeric to dimeric state is linked to the formation of a complex glycosylated form of PSMA and to the association of the formed PSMA homodimers with Lubrol WX-DRMs. Other biosynthetic forms of PSMA, such as the mannose-rich monomeric form favour a different membrane environment that is reflected by their association with Tween 20-DRMs in the ER and prior to their exit from this organelle (21). The dimeric form of PSMA is retained in Lubrol WX-DRMs at the cell surface (21) suggesting that dimerization and


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association with this type of DRMs are important for a full biological activity of PSMA particularly towards its interaction with its natural ligand. This interaction is mimicked in our study by cross-linking of homodimeric PSMA at the cell surface with anti-PSMA antibodies. As a consequence two major dramatic events occur: The first is an efficient internalization of PSMA with an almost complete disappearance of PSMA from the cell surface and appearance as discrete intracellular spots; the second is a shift of dimeric PSMA from Lubrol WX-DRMs to a different membrane environment that is enriched in cholesterol and sphingolipids and exemplified by Triton X-100-DRMs or lipid rafts. A major component that is associated with these rafts and PSMA is α-tubulin (Fig. 6). In endothelial cells internalization of PSMA has been shown to implicate its interaction with caveolin 1 (12) thus suggesting that lipid rafts may play a role in this event since caveolin is a major structural protein of caveolae and these in turn represent a special type of lipid rafts. In line with these observations our time-dependent endocytosis of PSMA provides an unequivocal support to the notion that lipid rafts are directly implicated in the clustering and internalization of PSMA. The internalization of PSMA involves its interaction with filamin A, an actin cross linking protein, and this association is involved in the localization of PSMA to the recycling endosomal compartment (11). Our data extends the role played by the cytoskeleton to include α-tubulin clearly indicating that microtubules are key players in PSMA endocytosis. While cross-linking of PSMA with antibodies and eliciting internalization could be considered to be an artificial system, cross-linking of membrane proteins is normally a physiological phenomenon that can lead to redistribution of these proteins into lipid rafts, resulting in novel protein interactions and initiation of cell signalling (24,30). Although occurring naturally via multivalent ligands, similar responses have been observed using antibodies (24). In previous studies we could demonstrate that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during this process of activation. As downstream effects we observed a strong induction of NF-kB activation associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically (22). Comparable effects are already described for the Myelin Oligodendrocyte Glycoprotein (31,32) and the T-cell receptor (31). In both cases antibody-induced cross-linking leads to redistribution to Triton X-100-DRMs coupled to downstream signalling cascades. Along these lines we strongly propose an essential role for lipid rafts or DRMs in endocytosis and signalling pathways involving PSMA. The microtubule cytoskeleton is particularly important for polarized targeting of apical cargo. Microtubule depolymerization results in aberrant delivery of several apical proteins, including PSMA, to the basolateral surface (33). But until present few information, if any, is available on the role of microtubules during internalization of PSMA. We could show that α-tubulin as well as partially β-tubulin are together with PSMA redistributed to Triton X-100-DRMs upon activation of PSMA by antibody-induced cross-linking. Immunofluorescence revealed that PSMA undergoes internalization along α-tubulin, suggesting necessity of microtubules for internalization of PSMA. In essence, understanding the molecular mechanisms underlying the antibody-induced cross-linking, deciphering the membrane structures and components governing this event as well as the internalization of PSMA constitute essential prerequisites for utilization of PSMA as a therapeutically suitable target in prostate cancer.


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ACKNOWLEDGEMENTS AND FUNDING We thank Falk Büttner and Dennis Kahlisch (University of Veterinary Medicine Hannover, Germany) for mass spectrometry analysis. This work was supported by AIRC 5x1000 project Application of Advanced Nanotechnology in the Development of Innovative Cancer Diagnostics Tools (funding attributed to M.C.), by the University of Verona, by Fondazione Cariverona project "Verona Nanomedicine Initiative" (to M.C.) and by Fondazione Cariverona/AIRC, Progetto Regione Veneto (to M.C.).


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FIGURE LEGENDS Figure 2-1 PSMA associates with Lubrol WX-DRMs as a dimeric protein (A) LNCaP cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. Lysates were subjected to centrifugation at 100.000 x g for 90 min in order to separate the insoluble (P) DRM-fraction and the soluble (S) material. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of total protein from both fractions were loaded for each lane on SDS gels and blotted. (B) After centrifugation the insoluble fraction (P) was solubilized with 1% (w/v) Triton X-100 and 1ml of both insoluble (P) and soluble (S) fraction was loaded on top of a 5-25% continuous sucrose gradient. After 18 h of centrifugation at 100.000 x g, 24 fractions of 500µl each were collected from bottom to top. Half of each fraction was precipitated using ethanol and PSMA was detected by western blot analysis. Figure 2-2 PSMA associates with Triton X-100-DRMs upon activation (A) LNCaP cells were activated by antibody-induced cross-linking of PSMA at 37°C or 4°C. Subsequently non activated and activated cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. DRMs (P) and soluble (S) fractions were isolated as described in Fig. 1. Equal amounts of total protein from all fractions were loaded for each lane on SDS gels and blotted. The distribution of PSMA activated with 7e11c mAb, canine PSMA activated with D2B mAb as well as LPH activated with 909 mAb were analysed as negative controls. Western blots were developed using D2B mAb for PSMA and anti-GFP mAb for canine PSMA and LPH. (B) Statistical analysis of results was performed using GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was accepted when p<0.05. Figure 2-3 Time course of association of PSMA with Triton X-100-DRMs (A) LNCaP cells were activated by antibody-induced cross-linking of PSMA for different time periods. Subsequently non activated and activated cells were solubilized with 1% (w/v) Triton X-100 in PBS. Lysates were subjected to centrifugation at 100.000 x g for 90 min in order to separate the insoluble (P) DRM-fractions. Equal amounts of total protein from each fraction were further analysed by western blotting. (B) Statistical analysis of results was performed using GraphPad Prism5 applying the parametric paired t test. Significance was accepted when p<0.05. Figure 2-4 Internalization of PSMA is followed by its association with Triton X-100-DRMs (A) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were either activated by antibody-induced cross-linking of PSMA or directly visualized using a Leica TCS SP5 confocal laser microscope. (B) LNCaP cells were cell-surface biotinylated for 30 min on ice. Cells were washed and quenched twice with 0.1% bovine serum albumin. Afterwards, internalization of PSMA was induced by antibody cross-linking. Remaining biotin on surface proteins was then cleaved by glutathione. Cells were


54

washed again, solubilized with Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and PSMA was immunoprecipitated from both insoluble (P) and soluble (S) fractions. Immunoprecipitates were subjected to SDS/PAGE and western blot analysis. The chemiluminescent detection of biotinylated PSMA was carried out using streptavidin horseradish peroxidase. * PSMAC ^ PSMAM Figure 2-5 Identification of proteins potentially interacting with PSMA (A) LNCaP cells were either activated by antibody-induced cross-linking of PSMA or subsequently solubilized with 1% Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of protein (400 µg) were precipitated with ethanol over night. Pellets were resuspended in rehydration buffer and 2-dimensional gel electrophoresis was performed. Gels were stained with colloidal coomassie G-250 and protein spots were quantified by using ImageJ. (B) LNCaP cells were either activated by antibody-induced cross-linking of PSMA or subsequently solubilized with 1% Triton X-100 and DRMs were extracted. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate. Equal amounts of total protein from all fractions were loaded for each lane on SDS gels and α- as well as β-tubulin concentrations were detected by western blot analysis. (C) Statistical analysis of results was performed using GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was considered at p<0.05. (D) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were fixed and α-tubulin was detected by indirect immunofluorescence. (E) COS-1 cells were transiently transfected with cDNA encoding YFP-PSMA. Two days after transfection cells were activated by antibody-induced cross-linking of PSMA, fixed and α-tubulin was detected by indirect immunofluorescence. Figure 2-6 Scheme of DRM-associated internalization of PSMA PSMA is transported to the Golgi where it associates with Lubrol WX-DRMs. Likewise, homodimeric forms as well as cell surface forms of PSMA associate with similar type of DRMs at the cell surface. Antibody-induced cross-linking of PSMA homodimers results in a partitioning of PSMA and α-tubulin into Triton X-100-DRMs. Concomitantly internalization of PSMA occurs along α-tubulin filaments.


55

FIGURES Figure 2-1 A

B

222120monomersdimers121110Bottom

22212019181716151413121110Bottom

222120monomersdimers121110Bottom

22212019181716151413121110Bottom

PS

MA

fr

om c

ell

lysa

te

PS

MA

from

Lu

brol

WX

-D

RM

s

PS

MA

so

lubl

e in

Lu

brol

WX

PSMAC

PSMAM

PSMAC

PSMAM

PSMAC

P S

Triton X-100

P S

Lubrol WX


56

Figure 2-2 A

SS PP

nonactivated activated

PSMA

PSMA

PSMA

caninePSMA

LPH

PSMA

Lubrol WX-DRMs

Triton X-100-DRMs

Triton X-100-DRMs

4°C

antibody for activation

D2B

D2B

D2B

7e11c

909

D2B

SS PP


PSMA

PSMA

PSMA

caninePSMA

LPH

PSMA

Lubrol WX-DRMs

Triton X-100-DRMs

Triton X-100-DRMs

4°C

antibody for activation

D2B

D2B

D2B

7e11c

909

D2B


57

B

LPH activated with 909

P nAct

S nAct

P Act

S Act

0

20

40

60

80

Triton X-100-DRMs

%

PSMA activated with D2B,4°C

P nAc t

S nAc t

P Act

S Act

0

50

100

150

*

Triton X-100-DRMs

%

canine PSMA activatedwith D2B

P nAct

S nAct

P Act

S Act

0

20

40

60

80

100

Triton X-100-DRMs

%

PSMA activated with 7e11c

P nAct

S nAct

P Act

S Act

0

20

40

60

80

100

Triton X-100-DRMs

%

PSMA activated with D2B

P nAct

S nAct

P Act

S Act

0

50

100

150

Lubrol WX-DRMs

%

PSMA activated with D2B

P nAct

S nAct

P Act

S Act

0

50

100

150

*

Triton X-100-DRMs%


58

Figure 2-3

PSMA in Triton X-100 DRMs

0 5 10 30 45 600

1

2

3

4

5

activation time (min)

%Activation Time (min)

PSMA in Triton X-100-DRMs

0 5 10 30 45 60

A

B

***

***

PSMA in Triton X-100 DRMs

0 5 10 30 45 600

1

2

3

4

5

activation time (min)

%Activation Time (min)


0 5 10 30 45 60

A

Activation Time (min)


0 5 10 30 45 60

Activation Time (min)


0 5 10 30 45 60

A

B

***

***


59

Figure 2-4

2 days after transfectionnon activated cells

2 days after transfectionactivated cells

-GlutathioneGlutathione

activatedactivatednon activated

Biotinylation

P S P S P S

biotinylatedPSMA

biotinylatedPSMA

DRMs

A

B

*^

*̂

2 days after transfectionnon activated cells

2 days after transfectionactivated cells

-GlutathioneGlutathione

activatedactivatednon activated

Biotinylation

P S P S P S

biotinylatedPSMA

biotinylatedPSMA

DRMs

A

B

*^

*̂


60

Figure 2-5 A

Triton X-100-DRMs, non activated

Triton X-100-DRMs, activated

tubulin

Triton X-100-DRMs, non activated

Triton X-100-DRMs, activated

tubulin

α-tubulin

β-tubulin

SS PP


B Cαααα-tubulin

P nAct

S nAct

P Act

S Act

0

20

40

60

80

100

*

DRMs

%

ββββ-tubulin

P nAct

S nAct

P Act

S Act

0

50

100

150

DRMs

%

α-tubulin

β-tubulin

SS PP


α-tubulin

β-tubulin

SS PP


SS PP


B Cαααα-tubulin

P nAct

S nAct

P Act

S Act

0

20

40

60

80

100

*

DRMs

%

ββββ-tubulin

P nAct

S nAct

P Act

S Act

0

50

100

150

DRMs

%


61

D

YFP-PSMA α-tubulin mergeYFP-PSMA α-tubulin merge

E

YFP-PSMA α-tubulin mergeYFP-PSMA α-tubulin merge


62

Figure 2-6

PSMA in Lubrol WX-DRMs of the cell membrane

PSMA in Triton X-100-DRMs of the cell membrane

Downstream signalling

PSMA and α-tubulin in Triton X-100-DRMs of endosomes

α-tu

bulin

endosome

PSMA homodimer

cholesterol

sphingolipids

phospholipids

CHAPTER III:CHAPTER III:CHAPTER III:CHAPTER III:

Cloning and characterisation of canine Cloning and characterisation of canine Cloning and characterisation of canine Cloning and characterisation of canine

prostateprostateprostateprostate----specispecispecispecific membrane antigenfic membrane antigenfic membrane antigenfic membrane antigen

Running head:

PSMA in dogs

PSMA IN DOGS

65

Cloning and characterisation of canine prostate-specific membrane antigen

Sonja Schmidt1, Giulio Fracasso2, Marco Colombatti2 and Hassan Y. Naim1

1Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Germany

2 Department of Pathology and Diagnostics, University of Verona, Italy

To whom correspondence should be addressed: Hassan Y. Naim, PhD, Department of

Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-

30559 Hannover, Germany, Tel.: 0049511 / 953-8780; Fax: 0049511 / 953-8585; Email:

[email protected]

Running head: PSMA in dogs

Acknowledgments

This work was supported by AIRC 5x1000 project Application of Advanced Nanotechnology

in the Development of Innovative Cancer Diagnostics Tools (funding attributed to M.C.),

by the University of Verona, by Fondazione Cariverona project "Verona Nanomedicine

Initiative" (to M.C.) and by Fondazione Cariverona/AIRC, Progetto Regione Veneto (to

M.C.).

PSMA IN DOGS

66

Abstract

BACKGROUND. Prostate-specific membrane antigen (PSMA) is a promising biomarker in

the diagnosis of prostate cancer and a potential target for antibody-based therapeutic

strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical

characteristics of the recombinant protein as a potential target for animal preclinical studies of

antibody based-therapies.

METHODS. Canine PSMA cDNA was isolated by PCR, cloned into expression vectors and

transfected into COS-1 and MDCK cells. The biosynthesis and glycosylation of the

recombinant protein were investigated in pulse-chase experiments, the cellular localization by

confocal laser microscopy, the mode of association of PSMA with the membrane with

solubilization in different detergents and its quaternary structure in sucrose-density gradients.

RESULTS. Canine PSMA shows 91% amino acid homology to human PSMA, whereby the

major difference is a longer cytoplasmic tail of canine PSMA compared to its human

counterpart. Canine PSMA is trafficked efficiently along the secretory pathway, undergoes

homodimerization when it acquires complex glycosylated mature form. It associates with

detergent-resistant membranes, which act as platforms along its intracellular trafficking.

Confocal analysis revealed canine PSMA at the cell surface, Golgi and the endoplasmic

reticulum. A similar distribution is revealed for human PSMA, yet with reduced cell surface

levels.

CONCLUSIONS. The cloning, expression, biosynthesis, processing and localization of

canine PSMA in mammalian cells is described. We demonstrate that canine PSMA reveals

similar characteristics to human PSMA rendering this protein useful as a translational model

for investigations of prostate cancer as well as a suitable antigen for targeted therapy studies

in dogs.

Keywords: biomarker; dog; prostate cancer

PSMA IN DOGS

67

Introduction

Prostate cancer is the most common malignancy in men and the second most common type of

cancer death in men in Europe. Besides humans, dogs are the only mammalian species

developing prostate tumours spontaneously. The incidence of tumours in dogs is lower than in

humans, but the number of affected dog is growing up. Similarly to what happens in humans

most of these tumours are adenocarcinomas metastasizing especially in lungs, lymph nodes

and bones with high probability. As well as in men also in dogs prostate cancer is most

commonly found in elderly patients (1). In 2008 Lai et al. showed that canine prostate cancer

is very aggressive and of a less differentiated type than most common human prostate

cancers, therefore it mostly resembles human, androgen refractory, poorly differentiated

prostate cancer with usually bad prognosis. Due to the above mentioned characteristics

studying prostate cancer in dogs could be of great importance to develop new animal models

for preclinical studies (e.g. pharmacokinetics, cross-reactivity, non specific toxicity), filling a

gap between rodent animal models and human clinical trials.

Successful therapy and elimination of prostate cancer requires an early diagnosis. Therefore

different prostate-specific proteins have been analysed as potential cancer biomarkers.


folate hydrolase and carboxypeptidase activity (2), initially identified in LNCaP cells by

immunoprecipitation (3). PSMA is expressed in epithelial cells of the prostate and at low

levels also in kidney, brain and intestine. Human PSMA (hPSMA) is overexpressed in

prostate cancer and levels of PSMA are directly proportional to disease grade and stage (4).

Also in neovasculature of other nonprostatic tumours PSMA expression has been detected,

but it is not present in healthy vasculature (5,6). PSMA is therefore one of the most promising

biomarkers in the diagnosis of prostate cancer and a potential target for antibody-based

therapeutic strategies (7). Characterisation of the canine isoform in relation to the human

counterpart could better support the future applications of this animal model in the

investigation of antibody based-treatments. Recent studies utilizing immunohistochemistry

revealed controversial results regarding expression of PSMA in prostatic tissue of dogs (8,9).

However results obtained by RT-PCR show clear expression of PSMA transcripts in the

canine prostate that was fivefold enhanced in carcinomas (8).

In this study we isolated canine PSMA and expressed it in either COS-1 cells or MDCK cells.

Thereby we observed that the canine and human PSMA proteins share many biochemical and

cellular characteristics. In addition to very strong amino acid similarities (91%), an equal

PSMA IN DOGS

68

distribution to cellular compartments, similar glycosylation pattern and a slightly more rapid

trafficking pattern were revealed. In a fashion similar to human PSMA canine PSMA also

forms homodimers and is associated with distinct lipid microdomains.

PSMA IN DOGS

69

Material and Methods

Reagents and antibodies

Tissue culture material was purchased from Sarstedt, RPMI-Medium and Dulbecco´s

Modified Eagle´s Medium and supplemented reagents (penicillin-streptomycin, fetal calf

serum, G418) were from PAA Laboratories. Triton X-100, proteinase inhibitors, chloroquine,

protein A-sepharose and bovine serum albumin were from Sigma. Acrylamide, TEMED,

SDS, Tris, sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium di-

hydrogen phosphate, sodium deoxycholate, paraformaldehyde, saponin, glutathione, Tween

20, DTT and PVDF membranes were purchased from Carl Roth. The ECL plus Western

Blotting Detection System and Biotin were from Thermo Scientific. Lubrol WX was from MP

Biomedicals, ammonium chloride from Merck and Pfu Ultra II Polymerase was from

Agiland. The anti-PSMA mAb D2B recognizing a luminal epitope of PSMA was produced in

our laboratory (10). The monoclonal ANTI-FLAG M2 antibody was purchased from Sigma

and anti-GFP monoclonal and polyclonal antibodies were from Clontech. Horseradish

peroxidase-coupled goat anti-mouse antibody was from Dako and secondary antibodies

coupled to Alexa Flour dyes were purchased from Invitrogen.

Cloning of canine PSMA

Total RNA was isolated from prostatic tissue of three healthy dogs using High Pur RNA

Tissue Kit from Roche. Total RNA was reverse transcribed to obtain first strand cDNA using

RevertAid First Strand cDNA Synthesis Kit from Fermentas and oligodT primers following

the manufacturer's instructions. Based on the predicted sequence of canine PSMA a set of

primers were designed and synthesized by Sigma. Using different primer pairs different

cDNA fragments were amplified by PCR and analysed by sequencing (eurofins MWG

operon). The obtained sequence was compared with the predicted gene and the open reading

frame was defined. The first 184 base pairs of the open reading frame of canine PSMA were

synthesized by eurofins MWG operon. Together with an amplicon of the last 185 - 2339 base

pairs of the open reading frame of canine PSMA the gene was cloned in a GFP-C1 expression

vector. Remaining amino acids between the synthesized part and the amplicon which belong

to the multiple cloning site of the vector were eliminated by mutagenesis PCR. Subsequently

the whole construct was sequenced to analyse its identity with the sequence obtained before.

PSMA IN DOGS

70

Another construct was designed by sub-cloning of the canine PSMA in a pCMV-3xFLAG-

Tag-1A expression vector.

Cell culture

COS-1 cells and MDCK cells were cultured in humidified atmosphere containing 5% CO2 in

air at 37°C in the presence of fetal calf serum (10% v/v) and penicillin-streptomycin (100

U/ml and 0.1 mg/ml respectively).

LNCaP cells were cultured under equal conditions in RPMI-Medium containing 2 g/L

glucose, fetal calf serum and penicillin-streptomycin.

Transient transfection of COS-1 cells

When cells reached a confluence of 30-40%, they were transiently transfected with 5 µg of

cDNA encoding GFP-cPSMA, 3xFLAG-cPSMA or YFP-hPSMA, respectively. The

transfection was carried out according to the DEAE-dextran-based method described

previously (11). Briefly cells were incubated in the presence of DNA and DEAE-dextran for 1

h at the standard culturing conditions and then left for further 2 h in complete DMEM

supplemented with 1 µg/ml chloroquine.

Generation of MDCK stable cell line expressing GFP-canine PSMA

MDCK cells stably expressing GFP-canine PSMA were obtained using the

Polybrene/dimethyl sulfoxide-based method. Therefore 10 µg of DNA was mixed with 3 ml

of DMEM containing 10% FCS and 30 µg of Polybrene. This transfection medium was used

to transfect 50% confluent MDCK cells. After incubation at 37 °C for 10 h the cells were

treated for 5 min with the transfection medium containing 25% (v/v) dimethyl sulfoxide. The

cells were washed once with DMEM containing 10% FCS and incubated in the same medium

overnight. The next day the cells were split 1:30 and grown in selective medium containing

G418 (500µg/ml). PSMA expression was finally confirmed by confocal microscopy and

western blot analysis.

DRM extraction

Cells were washed twice in ice-cold PBS and solubilized in PBS containing 1% (w/v)

detergent (Tween 20, Lubrol WX or Triton X-100 respectively) supplemented with a mixture

of proteinase inhibitors. Cells were homogenized with a Luer-21 Gauge needle 20 times/ml

and then maintained on ice for 2 hours. Afterwards, samples were pre-centrifuged 15 minutes

PSMA IN DOGS

71

at 8.000 x g and cell debris was discarded followed by a centrifugation at 100.000 x g, 4°C for

90 minutes (Beckman Optima LE-80, SW 55Ti-rotor). The supernatant and pellet obtained,

corresponding to the soluble and insoluble fractions respectively, were separately analysed.

The pellet fractions were lysed over night in a lysis-buffer containing 25 mM Tris, 50 mM

sodium chloride, 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate

supplemented with a mixture of proteinase inhibitors. On the next day the protein amounts of

all fractions were determined using Bradford protein assay (Bio-Rad) and 50 µg protein of

each fraction was analysed by SDS/PAGE and western blot analysis.

Immunoassays

MDCK cells and LNCaP cells were washed twice with PBS. Then cells were lysed in a lysis-

buffer containing 25 mM Tris, 50 mM sodium chloride, 0.5% (w/v) Triton X-100 and 0.5%

(w/v) sodium-deoxycholate supplemented with a mixture of proteinase inhibitors. After

removal of cell debris by centrifugation and pre-clearing with protein A-sepharose, the

supernatant was immunoprecipitated by using an anti-GFP mAb or D2B anti-PSMA mAb,

respectively for 1 hour at 4°C. Antigen-antibody complexes were then recovered with protein

A-sepharose over night, denatured and loaded on SDS/PAGE followed by western blot

analysis. When required, immunoprecipitated proteins were subjected to enzymatic digestion

with either Endoglycosidase H (Endo H) or with Endoglycosidase F (Endo F) according to a

method described previously (12).

Specific bands on PVDF membranes detected by the appropriate antibodies were visualized

by a ChemiDoc XRS Molecular Imager (Bio-Rad) device. Digital images obtained were

quantified by using image processing and analysis software ImageJ.

For indirect immunofluorescence, cells were fixed in 4% paraformaldehyde in PBS.

Quenching was performed twice for 10 minutes in 50 mM ammonium chloride in PBS.

Blocking and addition of the antibodies were performed in PBS containing 1% bovine serum

albumin and 0.5% saponin. Preparations were visualized using a Leica TCS SP5 confocal

laser microscope with an x 63 oil planapochromat lens (Leica Microsystems, Wetzlar).

Analysis of the quaternary structure

Monomers and dimers of PSMA were separated by sucrose density gradients under native

conditions. Cells were lysed for 2h in PBS containing 1% (w/v) of Triton X-100

supplemented with a mixture of proteinase inhibitors. After a centrifugation step at 8.000 x g

to remove cell debris, cell lysates were loaded on the top of 5 - 25% (w/v) continuous sucrose

PSMA IN DOGS

72

gradients which were centrifuged at 100.000 x g for 18h at 4°C (Beckman Optima LE-80, SW

40Ti-rotor). 24 fractions of 500 µl each were collected from bottom to top. Half of each

fraction was precipitated using ethanol and PSMA was detected by western blot analysis.

Biosynthetic labelling

MDCK cells stably expressing GFP-cPSMA or YFP-hPSMA were washed twice and

biosynthetically labelled with 100 µCi of [35S]methionine (from PerkinElmar) for 30 min at

37 °C. Then cells were chased, meaning incubated for further 1, 2, 3, 4, 5 h at 37°C in the

presence of fresh medium containing cold methionine. After cell lysis and

immunoprecipitation using anti-GFP mAb, proteins were analysed by SDS-PAGE and

autoradiography.

Statistical analysis

Statistical analysis of results was performed using GraphPad Prism5 applying the parametric

paired t test. Significance was accepted when p<0.05.

PSMA IN DOGS

73

Results

Isolation of canine PSMA cDNA and transfection into mammalian cells

To identify the genomic sequence of canine PSMA we isolated total RNA from prostatic

tissue of dogs followed by amplification of the cDNA encoding canine PSMA by PCR. To

amplify the cDNA it was necessary to perform PCRs of several small fragments of canine

PSMA with a size of 300 - 1000 base pairs. The cDNA fragments were sequenced and the

open reading frame of the protein was assessed (Accession number: HE820284).

Interestingly, the sequence we obtained differs from the predicted sequence of the dog in one

additional cytosine at position 114. This leads to a frame shift of the predicted sequence. The

deduced amino acid sequence revealed 47 amino acids that belong to the NH2-terminal

cytoplasmic tail of canine PSMA, whereas the cytoplasmic tail of human PSMA is

substantially shorter and contains 19 amino acids. Similar to the human species, cPSMA

spans the membrane once via a transmembrane domain that consists of 24 amino acids. The

large extracellular domain of cPSMA is 708 amino acids long and is therefore one amino acid

longer than its human counterpart. An alignment of the amino acid sequences of cPSMA and

hPSMA revealed 91 % amino acid homology whereby the main differences are found in the

cytoplasmic tail (Figure 1).

We next expressed GFP- and FLAG-tagged forms of cPSMA in COS-1 cells. Both protein

chimeras were transported to the cell surface along the constitutive secretory pathway from

the ER via the Golgi apparatus (2A, 3B). For further experiments we generated a MDCK cell

line stably transfected with GFP-cPSMA. For comparison we used a MDCK cell line stably

expressing YFP-hPSMA which was established in our laboratory before. The expressed

cPSMA was analysed at protein and cellular levels (Figure 2C – E). GFP-tagged cPSMA was

identified as a protein band that is slightly higher than its YFP-tagged human counterpart

(Figure 2E). This is explained by the additional amino acids of the cytoplasmic tail of

cPSMA.

We then examined the intracellular localization of cPSMA in transfected COS-1 cells and

compared it to that of hPSMA. Figure 3 shows that both proteins co-localize in the in the ER,

the Golgi and at the cell surface indicating that cPSMA is trafficked constitutively along the

secretory pathway as the human isoform (Figure 3C and F). To analyse differences between

canine and human PSMA in the distribution throughout the Golgi, we incubated co-

transfected cells at 20°C for 4h to accumulate proteins in the Golgi. Figure 3G – I show no

PSMA IN DOGS

74

difference in the distribution of both proteins when accumulating in the Golgi suggesting that

they are processed equally.

Glycosylation and trafficking of GFP-tagged canine PSMA

The extracellular domain of cPSMA reveals ten potential N-glycosylated sites. To investigate

its glycosylation in details, MDCK cells stably expressing GFP-cPSMA were used. LNCaP

cells endogenously expressing hPSMA were used as a control. After immunoprecipitation

proteins were either treated with Endo H, PNGase F or left untreated and analysed by western

blotting. Both cPSMA and hPSMA show a slightly smaller and diffused band pattern after

treatment with Endo H and a drastic shift in the apparent molecular weight after treatment

with PNGase F (Figure 4A). Both proteins are therefore highly glycosylated and the

glycosylation pattern we obtained were similar.

An interesting point in the biosynthetic features of PSMA is the presence of high proportions

of PSMA in an early immature mannose-rich glycosylated polypeptide through out its life

cycle compatible with a slow transport rate between the ER and the Golgi (13). To investigate

the trafficking of canine PSMA MDCK cells stably expressing GFP-cPSMA and YFP-

hPSMA were biosynthetically labelled for 30 min at 37 °C and chased for the indicated time

intervals. After immunoprecipitation using anti-GFP mAb, proteins were analysed by SDS-

PAGE and autoradiography. Subsequently after labelling only a mannose-rich glycosylated

form of canine and human PSMA (cPSMAM/hPSMAM) is detectable. After one hour of chase

an additional complex glycosylated form of canine PSMA occurs (cPSMAC) and after two

hours of chase only complex glycosylated forms of canine PSMA are visible. In comparison

the complex glycosylated form of human PSMA (hPSMAC) occurs not until a chase time of

two hours and mannose-rich glycosylated forms are visible during the whole experiment

(Figure 4B). This indicates a more rapid transport of canine PSMA between the ER and Golgi

seen by the earlier appearance of its mature complex glycosylated form compared to its

human counterpart.

Association of GFP-canine PSMA with distinct DRMs

We could show before that human PSMA is transported via different microdomains along the

secretory pathway (14). These rafts are described to be involved in signalling processes,

trafficking and endocytosis. The mannose-rich form of PSMA associates predominantly with

Tween 20-DRMs in the ER and the complex glycosylated form of later compartments is

mainly insoluble in Lubrol WX, but an interaction with Triton X-100-DRMs only occurs

PSMA IN DOGS

75

upon antibody-induced cross-linking of PSMA (data not shown). Experimentally, these

microdomains can be isolated based on their insolubility in cold, non ionic detergents (15).

Upon solubilization of cellular extracts with a detergent, DRMs can be recovered by

sedimentation. Here, we examined the association of canine PSMA with different types of

microdomains. Figure 5A shows that canine PSMA is partially associated with Lubrol WX-

and Tween 20-DRMs and cPSMA is completely soluble in Triton X-100 as the human

isoform. We could not detect any significant differences regarding detergent solubility

properties between the human and the canine form of PSMA (Fig 5B), indicating that as well

as human PSMA also canine PSMA is transported via distinct microdomains along the

secretory pathway.

Quaternary structure of GFP-canine PSMA

To examine the quaternary structure of canine PSMA cellular lysates were subjected to

continuous sucrose gradients given that dimeric or higher order quaternary structures are

retained in fractions with higher sucrose density than monomers. In a similar experimental set

up we have previously shown that hPSMA dimerizes. As shown in Figure 6 the monomeric

form of hPSMA peaks in fractions 16-19, which contain the mannose-rich and the complex

glycosylated form of PSMA. On the other hand, the dimeric forms of hPSMA are located in

fractions 13-15 and comprise exclusively the complex glycosylated glycoform. Since cPSMA

is found in fractions 13-15 and in comparison with the dimeric forms of hPSMA we conclude

that these fractions contain dimeric cPSMA forms.

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76

Discussion

PSMA has emerged as an important biomarker for the management and therapy of prostate

cancer in man, since its expression is largely restricted to cells of the prostatic epithelium with

protein levels proportional to tumour grade. In addition to humans, dogs are the only

mammalian species that develop prostate cancer spontaneously. Given that canine PSMA

shares strong homology to human PSMA, the dog can be considered to be a suitable animal

model to study PSMA physiology and also to evaluate the efficacy of PSMA-based targeted

therapies. Here we have isolated a cDNA clone encoding canine PSMA, and analysed its

expression, processing, glycosylation and trafficking in a heterologous cellular system.

cPSMA is a type-II-membrane-glycoprotein comprising 779 amino acids. The sequence

similarity between cPSMA and the human counterpart, hPSMA, is striking and amounts to

about 91% at the amino acid level. The major sequence differences between the canine and

human species are found in the cytosolic tails. The cytosolic tail of cPSMA is 29 amino acids

longer than the human counterpart and accounts thus exclusively for the longer canine version

of PSMA. Interestingly, cPSMA also exposes a cryptic endocytic signal of the type MXXXL

(16) that is found in hPSMA, but in the canine isoform it is not located directly at the N-

terminus. This is likely to be compatible with reduced or even absent endocytosis of cPSMA

with subsequent increased expression of the protein at the cell surface. In fact, increased

levels of cPSMA at the cell surface can be observed as compared to hPSMA. Nevertheless, a

detailed assessment of this hypothesis requires the generation of a suitable antibody that

recognizes the luminal part of cPSMA.

Our study has shown that recombinant cPSMA is processed and transported constitutively and

competently along the secretory pathway and is ultimately localized at the plasma membrane

in transfected COS-1 and MDCK cells. We also compared directly in the same cell the

localization of cPSMA with that of hPSMA. Both species co-localize in similar cellular

compartments and are revealed in the ER, the Golgi and on the plasma membrane.

Interestingly, however, cPSMA traverses the ER to the Golgi more rapidly than hPSMA by

virtue of the earlier appearance of its mature complex glycosylated forms. The variations in

the trafficking kinetics between both species are presumably due to differences in their

cytosolic domains, since the luminal parts of both molecules are strongly homologous.

Cytosolic tails play a role in controlling protein trafficking as stabilizers of a particular

quaternary structure. The trimerization of the hemagglutinin of the influenza virus, for

example, is necessary for its ER exit, whereby the trimers are stabilized by the cytoplasmic

PSMA IN DOGS

77

tail of the hemagglutinin (17). Likewise dimerization of intestinal lactase-phlorizin hydrolase

is a prerequisite for an efficient intracellular trafficking of the protein and truncation of the

cytosolic tail leads to a substantial reduction of the trafficking efficiency of lactase-phlorizin

hydrolase and its maturation in the Golgi (18). Along these lines it appears that the dimeric

forms of hPSMA are presumably not as stable and transport competent as their cPSMA

counterparts due to the substantially shorter cytosolic tail of hPSMA.

The trafficking of cPSMA requires its interaction with different DRMs. Obviously this

interaction specifies the various trafficking stages and biosynthetic forms of PSMA. Thus,

different biosynthetic forms of PSMA utilize several platforms of DRMs along the secretory

pathway. The mannose-rich form, for example, is associated with microdomains in the ER

that are resistant to the mild detergent Tween 20. These DRMs discriminate the ER-located

PSMA from the mature form that is processed in the Golgi and associates with Lubrol WX-

DRMs, a slightly stronger detergent than Tween 20. Obviously a detergent-hierarchy does

exist that discriminates between various biosynthetic protein forms based on their degree of

maturation and mode of association with the membrane. What is the structural basis of the

altered mode of association of the cPSMA biosynthetic forms with the membrane and

transition into a different type of membrane? One likely explanation relates the quaternary

structure of cPSMA to its pattern of solubility with a particular detergent. cPSMA dimerizes

only after it has acquired complex glycosylated forms in the Golgi. It is conceivable that a

single transmembrane segment in monomeric mannose-rich cPSMA would fit within a

different membrane environment as compared to the two associating segments in dimeric

complex glycosylated cPSMA.

Recent studies using immunohistochemistry revealed controversial results regarding

expression of PSMA in prostate tissue of dogs, for example only in castrated animals (8,9).

One likely explanation for these observations are differences in the epitopes of cPSMA as

compared to hPSMA. In fact, our own attempts to detect cPSMA with five monoclonal

antibodies directed against hPSMA were also not successful. However, results obtained by

RT-PCR revealed cPSMA in canine prostate and the expression levels were substantially

increased by a factor of five in prostate carcinoma (8).

Our current study aims to better elucidate the cellular behaviour of cPSMA in order to

validate this isoform for preclinical trials of PSMA-targeted therapies; moreover we would

like to increase the interest in developing antibodies recognizing cPSMA which could be

applied for management of prostate cancer in dogs.

PSMA IN DOGS

78

Conclusions

In conclusion, the current study has provided novel information on the biosynthesis and

processing of cPSMA. This information about the physiology of cPSMA antigen could be of

great importance in developing diagnostic/therapeutical approaches against prostate cancer in

dogs. Furthermore, the striking homologies between cPSMA and hPSMA render the canine

protein as an excellent target antigen to in vivo validate new anti-PSMA drugs, particularly

for the evaluation of pharmacokinetic parameters and nonspecific toxicity. Finally, the study

offers exquisite opportunities to generate high quality antibodies against cPSMA as

therapeutic tools in the treatment of prostate cancer.

PSMA IN DOGS

79

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2. Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP, May F, Mukherjee B,

Heston WD. Prostate-specific membrane antigen: a novel folate hydrolase in human

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3. Horoszewicz JS, Kawinski E, Murphy GP. Monoclonal antibodies to a new antigenic

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4. Ross JS, Sheehan CE, Fisher HA, Kaufman RP, Jr., Kaur P, Gray K, Webb I, Gray

GS, Mosher R, Kallakury BV. Correlation of primary tumor prostate-specific

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5. Chang SS, O'Keefe DS, Bacich DJ, Reuter VE, Heston WD, Gaudin PB. Prostate-

specific membrane antigen is produced in tumor-associated neovasculature. Clin

Cancer Res 1999;5(10):2674-2681.

6. Chang SS, Reuter VE, Heston WD, Bander NH, Grauer LS, Gaudin PB. Five different

anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA

expression in tumor-associated neovasculature. Cancer research 1999;59(13):3192-

3198.

7. Fracasso G, Bellisola G, Cingarlini S, Castelletti D, Prayer-Galetti T, Pagano F,

Tridente G, Colombatti M. Anti-tumor effects of toxins targeted to the prostate

specific membrane antigen. The Prostate 2002;53(1):9-23.

8. Lai CL, van den Ham R, van Leenders G, van der Lugt J, Mol JA, Teske E.

Histopathological and immunohistochemical characterization of canine prostate

cancer. The Prostate 2008;68(5):477-488.

9. Aggarwal S, Ricklis RM, Williams SA, Denmeade SR. Comparative study of PSMA

expression in the prostate of mouse, dog, monkey, and human. The Prostate

2006;66(9):903-910.

10. Colombatti M, Grasso S, Porzia A, Fracasso G, Scupoli MT, Cingarlini S, Poffe O,

Naim HY, Heine M, Tridente G, Mainiero F, Ramarli D. The prostate specific

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membrane antigen regulates the expression of IL-6 and CCL5 in prostate tumour cells

by activating the MAPK pathways. PloS one 2009;4(2):e4608.

11. Naim HY, Lacey SW, Sambrook JF, Gething MJ. Expression of a full-length cDNA

coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved,

enzymatically active, and transport-competent protein. The Journal of biological

chemistry 1991;266(19):12313-12320.

12. Naim HY, Sterchi EE, Lentze MJ. Biosynthesis and maturation of lactase-phlorizin

hydrolase in the human small intestinal epithelial cells. The Biochemical journal

1987;241(2):427-434.

13. Castelletti D, Fracasso G, Alfalah M, Cingarlini S, Colombatti M, Naim HY. Apical

transport and folding of prostate-specific membrane antigen occurs independent of

glycan processing. The Journal of biological chemistry 2006;281(6):3505-3512.

14. Castelletti D, Alfalah M, Heine M, Hein Z, Schmitte R, Fracasso G, Colombatti M,

Naim HY. Different glycoforms of prostate-specific membrane antigen are

intracellularly transported through their association with distinct detergent-resistant

membranes. The Biochemical journal 2008;409(1):149-157.

15. Schuck S, Honsho M, Ekroos K, Shevchenko A, Simons K. Resistance of cell

membranes to different detergents. Proceedings of the National Academy of Sciences

of the United States of America 2003;100(10):5795-5800.

16. Rajasekaran SA, Anilkumar G, Oshima E, Bowie JU, Liu H, Heston W, Bander NH,

Rajasekaran AK. A novel cytoplasmic tail MXXXL motif mediates the internalization

of prostate-specific membrane antigen. Molecular biology of the cell

2003;14(12):4835-4845.

17. Copeland CS, Zimmer KP, Wagner KR, Healey GA, Mellman I, Helenius A. Folding,

trimerization, and transport are sequential events in the biogenesis of influenza virus

hemagglutinin. Cell 1988;53(2):197-209.

18. Behrendt M, Polaina J, Naim HY. Structural hierarchy of regulatory elements in the

folding and transport of an intestinal multidomain protein. The Journal of biological

chemistry;285(6):4143-4152.

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81

Figures

Figure 3-1. Alignment of canine PSMA and human PSMA show 91% amino acid

homology with the biggest difference in a longer cytoplasmic tail of canine PSMA

RNA of prostatic tissue from three independent dogs was isolated and the cDNA of canine PSMA was amplified by different PCRs. After analysing the open reading frame an alignment of the amino acid sequences of canine PSMA versus human PSMA was performed. Only the first 180 amino acids of canine PSMA are shown. cPSMA – canine PSMA; hPSMA – human PSMA

PSMA IN DOGS

82

Figure 3-2. GFP-cPSMA is transported to the cell surface and has a molecular weight of

approx. 140 kDa

(A, B) COS-1 cells were transiently transfected with cDNA encoding GFP-canine PSMA (GFP-cPSMA) (A) or YFP-human PSMA (YFP-hPSMA) (B), respectively. Two days after transfection cells were visualized using a Leica TCS SP5 confocal laser microscope. (C, D) MDCK cells were stably transfected with cDNA encoding GFP-canine PSMA (C) or YFP-human PSMA (D), respectively. Selections of cells containing GFP-canine PSMA (C) or YFP-human PSMA (D) were positively selected by adding G418 to the DMEM medium. PSMA expression was confirmed by using a Leica TCS SP5 confocal laser microscope. (E) Finally PSMA expression was analysed by western blot analysis.

PSMA IN DOGS

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Figure 3-3. Expression and co-localization of pCMV-3T1-cPSMA and YFP-hPSMA in

COS-1 cells

COS-1 cells were transiently co-transfected with cDNA encoding YFP-hPSMA (A, D, G) and cDNA encoding 3xFLAG-cPSMA (B, E, H). Two days after transfection 3xFLAG-cPSMA was detected by indirect immunofluorescence and cells were visualized by using a Leica TCS SP5 confocal laser microscope. (G-I) To accumulate proteins in the Golgi cells were incubated at 20°C for 4h before fixing them for immunofluorescence and confocal laser microscopy. cPSMA – canine PSMA; hPSMA – human PSMA

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Figure 3-4. Glycosylation and trafficking pattern of GFP-canine PSMA are similar to

human PSMA

(A) To investigate glycosylation, MDCK cells stably expressing GFP-cPSMA and LNCaP cells endogenously expressing hPSMA were lysed and after immunoprecipitation using anti-GFP mAb or D2B anti-PSMA mAb, respectively, proteins were either treated with Endo H, Endo F or left untreated and then analysed by SDS-PAGE and western blot analysis. (B) MDCK cells stably expressing GFP-cPSMA or YFP-hPSMA were biosynthetically labelled for 30 min at 37 °C and chased for the indicated time intervals. After immunoprecipitation using anti-GFP mAb, proteins were analysed by SDS-PAGE and autoradiography. cPSMAM – mannose-rich glycosylated form of canine PSMA; cPSMAC – complex glycosylated form of canine PSMA; hPSMAM - mannose-rich glycosylated form of human PSMA; hPSMAC - complex glycosylated form of human PSMA

PSMA IN DOGS

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Figure 3-5. GFP-cPSMA is partially associated with Tween 20- and Lubrol WX-DRMs,

but not with Triton X-100-DRMs

(A) MDCK cells stably expressing GFP-cPSMA or YFP-hPSMA, respectively, and LNCaP cells endogenously expressing hPSMA were solubilized with either 1% Triton X-100, 1% Lubrol WX or 1% Tween 20 (w/v) in PBS. Lysates were subjected to centrifugation at 100.000 x g for 90 min in order to separate the insoluble (P) DRM-fraction and the soluble (S) material. After centrifugation the insoluble fraction (P) was solubilized with a lysis-buffer containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) sodium-deoxycholate and equal amounts of total protein from both fractions were loaded for each lane on SDS gels and blotted. (B) Statistical analysis of results was performed using ImageJ and GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was accepted when p<0.05.

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Figure 3-6. GFP-cPSMA forms dimers

MDCK cells stably expressing GFP-cPSMA or YFP-hPSMA, respectively, and LNCaP cells were solubilized with 1% (w/v) Triton X-100 in PBS and 1 ml of each lysate was loaded on top of a 5 - 25% continuous sucrose gradient. After 18 h of centrifugation at 100.000 x g, 24 fractions of 500 µl each were collected from bottom to top. Half of each fraction was precipitated using ethanol and PSMA was detected by western blot analysis.

CHAPTER ICHAPTER ICHAPTER ICHAPTER IVVVV::::

DDDDiscussioniscussioniscussioniscussion

DISCUSSION

89

IV. DISCUSSION

PSMA is an important biomarker for the management and therapy of prostate cancer in men,

since its expression is largely restricted to cells of the prostatic epithelium with increased

protein levels in prostate cancer cells proportional to the grade of the tumour.

Within this thesis I addressed the role of lipid rafts in two major physiological events in the

life cycle of PSMA, homodimerization and internalization. The results clearly propose an

essential role for different microdomains in both dimerization and internalization.

Furthermore, I investigated possible interaction partners of PSMA in lipid rafts, which trigger

the signalling capacity of PSMA. Thereby my data clearly indicate that microtubules are key

players in PSMA endocytosis. Additionally, a high affinity laminin receptor (67LR) was

identified as an interacting partner of PSMA.

Moreover, I could demonstrate a homologous form of PSMA in prostatic tissue of dogs

(cPSMA) and I could provide information on the expression, biosynthesis and processing of

this canine PSMA protein. Striking homologies between the canine and the human protein

establish the canine version as an excellent model for the human prostate.

4.1. Quaternary structure of PSMA Dimerization is a mechanism responsible for the activation of many transmembrane receptors

(SCHLESSINGER 2000). For these receptors, dimerization follows ligand binding and leads

to the activation of protein kinase activity (SCHLESSINGER 2000).

For PSMA, homodimerization is required for its enzymatic activity (SCHULKE et al. 2003)

and occurs late along the secretory pathway (CASTELLETTI et al. 2008). The transition from

a monomeric to a dimeric state is linked to the formation of a complex glycosylated form of

PSMA in the Golgi (CASTELLETTI et al. 2008). The extracellular domain is critical for

dimerization of PSMA, since truncated versions lacking the cytoplasmic as well as the

transmembrane domain are still able to form homodimers (SCHULKE et al. 2003).

DISCUSSION

90

PSMA associates with different membrane domains depending on its state of dimerization.

The mannose-rich and monomeric form of PSMA in the ER favours an intrinsic membrane

environment that is reflected by its association with Tween 20-DRMs (CASTELLETTI et al.

2008). Only the dimeric form of PSMA is retained in Lubrol WX-DRMs (Fig. 2-1) suggesting

that dimerization and association with this type of DRMs are important for a full biological

activity of PSMA, particularly towards its interaction with its natural ligand.

GPI-anchored receptors cluster in particular detergent-resistant membrane microdomains,

where they may make contact with various signalling molecules (SIMONS and TOOMRE

2000). It has been hypothesized that their association with these membrane domains is

regulated by oligomerization of these proteins (SIMONS and TOOMRE 2000).

Dimerization of PSMA may therefore not only determine its association with other proteins,

but also direct these interactions to discrete membrane domains.

Similar events are described for the Toll-like receptor 4 (TLR4) where recruitment of TLR4

into lipid rafts is coupled with dimerization of the protein. Both receptor dimerization and

recruitment of TLR4 into lipid rafts appear to be initial events leading to the activation of

TLR4 signalling pathways induced by dietary saturated fatty acids (WONG et al. 2009).

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a GPI-anchored

membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell

migration and proliferation. As well as PSMA also cell surface uPAR dimerizes and dimeric

uPAR partitions preferentially to detergent-resistant lipid rafts. But dimerization of uPAR

does not necessarily require raft partitioning (CUNNINGHAM et al. 2003).

SIMONS and TOOMRE (2000) describe at least three different ways for receptors to behave

in lipid rafts.

1. Individual receptors with weak raft affinity could oligomerize on ligand binding, and

this would lead to an increased residency time in rafts.

2. Receptors associated at steady state with lipid rafts could be activated through ligand

binding.

DISCUSSION

91

3. Activated receptors could recruit cross-linking proteins that bind to proteins in other

rafts, and this would result in raft coalescence (SIMONS and TOOMRE 2000).

For maturation and activation of PSMA, an importance of all three ways can be hypothesized.

1. Homodimerization of PSMA is required for its association with Lubrol WX-DRMs.

2. PSMA dimers linked to Lubrol WX-DRMs can be activated through ligand/antibody

binding.

3. This activation triggered by ligand/antibody binding leads to redistribution of PSMA

to another type of lipid rafts or raft coalescence and subsequent internalization of the

protein via microtubules.

4.2. Internalization of PSMA via microtubules Upon ligand or antibody cross-linking, some plasma membrane receptors undergo enhanced

partitioning into sphingolipid-cholesterol membrane microdomains as an obligatory first step

toward participation in early signal transduction events (SIMONS and IKONEN 1997;

SIMONS and TOOMRE 2000; FRIEDRICHSON and KURZCHALIA 1998).

In this study, ligand binding is mimicked by cross-linking of homodimeric PSMA at the cell

surface with anti-PSMA antibodies. As a consequence two major dramatic events occur: The

first is an efficient internalization of PSMA with an almost complete disappearance of PSMA

from the cell surface and appearance as discrete intracellular spots (Fig. 2-4); the second is a

shift of dimeric PSMA from Lubrol WX-DRMs to a different membrane environment that is

enriched in cholesterol and sphingolipids (Fig. 2-2). The clustering and subsequent

association of PSMA with these Triton X-100-DRMs occur in a time-dependent manner (Fig.

2-3).

To examine a possible correlation between clustering into Triton X-100-DRMs and

internalization of PSMA after antibody cross-linking, the activation assay was performed at

4°C where internalization is blocked. Redistribution of PSMA to Triton X-100-DRMs occurs

also after activation at 4°C (Fig. 2-2). Therefore redistribution of PSMA into Triton X-100-

DRMs is likely to be independent of internalization. On the other hand biotinylation

experiments revealed that PSMA, which undergoes internalization, is afterwards present in

DISCUSSION

92

Triton X-100-DRMs (Fig. 2-4 B). This shows that upon antibody-induced activation surface

PSMA is recruited to another micro-environment, which is resistant to Triton X-100, followed

by subsequent internalization of PSMA molecules in the same type of Triton X-100-resistant

microdomains.

In endothelial cells internalization of PSMA has been shown to implicate its interaction with

caveolin 1 (ANILKUMAR et al. 2006), thus suggesting that lipid rafts may play a role in this

event since caveolin is a major structural protein of caveolae and these in turn represent a

special type of lipid rafts. In line with these observations, the time-dependent endocytosis of

PSMA and recruiting to Triton X-100-insoluble membrane domains provides unequivocal

support to the notion that lipid rafts are directly implicated in the clustering and

internalization of PSMA.

Furthermore, antibody-induced internalization of PSMA is involved in signalling processes

(COLOMBATTI et al. 2009). In previous studies our group could demonstrate an activation

of the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 during the process of

antibody-induced activation of PSMA, resulting in an enhanced proliferative potential of

LNCaP cells (COLOMBATTI et al. 2009). Our group could also show interactions of PSMA

with phosphorylated p130 CAS as well as phosphorylated src-kinases in lipid rafts, and that

these interactions are strongly enhanced upon antibody-induced activation of PSMA (data not

shown).

Comparable effects of recruiting proteins into lipid rafts followed by downstream-signalling

are already described for the myelin oligodendrocyte glycoprotein (MOG), a component of

the myelin membrane, which is involved in the pathology of multiple sclerosis (MARTA et

al. 2003; MARTA et al. 2005). In particular, antibodies against MOG are elevated in multiple

sclerosis patients and they have been implicated as mediators of demyelination. In cultured

oligodendrocytes MOG is not associated with Triton X-100-DRMs; however antibody cross-

linking of MOG on the surface of oligodendrocytes results in the repartitioning of around

95% of MOG into the Triton X-100-insoluble fraction. This redistribution occurs rapidly in

less than 1 minute. Furthermore it is antibody dose-dependent, requires an intact cytoskeleton,

DISCUSSION

93

leads to phosphorylation or dephosphorylation of specific proteins like β-tubulin and the Gβ

subunit of the G-protein complex and an activation of the MAPK/Akt pathways. Therefore it

is hypothesized that antibody-mediated redistribution of MOG into Triton X-100-insoluble

microdomains initiates specific cellular signalling that could be related to initial steps of

MOG-mediated demyelination of oligodendrocytes (MARTA et al. 2003; MARTA et al.

2005).

Also T-cell receptors (TCRs) are recruited into Triton X-100-insoluble membranes upon

receptor stimulation. TCR recruitment is accompanied by the accumulation of a series of

prominent tyrosine-phosphorylated substrates and by an increase of Lck activity in Triton X-

100-DRMs, indicating possible raft association (MONTIXI et al. 1998; JANES et al. 2000;

GAUS et al. 2005).

Lipid rafts are also involved in IgE signalling. As well as PSMA, MOG and TCRs also FcεRI

is soluble in Triton X-100 at steady state, but becomes insoluble in low concentrations of this

detergent after cross-linking. Moreover src-family tyrosine kinases also aggregate in these

microdomains (FIELD et al. 1995). Furthermore IgE signalling is abolished if surface

cholesterol is depleted (SHEETS et al. 1999). XU et al. (1998) could show the more

participants are collected into the raft platforms, the higher the signalling response. Therefore

uncontrolled amplification of the signalling cascade by raft clustering might trigger

hyperactivation, resulting in an allergic shock (XU et al. 1998).

In general, all these studies show that activation of specific proteins leads to recruitment of

these and additional proteins to a new micro-environment, which is detergent resistant and

where the phosphorylation state can be modified by local kinases and phosphatases, resulting

in downstream signalling.

Along these lines an essential role for lipid rafts or DRMs in endocytosis and signalling

pathways involving PSMA can be proposed.

While cross-linking of PSMA with antibodies and eliciting internalization could be

considered to be an artificial system, cross-linking of membrane proteins is normally a

physiological phenomenon that can lead to the redistribution of these proteins into lipid rafts,

resulting in novel protein interactions and initiation of cell signalling (SIMONS and

DISCUSSION

94

TOOMRE 2000; IKONEN 2001). Although occurring naturally via multivalent ligands,

similar responses have been observed using antibodies (SIMONS and TOOMRE 2000).

Interactions of PSMA with different proteins like the actin cross-linking protein filamin A

(ANILKUMAR et al. 2003) and clathrin as well as the adaptor protein complex-2

(GOODMAN et al. 2007) have been shown to participate in internalization of PSMA. To

identify additional proteins potentially interacting with PSMA, the proteins of Triton X-100-

DRMs in non activated cells were compared with those of activated cells by 2-dimensional

gel electrophoresis. Mean relative spot volumes and differences in expression were calculated

using ImageJ and the differing spots were analysed by mass spectrometry followed by Swiss-

Prot database search with MASCOT to assign identities.

The expression levels of several proteins varied in Triton X-100-DRMs due to antibody-

induced activation of PSMA. One of these proteins which increased substantially in Triton X-

100-DRMs isolated from activated cells is tubulin (Fig. 2-5 A).

Western Blot experiments confirmed the significant clustering of α-tubulin in Triton X-100-

DRMs upon activation. Also the levels of β-tubulin increase in these DRMs, but not to the

same extent as α-tubulin (Fig. 2-5 B). These findings suggest that microtubules are involved

in internalization of PSMA.

Co-localization experiments of PSMA and α-tubulin before and after cross-linking and

internalization of PSMA were performed in COS-1 cells that transiently express the YFP-

tagged form of PSMA. Upon antibody-induced cross-linking of PSMA, internalization

occurred and several endosomes containing YFP-PSMA could be observed to co-localize

with α-tubulin along or in close vicinity to the cell surface (Fig. 2-5 D and E).

These observations, when taken together, strongly suggest that YFP-PSMA is transported

along the tubulin filaments and lipid rafts play an important role in this event.

In general, the microtubule cytoskeleton is particularly important for polarized targeting of

apical cargo. Microtubule depolymerization results in aberrant delivery of several apical

proteins, including PSMA, to the basolateral surface (CHRISTIANSEN et al. 2005).

CHRISTIANSEN et al. (2006) could show a functional role for syntaxin 3 in the microtubule-

dependent apical targeting of PSMA, since point mutations into syntaxin 3 abolish its

polarized distribution and causes PSMA to be targeted in a non polarized fashion. Similar

DISCUSSION

95

results showing a non polarized targeting of both proteins were obtained after microtubule

depolymerization (CHRISTIANSEN et al. 2006). However, at the time of writing only little

information is available on the role of microtubules during internalization of PSMA. I could

show that α-tubulin as well as partially β-tubulin are together with PSMA redistributed to

Triton X-100-DRMs upon activation of PSMA by antibody-induced cross-linking.

Immunofluorescence revealed that PSMA undergoes internalization along α-tubulin,

suggesting necessity of microtubules for internalization of PSMA.

Tubulin typically is described as a non-raft protein; however upon signalling events it may

become raft-associated. Recent studies have focused on the function of raft-associated tubulin

and microtubules (HEAD et al. 2006; JOLLY et al. 2007; MARTA et al. 2003; MARTA et al.

2005), showing that microtubules play a role in maintaining lipid raft structure and function.

HIV type 1 requires lipid rafts and disruption of the microtubule cytoskeleton with colchicine

or nanodazole can disrupt the spread of HIV-1 in T-cells (JOLLY et al. 2007). As mentioned

before, antibody-cross-linking of MOG leads to recruiting of MOG into lipid rafts and

subsequent dephosphorylation of raft-associated β-tubulin (MARTA et al. 2003; MARTA et

al. 2005).

Also connections between the cytoskeleton and caveolar-regulated cellular events are known

(HEAD et al. 2006), indicating that microtubules and actin filaments contribute to the

presence of caveolae. A protein-complex of caveolin and the actin cross-linking protein

filamin A could be detected. Disruption of the cytoskeleton with colchicine or cytochalasin D

results in a loss of morphological caveolae and redistribution of several caveolar/lipid raft-

resident proteins into non raft-fractions (HEAD et al. 2006). Furthermore, treatment with

colchicine or cytochalasin D decreases phosphorylation of caveolin-1, MAPK p38 and src

(HEAD et al. 2006). Since PSMA shows interactions with both caveolin-1 and filamin A, it is

likely that these proteins build a multiprotein-complex at least in endothelial cells, which is

possibly bound to microtubules.

The internalization of PSMA involves its interaction with filamin A, an actin cross linking

protein, and this association is involved in the localization of PSMA to the recycling

endosomal compartment (ANILKUMAR et al. 2003). My data extend the role played by the

DISCUSSION

96

cytoskeleton to include α-tubulin, clearly indicating that microtubules are key players in

PSMA endocytosis. Furthermore, lipid rafts have been shown to be directly involved in

endocytosis of PSMA and following signalling events.

Understanding the molecular mechanisms underlying the antibody-induced cross-linking,

deciphering the membrane structures and components governing this event, as well as the

internalization of PSMA, represents essential prerequisites for utilizing PSMA as a

therapeutically suitable target in prostate cancer.

4.3. Association of PSMA with other proteins In order to identify additional proteins potentially interacting with PSMA in lipid rafts or

DRMs I compared again protein amounts of Triton X-100-DRMs between non activated and

activated LNCaP cells. Again I determined protein profiling by 2-dimesional gel

electrophoresis and differing proteins were analysed using mass spectrometry followed by

Swiss-Prot database search with MASCOT to assign identities.

Besides tubulin, laminin receptor-1 (67LR) was one of the proteins varying in DRMs upon

antibody-induced cross-linking of PSMA. It clearly shows a trend to decrease in Triton X-

100-DRMs upon activation of PSMA (Data not shown).

67LR is a non-integrin laminin receptor, derived from the dimerization of a 37 kDa cytosolic

precursor. 67LR binds to matrix proteins and transduces signals to the interior of the cell. The

amino acid sequence of 67LR is highly conserved through evolution, suggesting a key

biological function. In addition to acting as a laminin receptor, mediating high-affinity

interactions between cells and laminin, this raft-associated 67LR acts as a receptor for the

internalization of some viruses, prions and bacteria (WANG et al. 1992; GAUCZYNSKI et al.

2001; HUNDT et al. 2001; KIM et al. 2005). 67LR is highly expressed in cancer tissue

(CIOCE et al. 1991), and widely recognized as a molecular marker of metastatic

aggressiveness (MARTIGNONE et al. 1993; SANJUAN et al. 1996).

Several interaction partners of 67LR are known, e.g. phosphoprotein enriched in

diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15), which is an anti-apoptotic

DISCUSSION

97

protein whose expression is increased in several human cancers. PED/PEA-15 is involved in

the regulation of major cellular functions, including cell adhesion, migration, apoptosis,

proliferation and glucose metabolism. It shows the ability to induce cell responses to

extracellular matrix-derived signals through interaction with 67LR. This may be of crucial

importance for tumour cell survival in a poor microenvironment, thus favouring the metastatic

spread and colonization (FORMISANO et al. 2011).

To test for an interaction between PSMA and 67LR we lacked an appropriate cell-line.

MDCK cells stably transfected with a cDNA encoding YFP-PSMA turned out to be a

convincing model for these studies, because non transfected MDCK cells could be used as

negative controls.

Indirect immunofluorescence of 67LR analysed by confocal microscopy revealed co-

localizations between 67LR and PSMA in the Golgi-region and on the cell surface (Fig. 4-1

A). Co-immunoprecipitation was performed to acquire more evidence, which showed a direct

interaction between 67LR and YFP-PSMA (Fig. 4-1 B).

DISCUSSION

98

67LR

YFP-PSMA

Cell lysatesImmunoprecipitationD2B (anti-PSMA)

MDCK YFP-PSMA

Activation - + +- --

WT MDCK YFP-PSMA WT

YFP-PSMA

67LR

Cell surface

Golgi

mergeA

B

67LR

YFP-PSMA


MDCK YFP-PSMA


WT MDCK YFP-PSMA WT

YFP-PSMA

67LR

Cell surface

Golgi

merge

67LR

YFP-PSMA


MDCK YFP-PSMA


WT MDCK YFP-PSMA WT

YFP-PSMA

67LR

Cell surface

Golgi

mergeYFP-PSMA

67LR

Cell surface

Golgi

mergeA

B

Figure 4-1. PSMA co-localizes and co-immunoprecipitates with 67LR. (A) MDCK cells stably expressing YFP-PSMA were fixed and 67LR was detected by indirect immunofluorescence. (B) Both non transfected MDCK cells and MDCK cells stably expressing YFP-PSMA were lysed with 1% Triton X-100 and PSMA was immunoprecipitated. Immunoprecipitates as well as 20µl of total cell lysates were subjected to SDS/PAGE and PSMA as well as 67 LR were detected by western blot analysis.

The interaction between PSMA and 67LR, together with the down-regulation of 67LR in

Triton X-100-DRMs upon activation of PSMA, might be of particular interest because 67LR

plays an important role in cell adhesion to the basement membrane and in the consequent

activation of signalling transduction pathways. Reduced levels of 67LR within lipid rafts or

DRMs of the plasma membrane may result in less adhesion and therefore increased risk of

metastasis. This is in line with the fact that both proteins, PSMA as well as 67LR, are highly

DISCUSSION

99

expressed in prostate cancer cells and their expression levels are proportional to tumour

aggressiveness and risk of metastasis. An increased expression of PSMA in prostate cancer

cells, together with an increased activation of PSMA by a putative ligand may result in less

expression of 67LR within the plasma membrane, explaining more aggressive prostate cancer

with a higher risk of metastasis. After detaching from the basement membrane the

redistribution of 67LR out of Triton X-100-DRMs might be reversible. Then 67LR may

return into the plasma membrane and the high number of free laminin receptors on the tumour

cell surface may facilitate interaction of these cells, through laminin within the vascular

basement membrane (TERRANOVA et al. 1983). Interactions between cancer cells and

laminin have been shown to play a critical role during this process. Indeed, the ability of

cancer cells to attach to laminin has been correlated with their metastatic potential (BARSKY

et al. 1984).

4.4. Canine PSMA Recent studies utilizing immunohistochemistry revealed controversial results regarding

expression of PSMA in prostate tissue of dogs, for example an expression of the protein was

only obtained in castrated animals (AGGARWAL et al. 2006; LAI et al. 2008). However,

results obtained by RT-PCR revealed cPSMA in the canine prostate and the expression levels

were substantially increased by a factor of five in prostate carcinoma (LAI et al. 2008).

To identify the genomic sequence of canine PSMA total RNA from prostatic tissue of

different dogs was isolated followed by amplification of the cDNA encoding canine PSMA

by PCR. Afterwards the cDNA was sequenced and the open reading frame of the protein was

assessed. To investigate the expression, processing, glycosylation and trafficking pattern of

cPSMA GFP- and FLAG-tagged forms of cPSMA were generated and expressed in either

COS-1 or MDCK cells.

As well as human PSMA canine PSMA is a type-II-transmembrane-glycoprotein comprising

779 amino acids. The sequence similarity between cPSMA and hPSMA is striking with

approximately 91% similarity at the amino acid level. The major sequence differences

between the canine and human protein are found in the cytosolic tails. The cytosolic tail of

DISCUSSION

100

cPSMA is 29 amino acids longer than the human counterpart and accounts thus exclusively

for the longer canine version of PSMA. Interestingly, cPSMA also exposes a cryptic

endocytic signal of the type MXXXL (RAJASEKARAN et al. 2003) that is found in hPSMA,

but in the canine protein it is not located directly at the N-terminus (Fig. 3-1). This is likely to

be compatible with reduced or even absent endocytosis of cPSMA with subsequent increased

expression of the protein at the cell surface. This hypothesis could not be examined, due to the

lack of an antibody that recognizes the luminal part of cPSMA.

Both PSMA proteins are highly glycosylated with ten potential N-glycosylated sites each and

the glycosylation pattern I obtained utilizing endoglycosidase H and F as well as pulse-chase

experiments were similar (Fig. 3-4).

The study has shown that recombinant cPSMA is processed and transported constitutively and

competently along the secretory pathway and is ultimately localized at the plasma membrane

in transfected COS-1 and MDCK cells (Fig. 3-2). Both species, cPSMA and hPSMA co-

localize in similar cellular compartments and are revealed in the ER, the Golgi and on the

plasma membrane (Fig. 3-3). Interestingly, however, cPSMA traverses the ER to the Golgi

more rapidly than hPSMA by virtue of the earlier appearance of its mature complex

glycosylated forms (Fig. 3-4 B). The variations in the trafficking kinetics between both

species are presumably due to differences in their cytosolic domains, since the luminal parts

of both molecules are strongly homologous. Cytosolic tails play a role in controlling protein

trafficking as stabilizers of a particular quaternary structure, as described for lactase-phlorizin

hydrolase, where dimerization is mediated by its cytosolic tail and this is required for an

efficient intracellular trafficking of the protein (BEHRENDT et al. 2010). Also trimers of the

hemagglutinin of the influenza virus are stabilized by the cytoplasmic tail of the

hemagglutinin and this trimerization is crucial for its ER exit (COPELAND et al. 1988). As

well as hPSMA also cPSMA shows a dimeric quaternary structure. Interestingly more than

80% of cPSMA is present in a dimeric form and in comparison the dimeric form of hPSMA

represents about 50-60% (Fig. 3-6). Therefore it is likely that cPSMA dimerizes more

efficiently than the human protein. This phenomenon can also be explained by the differences

in trafficking kinetics. Due to a more rapid trafficking of cPSMA between the ER and the

DISCUSSION

101

Golgi, more complex glycosylated cPSMA is available in the Golgi to form homodimers. It

appears that the dimeric forms of cPSMA are presumably more stable and transport

competent than their hPSMA counterparts due to the substantially longer cytosolic tail of

cPSMA.

Regarding detergent solubility properties I could not detect any significant differences

between the human and the canine form of PSMA. As well as hPSMA, cPSMA partially

associates with Tween 20- and Lubrol WX-DRMs and cPSMA is completely soluble in

Triton X-100 as the human protein (Fig. 3-5). This finding indicates that as well as human

PSMA, canine PSMA is also transported via distinct microdomains along the secretory

pathway.

This information about the physiology of PSMA in dogs could be of great importance in

developing diagnostic and therapeutic approaches against prostate cancer in dogs.

Furthermore, the striking homologies between cPSMA and hPSMA render the canine protein

as an excellent target antigen to validate new anti-PSMA drugs in vivo, particularly for the

evaluation of pharmacokinetic parameters and nonspecific toxicity.

CHAPTER VCHAPTER VCHAPTER VCHAPTER V::::

SSSSummaryummaryummaryummary

SUMMARY

105

V. SUMMARY

Sonja Schmidt - PSMA, a marker for prostate cancer: Molecular signalling

and transport mechanisms in humans and their transferability to dogs


folate hydrolase and carboxypeptidase activity. PSMA is mainly expressed in epithelial cells

of the prostate and elevated levels of PSMA are detected in prostate cancer cells including

those that are metastatic, and in neovasculature of further tumours. Thereby levels of PSMA

are directly proportional to disease grade and stage. It is therefore a promising biomarker in

the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies

in men.

PSMA is associated with detergent-resistant membranes (DRMs) and transported via different

DRMs along the secretory pathway. In this study we addressed the role of DRMs or lipid rafts

in two major physiological events that are of pivotal importance in the life cycle of PSMA,

homodimerization and internalization. The mature and homodimeric forms of PSMA are

mainly insoluble in Lubrol WX, but do not associate with Triton X-100-DRMs. Antibody-

induced cross-linking of PSMA leads to increased internalization of PSMA, activation of

different signalling cascades like the MAPK-pathway, redistribution of PSMA to Triton X-

100-DRMs in a time-dependent manner as well as alterations in the expression levels of

several proteins in these DRMs. One of the proteins also clustering in Triton X-100-DRMs is

α-tubulin, and this study shows that PSMA is internalized via microtubules. Another protein

showing a trend of downregulation in Triton X-100-DRMs upon activation of PSMA is

laminin receptor-1 (67LR). Indirect immunofluorescence and co-immunoprecipitation

experiments revealed a direct interaction between 67LR and YFP-PSMA. This interaction

might be of particular interest, because 67LR plays an important role in cell adhesion to the

basement membrane and less expression of 67LR within microdomains of the cell surface

might be associated with an increased risk of metastasis.

SUMMARY

106

Taken together, the results strongly propose an essential role for different types of membrane

microdomains in homodimerization, internalization and signalling pathways involving

PSMA.

As a potential target for animal preclinical studies of antibody based-therapies, the canine

PSMA protein was identified and investigated regarding its cellular distribution and the

biochemical characteristics in either COS-1 cells or MDCK cells. It appears that the canine

and human PSMA proteins share many biochemical and cellular characteristics. Canine

PSMA shows 91% amino acid homology to human PSMA, whereas the major difference is a

longer cytoplasmic tail of canine PSMA compared to its human counterpart. In addition to an

equal distribution to cellular compartments, similar glycosylation pattern and a slightly more

rapid trafficking pattern were revealed. In a fashion almost similar to human PSMA, canine

PSMA also forms homodimers and is associated with distinct lipid microdomains.

Therefore human and canine PSMA are very similar in their behaviour and canine PSMA

turned out to be a useful model-protein for prostate cancer and suitable antigen for targeted

therapy studies in dogs.

CHAPTER VI:CHAPTER VI:CHAPTER VI:CHAPTER VI:

ZusammenfassungZusammenfassungZusammenfassungZusammenfassung

ZUSAMMENFASSUNG

109

VI. ZUSAMMENFASSUNG

Sonja Schmidt - Prostatakrebsmarker PSMA: Molekulare Signal- und

Transportmechanismen beim Menschen und ihre Übertragbarkeit auf den

Hund

Das Prostata-spezifische Membran Antigen (PSMA) ist ein Typ-II-Transmembranprotein mit

Folathydrolase- und Carboxypeptidase-Aktivität. PSMA wird überwiegend in den

Epithelzellen der Prostata exprimiert, wobei die Expression in Prostatakrebszellen, deren

Metastasen und auch Blutgefäßen zahlreicher anderer Tumorarten proportional zum

Schweregrad der Erkrankung erhöht ist. Deshalb handelt es sich hierbei um ein

vielversprechendes Markerprotein, das für die Prostatakrebs-Diagnose sowie für verschiedene

Immuntherapien beim menschlichen Prostatakarzinom herangezogen werden kann.

Im Zuge seiner Biosynthese wird PSMA entlang des sekretorischen Transportweges in

unterschiedlichen Membranmikrodomänen transportiert. Im Rahmen dieser Arbeit sollte die

Funktion der Membranmikrodomänen während der Homodimerisierung und Endozytose von

PSMA untersucht werden. Die reife, homodimere Form von PSMA ist mit Lubrol WX-

resistenten Membranmikrodomänen assoziiert, nicht aber mit Triton X-100-resistenten

Membranen. Cross-linking von oberflächlichen PSMA-Molekülen mit Hilfe spezifischer

Antikörper führt zu einer erhöhten Endozytose-Rate des Proteins, zur Aktivierung

verschiedener Signalkaskaden wie dem MAPK-Signalweg, zu einer Umverteilung von PSMA

in Triton X-100-resistente Membranmikrodomänen und gleichzeitig zu einer veränderten

Expression anderer Proteine in diesen Triton X-100-resistenten Membranen. α-tubulin wird

aufgrund der Aktivierung von PSMA ebenfalls in Triton X-100-resistente

Membranmikrodomänen umsortiert und diese Studie ergab, dass die Endozytose von PSMA

entlang der Mikrotubulie stattfindet. Der Laminin Rezeptor-1 (67LR) ist nach der Aktivierung

von PSMA weniger stark mit den Triton X-100-resistenten Membranen assoziiert. Indirekte

Immunfluoreszenz und Co-Immunpräzipitation zeigten eine direkte Interaktion zwischen

67LR und YFP-PSMA. Da 67LR eine wichtige Rolle bei der Zelladhaesion spielt, könnte

ZUSAMMENFASSUNG

110

eine verminderte Expression in den Membranmikrodomänen der Zellmembran ein erhöhtes

Risiko für Krebsmetastasen darstellen.

Diese Ergebnisse demonstrieren die Wichtigkeit verschiedener Membranmikrodomänen bei

der Homodimerisierung, Endozytose und den Signalwegen von PSMA.

Da auf PSMA basierende Therapien auch in vorklinischen Studien am Tier getestet werden

müssen, wurde als mögliches Modell das Hunde-PSMA-Protein identifiziert um dessen

zelluläre Verteilung und die biochemischen Charakteristika in COS-1-Zellen und MDCK-

Zellen zu untersuchen. Hierbei zeigten sich viele identische biochemische und zelluläre

Eigenschaften. Die Aminosäure-Sequenz vom Hunde-PSMA ist zu 91% homolog zu der

entsprechenden Sequenz des Menschen, wobei der größte Unterschied zwischen beiden ein

längerer cytoplasmatischer Teil des Hunde-PSMA ist. Genauso wie die Verteilung beider

Proteine zwischen den verschiedenen zellulären Kompartimenten, ist auch die Glykosylierung

beider Proteine ähnlich. Der Transport vom Hunde-PSMA zwischen dem ER und dem Golgi-

Apparat findet etwas schneller statt, verglichen mit der Transportrate des humanen Proteins.

Sowohl die Homodimerisierung als auch die Assoziation mit verschiedenen

Membranmikrodomänen ist bei den PSMA-Proteinen beider Spezies vergleichbar.

Da PSMA von Mensch und Hund sehr ähnliche Charakteristika aufweisen, hat diese Studie

gezeigt, dass Hunde-PSMA ein geeignetes Modell-Protein für die menschliche Prostata und

ein geeignetes Ziel-Antigen für auf PSMA basierende Therapien darstellt.

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AAAAppppppppendixendixendixendix

APPENDIX

139

VIII. APPENDIX

8.1. Acknowledgements

Sincere thanks are given to Prof. Hassan Y. Naim for teaching me, for the interesting project I

was working on and for his time and ideas. I am also grateful for the financial support as well

as the opportunity to participate in several very interesting national and international

meetings.

I am much indebted to Prof. Rita Gerardy-Schahn and Prof. Wolfgang Bäumer for their

contribution as the members of my advisory committee. The discussions during our meetings

were very fruitful and helpful for me.

This is a great opportunity to express my respect and thanks to Prof. Marco Colombatti for

second evaluation of my thesis and together with Giulio Fracasso and Dunia Ramarli for the

very successful cooperation we had during the whole time.

I am also grateful for the assistance and collaboration of my colleagues at the Department of

Physiological Chemistry.

Finally, I would like to thank my family for their support during my whole academic studies.

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