µ2 Binding Directs the Cystic Fibrosis Transmembrane ... · µ2 Binding Directs the Cystic...

µµµµ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the

Clathrin Mediated Endocytic Pathway

Kelly M. Weixel and Neil A. Bradbury*

Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of

Pittsburgh School of Medicine Pittsburgh, PA 15261 USA.

Running Title: CFTR binds to µ2 subunit of AP-2 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; GST, glutathione-S-transferase, PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance. Address for correspondence: *Neil A. Bradbury Dept. of Cell Biology and Physiology University of Pittsburgh School of Medicine 3500 Terrace Street, Pittsburgh, PA 15261. Tel. No: 412-648-2845 Fax: 412-648-8330 E-mail: [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 17, 2001 as Manuscript M104545200

Weixel and Bradbury: CFTR Binds µ2

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SUMMARY

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-

based internalization motif, Y1424DSI, which interacts with the endocytic clathrin adaptor

complex, AP-2, and is required for its efficient endocytosis. Although direct interactions

between several endocytic sequences and the medium chain and endocytic clathrin adaptor

complexes has been shown by protein-protein interaction assays, whether all these interactions

occur in vivo or are physiologically important has not always been addressed. Here we show,

using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and

the µ subunit of AP-2. Crosslinking experiments were performed using photoreactive peptides

containing the YDSI motif and purified adaptor complexes. CFTR peptides crosslinked a 50 kDa

subunit of purified AP-2 complexes, the apparent molecular weight of µ2. Furthermore, isolated

µ2 bound to the sorting motif, YDSI, both in crosslinking experiments and GST-pulldown

experiments, confirming that µ2 mediates the interaction between CFTR and AP-2 complexes.

Inducible overexpression of dominant-negative µ2 in HeLa cells results in AP-2 complexes that

fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced

compared to wild type HeLa cells. These results indicate that the AP-2 endocytic complex

selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl

terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the µ2 subunit of AP-2

and mutations in µ2 that block its interaction with YDSI inhibit the incorporation of CFTR into

the clathrin-mediated endocytic pathway.


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INTRODUCTION

Cystic Fibrosis (CF), the most common lethal genetic disease of Caucasians, is caused by

mutations in the gene encoding the protein for the cystic fibrosis transmembrane conductance

regulator, (CFTR) (1). Alterations in the genetic sequence of CFTR result in the impairment of

transepithelial chloride secretion in response to the activation of a cAMP-mediated signal

transduction pathway in CF cells (2). Indeed, it is now established that CFTR functions at the

apical plasma membrane of polarized epithelial cells to regulate chloride permeability in

response to cAMP-dependent protein kinase (PKA)-mediated phosphorylation, ATP binding and

ATP hydrolysis (3-6).

Morphological, biochemical and functional evidence indicates that in addition to a cell

surface localization, CFTR is also found in endosomal and recycling compartments. The

presence of CFTR within endosomes was demonstrated functionally in several cell lines

expressing endogenous and exogenous CFTR. Thus PKA stimulated an anion conductance in

isolated as well as in situ endosomes, a conductance that was susceptible to inhibition by

monoclonal anti-CFTR antibodies (7). In addition, immunocytochemistry at the light

microscopy level has revealed co-localization of CFTR with rab4, a member of the small GTP-

binding protein family, and a component of recycling endosomes (8). Compelling evidence

indicates that CFTR enters endosomal compartments through the clathrin-mediated endocytic

pathway (9-12). Furthermore, perturbation of clathrin-coated vesicle (CCV) formation inhibits

the removal of CFTR from the plasma membrane (12,13).


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Clathrin mediated internalization of integral membrane proteins relies on the presence of

relatively short peptide sequences within their cytosolic tails. Although heterogeneous, most

sorting signals fall into two main classes (14-17). The first class is characterized by an essential

tyrosine residue, either as part of an NPXY motif (as initially identified in the LDL receptor), or

in the context of a YXXΦ motif, where X is any amino acid and Φ is a bulky hydrophobic amino

acid). The second class of internalization motifs typically contains a dileucine sequence, though

in some cases one of the leucines may be replaced by an isoleucine, valine, alanine or

methionine. Such endocytic sorting signals have been most extensively studied in type I and

type II membrane proteins. Depending upon the precise context of the sorting signal, such

motifs can be also recognized in sorting events within the trans Golgi network as well as

endosomes (18,19).

Clathrin adaptor complexes (APs) have been obvious candidates to recognize sorting

signals and to act as adaptors between integral membrane proteins and the clathrin lattice.

However, in only a very few cases have membrane proteins and adaptors co-immunoprecipitated

(20). Several in vitro assays, including bead pull-down and surface plasmon resonance had

shown interaction between tyrosine-based sorting signals and clathrin adaptor complexes (21-

25), yet surprisingly little data is available concerning whether such interactions occur in vivo or

are physiologically important. Although a direct interaction between YXXΦ internalization

sequences and µ2 subunits has been demonstrated, the binding of receptors to AP-2 does not


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necessarily correlate with the internalization capacity of proteins bearing YXXΦ motifs. For

example, the epidermal growth factor (EGF) receptor strongly binds AP-2 through a YRAL

sequence (26). However, mutations in the YRAL sequence that abolish the interaction of the

EGF receptor with AP-2 do not significantly affect internalization of the receptor (26) (27). In

contrast, transferrin receptors whose endocytic removal from the plasma membrane shows a

strong dependence upon a YTRF motif (28), show very weak, if any, detectable interaction with

the AP-2 endocytic adaptor complex (21,29). In addition, although direct interaction between

tyrosine based sequences and the µ subunit of the endocytic adaptor complex AP-2 have been

demonstrated by several types of protein-protein interaction assays (21) (23), a recent report

suggests that the NPXY motif binds directly to the terminal domain of the clathrin heavy chain

rather than directly to AP-2 (30).

In contrast to the many studies on endocytic signals in type I and II membrane proteins,

relatively little is known about the endocytic signals in polytopic membrane proteins such as

transporters and ion channels. The β2-adrenergic receptor contains a highly conserved tyrosine

residue (Tyr326) that is responsible for the ligand-induced internalization of the receptor (31).

GLUT4, the insulin-responsive sodium-glucose co-transporter is constitutively retrieved from the

plasma membrane via clathrin-mediated pathways in the absence of insulin and contains a

leucine endosomal targeting signal (32). Recently, a tyrosine (Tyr1424)-based motif was

identified as a potential endocytic targeting signal in the carboxyl cytoplasmic tail of CFTR

(10,33,34). Mutation of this sequence, either in the context of a chimera consisting of TfR and


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the carboxyl terminal tail of CFTR (10) or, in intact CFTR stably expressed in a heterologous

cell line (35), inhibits the retrieval of CFTR from the plasma membrane.

Thus although AP-2 adaptors, and their medium chain subunits, have been implicated in

the recruitment of several type of plasma membrane proteins into the clathrin-dependent

endocytic pathway, the molecular details and the physiological significance of such interactions

is poorly understood. Moreover, such information is completely lacking for ion channels. We

have used both in vitro protein-protein interaction studies and in vivo studies with inducible

dominant-negative µ2, to investigate the interaction of adaptor complexes with the tyrosine

endocytic sequence of a clinically important ion channel, CFTR. The analyses of in vitro

binding and endocytic trafficking reveal, for the first time in a polytopic ion channel, a unified

model for CFTR endocytosis showing a strong correlation between in vitro binding of CFTR to

µ2 and its endocytic capacity.

EXPERIMENTAL PROCEDURES

Monoclonal antibodies against α adaptin and β1/β2 adaptin were obtained from BD

Transduction Laboratories (Lexington, KY). Monoclonal antibody AC1-M11 against α-adaptin

was a generous gift from Dr. M.S. Robinson (University of Cambridge). A rabbit polyclonal


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antiserum to an amino-terminal sequence of the µ2 chain was custom generated by Affinity

BioReagents (Golden, CO). Antibodies were obtained by immunization with a peptide

corresponding to residues 11-29 of human µ2 (KGEVLISRVYRDDIGRNAV). This antibody

was specific for µ2 and did not recognize the related µ1 protein (data not shown). Polyclonal

anti-CFTR antibodies were from Affinity Bioreagents, Inc. (Golden, CO). Alexa-Flour 488 goat

anti-rabbit secondary antibodies were from Molecular Probes (Eugene, OR). Gelvatol was from

Monsanto Co. (Augusta, GA). TNT T7 Coupled Reticulocyte Lysate System was from

Promega (Madison, WI). The QuickChange site-directed mutagenesis kits were obtained from

Stratagene (La Jolla, CA). SuperSignal West Pico Chemiluminescent Substrate and GelCode

Blue Stain Reagent were from Pierce (Rockford, IL). Glutathione-sepharose 4B and Redivue

35S-methionine were from Amersham Pharmacia Biotech (Piscataway, NJ). CompleteEDTA-

free protease inhibitor tablets were obtained from Roche Molecular Biochemicals. Peptides were

synthesized by New England Peptide (Fitchburg, MA). Geneticin, LipofectAMINE 2000

Reagent, Primers and tissue culture media were from Life Technologies, Inc (MD). All other

antibiotics and materials were from Sigma and were of reagent grade quality.

Purification of AP complexes

Clathrin coated vesicles were obtained from bovine calf brains (PelFreeze) as previously

described. (36-38). Purified adaptor complexes were analyzed to establish that all four subunits


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were present in the preparation by protein staining with GelCodeBlue reagent, (Pierce) and

immunoblot with subunit specific antibodies.

Surface Plasmon Resonance

The interaction between CFTR carboxyl-terminus peptides and AP-1 or AP-2 was analyzed in

real-time by surface plasmon resonance (SPR) (39) using a Biacore X Biosensor (Biacore,

Piscataway, NJ). Peptides (KVIEENKVRQYDSIQ) were coupled via their amino terminal

biotin moiety to an SA5 sensor chip (streptavidin surface) according the manufacturers

instructions. All binding studies were performed with buffer containing 20 mM HEPES-NaOH

[pH 7.0], 150 mM NaCl, 10 mM KCl, 2 mM MgCl2, 0.2 mM dithiothreitol) at a flow rate of 20

µl/min. Purified adaptor and clathrin preparations were centrifuged at 250,000 g for 30 minutes

prior to the experiment to remove potential aggregates. Adaptors and clathrin were used at 100

nM unless otherwise noted. A short pulse injection (15 sec) of 20 mM NaOH/0.5% SDS was

used to regenerate the sensor chip surface after each experiment. The peptide-derivatized sensor

chip remained stable and retained its specific binding capacity throughout the experiments.

Peptide Synthesis

Several peptides were synthesized by New England Peptide, (Fitchburg, MA) and are shown in

Table I. The synthetic peptide *YQRL corresponds to the carboxyl-terminus of the cytoplasmic

tail of TGN38, and has been shown to bind to purified AP-2 (23). Photoreactive peptides *YDSI

and *ADSA correspond to the peptides YDSI and ADSA and have a photoreactive probe,


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benzoylphenylalanine (BPA) at position Y-3 and a biotin moiety added to the amino-terminal

lysine to facilitate detection of cross-linked products using streptavidin-HRP. Peptides were

stored at 4°C until use when they were diluted in water to a final concentration of 2µM.

Plasmid Constructs

The GST-CT construct (kindly provided by Drs R Frizzell and F. Sun, University of Pittsburgh)

contains the carboxyl terminus of CFTR (amino acids 1404-1480) amplified from pBQ4.7 CFTR

cDNA using the polymerase chain reaction and subcloned in to the pGEX 4T-1 vector. The

cDNA for GST-fusion proteins containing the human immunodeficiency virus type 1 p6 protein

(GST-p6) and equine infectious anemia virus p9 protein (GST-p9) were kindly provided by Dr.

R. Montellaro, (University of Pittsburgh). GST constructs containing µ1 and µ2 were provided

by Dr. J. Bonafacino, NIH. The cDNA of mouse µ2 was subcloned from a pACTµ2 construct

kindly provided by J. Bonafacino into pCDNA3.1 for in vitro translation reactions. Epitope

tagged wild type µ2 in pcDNA 3.1 was obtained from Dr. A. Sorkin, (University of Colorado).

UV –induced cross-linking reactions

Crosslinking experiments were performed as described (23); briefly, Purified AP

complexes were kept in Tris-buffer (250 mM Tris, pH 7.4 1 mM EGTA, 0.5 mM MgCl2, 0.5

mM dithiothreitol). AP concentrations ranging between 0.13-.6 mg/ml were incubated with 0.2

µM photoreactive peptides. For competition experiments, peptides YQRL, ADSA, or YDSI

were included at a final concentration of 200 µM. Crosslinking experiments were carried out in


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microtitre plates in a final volume of 20 µL. Plates were incubated on ice for 30 minutes in the

dark. Samples were irradiated at 80,000µJ/cm2 to crosslink samples. Following irradiation, 5µl

of 5X Laemmli sample buffer was added to the samples and boiled for 5 minutes.

Detection of crosslinked products

To identify the AP protein chains that were crosslinked to the biotinylated peptide, an aliquot of

the crosslinking reaction was subject to SDS-PAGE and transferred to nitrocellulose. Following

blocking (10 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween-20, 10% non-fat dry milk) for 1

hour at room temperature, the membrane was rinsed in TBST (10 mM Tris pH 8.0, 150 mM

NaCl, 0.05% Tween-20) and incubated for 30 minutes with streptavidin conjugated with

horseradish peroxidase (HRP, Zymed: 5 µl/ml) in blocking buffer. The membrane was then

washed 6 times for 5 minutes with TBST and processed for ECL using SuperSignal West Pico

ECL Reagent, (Pierce). For recapturing experiments, AP complexes were photolabeled with

*YSDI peptides and AP complexes were immunoprecipitated with AC1-M11 antibodies.

Immunoprecipitates were washed five times in RIPA buffer (1% Triton X-100, 0.3 M NaCl, 50

mM Tris-HCl [pH 7.0], 0.1% BSA), supplemented with CompleteEDTA-free Protease

Inhibitors (Roche). Samples were resuspended in SDS buffer (0.1 M Tris-HCl, [pH 7.4], 1%

SDS, 10 mM dithiothreitol). Samples were shaken vigorously for 20 minutes at 4° and boiled

for 5 minutes to release the Immunoprecipitates from the antibodies and to denature the AP

complex. The extract was diluted 20 fold with RIPA, clarified by centrifugation at 15,000 g,

4°C. Samples were then incubated with rotation at 4° for 1 hour with one of the following


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antibodies (anti-α, anti-β1/β2, or anti-µ). Immunoprecipitates were washed twice with RIPA

and once with RIPA minus detergent. Samples were analyzed by SDS-PAGE and immunoblot

using streptavidin-HRP as described above.

Mammalian Cell Culture and Transfection

HeLa cell lines expressing HA-tagged D176A/W421A mutant µ2 constructs under control of the

tetracycline-off system were kindly provided by Dr. A. Sorkin (University of Colorado). The

HeLa cell line been previously characterized and expresses an epitope-tagged µ2 containing

mutations at D176 and W421 under control of the Tet-Off System. Studies in this cell line

demonstrate that mutant HA-µ2 incorporates into AP-2 and is targeted to coated pits. Inducible

overexpression of the mutant µ2 resulted in the replacement of endogenous wild type µ2 in AP-2

complexes and the complete abrogation of AP-2 interactions with tyrosine-based internalization

motifs (40). Cells were grown in DMEM supplemented with 10% fetal calf serum, 400 µg/ml

G418, 200ng/ml puromycin, and 2ng/ml doxycycline, a tetracycline derivative. For experiments,

cells were plated in the growth medium without selection markers with or without doxycycline.

At 24 h after plating, medium was replaced with fresh medium with or without doxycycline;

minus doxycycline cells were supplemented with 2 mM sodium butyrate to ensure high levels of

HA-µ2 protein expression to replace the endogenous wild type µ2 in AP-2 complexes.

Experiments were performed 3-4 days after plating. For transient transfections cells were plated

at 50-60% confluency as outlined above. 48 h after plating cells were transfected with

pcDNA3.1 CFTR plasmids using the Lipofectamine 2000 reagent according to the


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manufacturer’s instructions (LifeTechnologies). Transfected cells were used 48 hours after

transfection.

GST pull-down experiments

HeLa cells cultured in the presence or absence of doxycycline were washed with PBS to remove

media and to cool the cells. Cells were lysed in TGH buffer (50 mM HEPES [pH 7.4,] 1%

Triton X-100, 10% glycerol, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA), supplemented with

CompleteEDTA-free protease inhibitor tablets (Roche Molecular Biochemicals) for 20

minutes at 4°C. Insoluble material and aggregates were removed by centrifugation for 45 min at

125,000 X g. Glutathione-sepharose loaded with 20 µg of GST or GST-CT, were incubated

with HeLa lysates for 3 hours to overnight at 4°C. Beads were washed three times with TGH

and once with HEPES buffer (20 mM HEPES, 150 mM KCl, 2 mM MgCl2 [pH 7.2]). Beads

were resuspended in Laemmli buffer and bound proteins were resolved by SDS-PAGE,

transferred to nitrocellulose, and processed for immunoblot according to standard protocols.

Detection of bound primary monoclonal antibodies was performed using horseradish peroxidase-

conjugated goat anti-mouse secondary antibodies and enhanced chemiluminescence (ECL) using

SuperSignal West Pico Reagents (Pierce).

pcDNA 3.1 constructs containing wild type or D176A/W421A µ2 were translated at

30°C for 1.5 hour in the presence of 35S-methionine using the TNT coupled protein translation

kit (Promega), according to the manufacturer’s instructions. Prior to pull-down experiments


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10% of the translation was reserved for SDS-PAGE analysis. For each pull-down experiment,

45 µl of the translation was diluted into 1 ml of TGH buffer and cleared by centrifugation for 45

min at 125,000g. 20 µg of GST-CT, GST-P6, GST-P9, and GST preabsorbed onto glutathione-

Sepharose were incubated with TGH diluted translation products for 3 hours to overnight at 4°C.

Beads were washed 3 times with TGH buffer and resuspended in Laemmli buffer. Proteins were

resolved by SDS-PAGE, gels were dried and processed for autoradiography.

Expression of GST fusion proteins

GST-fusion proteins were expressed in Escherichia coli BL21 de3pLysS strain. Protein

expression was induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 3 hours and

GST fusion proteins were purified on glutathione-Sepharose 4B (Amersham Pharmacia Biotech).

Immunofluorescence

Cells grown on glass coverslips were cooled to 4°C for 15 minutes and washed 3 times

with ice cold PBS. Cells transiently expressing CFTR were incubated with polyclonal anti-

CFTR antibodies diluted 1:500 in PBS containing 1% BSA for 1 hour at 4°C. Cells were then

rapidly warmed to 37°C with pre-warmed media and incubated for 15 minutes in a 37°C

incubator at 5% CO2. Cells were immediately cooled to 4°C with PBS-containing 1% BSA and

kept on ice. The cell surface was labeled with wheat germ agglutinin conjugated to Rhodamine


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(Molecular Probes) for 30 minutes at 4°C. Cells were then washed, fixed in 2%

paraformaldehyde and permeabilized in PBS containing 0.1% Triton X-100. The cells were

incubated with Alexa-Flour 488 goat anti-rabbit secondary antibodies for 1 hour. The cells were

washed and subsequently mounted onto glass slides using Gelvatol. Confocal microscopy was

performed on a TCS confocal microscope equipped with krypton, argon, and helium-neon lasers

(Leica, Deerfield, IL). Images were imported into Adobe Photoshop for final presentation. To

examine colocalization of CFTR and cell surface markers, the images were imported into

Metamorph imaging software (Universal Imaging Corporation Downingtown, PA). The stored

images were converted into binary images and a new series of images were generated by

performing a Boolean operation "AND" in the pairs of images representing Alexa-Fluor 488 and

Rhodamine signals within the same optical section. The final presentation of colocalized proteins

uses differential intensity spectral representation encompassing the black/white intensity range 0-

255.

Radioactive Binding Assay

The internalization efficiency of CFTR was measured by monitoring the cell-surface density of

CFTR with a rabbit polyclonal antibody against an extracellular epitope of CFTR and 125I-

labeled Protein A (NEN). Cell-surface CFTR was labeled for 1 hour with anti-CFTR antibodies

(3µg/ml) in Optimem at 4°C. Following incubation, the cells were washed with PBS and

internalization was initiated by incubating the cells in pre-warmed 37°C Optimem media

followed by incubation at 37°C for 15 minutes. Cells were rinsed with ice-cold PBS and anti-


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CFTR antibody remaining at the cell-surface was measured by with binding of 125I-Protein A

(30µCi total) in Optimem media for 1 hour at 4°C. The cells were rinsed in ice-cold PBS and

solubilized in 100 mM NaOH/0.1% v/v Triton X-100. The radioactivity of solubilized cells was

determined with a gamma counter. Non-specific binding was measured in mock transfected

cells. The internalization efficiency of CFTR in wild type HeLa cells and HeLa cells expressing

the dominant negative mutant µ2 was expressed as a percentage of the decrease in the surface

binding of 123I-Protein A between the zero time point and fifteen minutes. The results are a mean

of three individual experiments ± s.e.m.

RESULTS

The tyrosine-based sorting signal of CFTR selectively interacts with plasma membrane

adaptors, AP-2.

We employed surface plasmon resonance to monitor interaction of the carboxyl cytoplasmic tail

of CFTR with purified clathrin, AP-1 and AP-2. This method has been used in several studies to

analyze the interaction between purified adaptors and receptor tails. A typical binding

experiment using adaptors from bovine brain is shown in Figure 1. As the solution passed over a


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sensor surface immobilized with wild-type CFTR peptide was changed from buffer alone to

buffer containing AP-2, a rapid increase in signal was recorded reflecting AP-2 binding and an

increase in mass at the sensor surface. Upon returning to buffer after 3 minutes of AP-2

exposure, a loss of signal was observed, consistent with a loss of mass from the sensor surface.

The signal did not return entirely to the initial baseline, indicating that some protein remained

bound. After exposure to 20 mM NaOH/0.5% SDS for 2 minutes, the surface was completely

regenerated (data not shown). When AP-1 containing solution was passed over the CFTR

containing chip, a small binding signal as observed, which decreased upon AP-1 washout. Note,

however, that the signal obtained with AP-1 was observed at a ten-fold higher adaptor

concentration that that for AP-2 binding. As expected, clathrin showed no interaction with

CFTR at all, consistent with the notion that CFTR/clathrin showed no interaction with CFT at

all, consistent with the notion that CFTA/clathrin interactions are mediated via adaptor

complexes.

The tyrosine-based sorting signal in CFTR binds to the µµµµ2 subunit of AP-2

To characterize the subunit of AP-2 that interacts with the carboxyl terminus of CFTR, we

performed in vitro cross-linking assays using synthetic photoactivatable peptides that contained

the tyrosine motif identified in CFTR, *YDSI (Table I). We initially identified that *YDSI

peptides specifically associate with plasma membrane adaptors, AP-2, (Figure 2A, lane 4). The

*YDSI peptide crosslinks to a 50 kDa subunit of the AP-2 complex, the apparent molecular


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weight of µ2, but fails to recognize any of the subunits of the Golgi specific AP-1 complex,

(Figure 2A, lane 2). Furthermore, peptides containing alanine substitution at tyrosine 1424 and

isoluecine1427, *ADSA, abolished the ability of the peptide to crosslink to AP-2 (Figure 2A,

lane 3). Specificity of binding was confirmed by performing crosslinking studies in the presence

of competitor peptides. Crosslinking reactions were performed as described in Figure 2 with or

without the presence of 100 fold excess competitor peptides. Peptides containing mutations in

the tyrosine based sorting motif failed to compete with wild type peptides for AP-2 crosslinking,

(Figure 3, lane 2), whereas peptides containing the tyrosine based sorting motif from TGN38

successfully competed with CFTR peptide for AP-2 crosslinking, as did the wild type peptide

YDSI, (Figure 3, lanes 3 and 4 respectively). This is consistent with our hypothesis that the

interaction between CFTR and AP-2 is dependent on the tyrosine-based motif YDSI.

While the intact complex was present in the crosslinking reaction as detected by

immunoblot and Coomassie staining (Figure 2B and 2C respectively), a 50 kDa subunit was the

only crosslinked species detected. To confirm that the crosslinked protein was µ2, recapturing

experiments were performed. After photolabeling, AP-2 complexes were immunoprecipitated

with the AP-2 specific antibody, anti-α-adaptin. Isolated AP-2 complexes were dissociated into

individual subunits by denaturation and boiling followed by immunoprecipitation of the resultant

individual subunits. The recaptured subunits were then analyzed by SDS-PAGE and

immunoblot with streptavidin-HRP conjugates to determine which subunit had been crosslinked

by the biotinylated peptide. Confirming the results in Figure 2A, the only subunit of the AP-2

complex that was crosslinked by the *YDSI peptide was µ2 (Figure 4, lane 6). To confirm that


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the �2 subunit of AP-2 is solely responsible for mediating the interaction between CFTR and

AP-2, crosslinking experiments were performed with isolated µ2 subunits. A GST-fusion

protein containing the medium subunit of the AP-2 complex, µ2, was purified onto glutathione

Sepharose beads and incubated with the photoreactive peptides described above. After UV

illumination, crosslinked proteins were resolved by SDS-PAGE and detected by immunoblot.

*YDSI peptides crosslinked to GST-µ2, but failed to crosslink to GST alone (Figure 5, lanes 9, 4

and 7 respectively). Furthermore, GST-fusion proteins containing the medium subunit of AP-1,

µ1, were not crosslinked by *YDSI (Figure 5, lanes 5 and 8). As seen with the intact AP-2

complexes, crosslinking was specific for the tyrosine-based motif as peptide *ADSA failed to

crosslink to µ2 (Figure 5, lane 6). These results demonstrate that the interaction between plasma

membrane adaptors, AP-2, and CFTR requires the intact sorting signal, YDSI. Furthermore, this

interaction is mediated solely through the medium subunit of the AP-2 complex, µ2

Dominant Negative Mutant µµµµ2 Fails to Interact with CFTR

Binding assays and crystal structure data have identified that amino acid residues D176

and W421 of µ2 as essential for the binding of YXXΦ motifs. To characterize the binding site

of µ2 for YDSI, we performed pull-down assays using in vitro translated µ2 and GST-fusion

proteins containing the YDSI motif of CFTR, GST-CT. Wild type µ2 or µ2 with mutations

D176A/ W421A were translated in vitro in the presence of 35S-methionine and incubated with

either GST-CT or GST alone. GST-CT efficiently bound to wild type µ2, but failed to interact


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with mutant µ2, (Figure 6, Lanes 2 and 1 respectively). Similar experiments were performed as

controls using the p9 protein from equine infectious anemia virus that has previously been shown

to interact with µ2, and the human immunodeficiency virus type 1 p6 protein that does not

interact with µ2 (Figure 5, lanes 4 and 6 respectively) (41). Thus, mutagenesis and pull-down

assays demonstrate that the two regions on µ2 containing D176 and W421 are critical in µ2

interaction with the YDSI sorting signal of CFTR.

Inducible overexpression of dominant negative µµµµ2 inhibits the interaction of AP-2 and

CFTR

A Previously characterized HeLa cell line that expresses a dominant negative mutant µ2

(D176A/W421A) under control of the Tet-Off System (40) was utilized to investigate how the

replacement of endogenous µ2 by mutant µ2 affects the interaction between AP-2 and CFTR.

Lysates from HeLa cells expressing either the wild type µ2 or dominant negative µ2 were

incubated with GST-CT or GST alone. The amount of AP-2 that bound to GST-CT was

determined by immunoblot of GST precipitates using an antibody specific for the AP-2 complex,

α -adaptin. Wild-type AP-2 complexes efficiently bound to GST-CT, but not GST alone (Figure

6, lanes 3 and 4 respectively). In contrast, when the expression of mutant µ2 was induced by the

removal of doxycycline, binding of AP-2 and GST-CT was abolished (Figure 7, lane 2). The

amount of AP-2 in the input faction from cells expressing wt or dominant negative µ2 was the

same (data not shown). These results demonstrate that the µ2 subunit of the AP-2 complex


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alone mediates the interaction between coated pit adaptor and the internalization motif YDSI in

CFTR.

To examine the consequence of blocking AP-2 and CFTR interactions on the

internalization of CFTR, full-length CFTR was transiently expressed in wild type and dominant

negative µ2 expressing HeLa cells. The internalization of CFTR was followed by

immunofluorescence using a polyclonal antibody against an extracellular epitope of CFTR.

HeLa cells were labeled for 1 hour at 4° C with anti-CFTR antibodies, cells were then warmed to

37° for fifteen minutes to allow for internalization. Following internalization, cells were

returned rapidly to 4°C and the cell surface was labeled with Rhodamine conjugated Wheat

Germ Agglutinin (WGA). The internalized pool of CFTR was then detected by fixing and

permeabilizing the cells and incubating with Alexa-Flour 488 conjugated secondary antibodies.

In wild type cells following the internalization period CFTR was found primarily as discrete

punctate vesicles within the cytoplasm (Figure 8, panel B). CFTR signal (green) did not co-

localize with the WGA signal (red) at the cell surface in merged images demonstrating that

CFTR was rapidly internalized from the plasma membrane in cells expressing wild type µ2

(Figure 8, panels B and C). However, in cells expressing the dominant negative mutant µ2, the

immunolocalization pattern of CFTR was different, with the majority of the signal remaining at

the cell surface (Figure 8, panels E and F). The signal for CFTR in the mutant cells co-localizes

with that of the cell surface marker WGA, yielding a yellow cell surface staining pattern upon

merge of the separate signals (Figure 8, panel F). These observations suggest that CFTR


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internalization was inhibited in cells expressing the dominant negative mutant µ2. These images

were imported into Metamorph imaging software to compare the regions of colocalization in

HeLa cells expressing endogenous wild type µ2 or dominant negative mutant µ2. The degree of

colocalization of green (CFTR) and red (WGA) signal is depicted as a spectral plot

encompassing the black/white intensity range 0-255, with white being the most intense. Cells

expressing the dominant negative mutant µ2 show significantly greater co-localization of CFTR

with WGA (Figure 9 panel A) compared to cells expressing the endogenous wild type µ2 (Figure

9, panel B). In addition, the regions where CFTR and WGA overlap in HeLa cells expressing

the dominant negative mutant µ2 show a greater intensity of colocalization compared to cells

expressing endogenous wild type µ2 in differential intensity maps; (compare panels A and B in

Figure 9), suggesting that more CFTR remains at the cell surface in cells expressing the

dominant negative mutant µ2 compared to cells expressing wild type endogenous µ2.

A radioactive antibody-binding assay was implemented to compare quantitatively the

rates of endocytosis of CFTR in cells expressing wild-type and dominant negative µ2. The

extracellularly exposed epitope of CFTR was saturated with anti-CFTR antibodies at 4°C.

Endocytosis was subsequently initiated by shifting the temperature to 37°C. After 15 min, anti-

CFTR antibody remaining at the cell surface was measured by the specific binding of 125-I

labeled protein-A at 4°C. The rapid disappearance of cell surface anti-CFTR antibodies in HeLa

cells expressing wild-type µ2 indicated that CFTR is internalized with high efficiency in HeLa

cells (Figure 10), as has been shown for many other cell types. Approximately 60% of cell


22

surface CFTR was endocytosed during a 15-minute incubation. This implies an internalization

rate of 4.2 ± 0.3 %/min (n = 3) for CFTR in cells expressing wild-type µ2. In contrast, the

internalization rate of CFTR in cells expressing dominant negative µ2 was significantly slower

(1.8 ± 0.2 %/min; n = 3) (Figure 10).


23

DISCUSSION

Both in vitro (including co-immunoprecipitation, pull-down, SPR) and in vivo (yeast-2-

hybrid and dominant-negative cell lines) assays have been utilized to investigate the interaction

between internalized integral membrane proteins and subunits of the endocytic AP-2 clathrin

adaptor complex. However, although a direct interaction between YXXΦ internalization

sequences and µ2 subunits has been demonstrated, the binding of receptors to AP-2 does not

necessarily correlate with the internalization capacity of proteins bearing YXXΦ motifs. For

example, the epidermal growth factor (EGF) receptor strongly binds AP-2 through a YRAL

sequence (26). However, mutations in the YRAL sequence that abolish the interaction of the

EGF receptor with AP-2 do not significantly affect internalization of the receptor (26). In

contrast, transferrin receptors whose endocytic removal from the plasma membrane shows a

strong dependence upon a YTRF motif (28), show very weak, if any, detectable interaction with

the AP-2 endocytic adaptor complex (21,29). Moreover, while such studies as those described

above have been performed on monotopic abundantly expressed receptors, there is a paucity of

information regarding the interaction of clathrin adaptor proteins and polytopic low abundance

proteins, such as ion channels. Our data provide the first evidence linking in vitro binding data

with in vivo internalization data for a clinically important ion channel, the cystic fibrosis

transmembrane conductance regulator, CFTR.

While the primary function of CFTR is to regulate transepithelial chloride permeability

by functioning as an apical chloride channel, immunolocalization and functional studies


24

demonstrate that CFTR also resides in the endosomal and recycling compartments (8,42). Cell

surface biotinylation indicates that CFTR undergoes rapid and efficient internalization in both

polarized epithelial and non-polarized cells (9,10). In addition, several studies indicate that the

distribution of CFTR may be regulated by cAMP-dependent protein kinase A (PKA) by

promoting the translocation of CFTR from an intracellular pool to the plasma membrane and by

inhibiting the internalization of CFTR from the plasma membrane (43-47). These results suggest

that the amount of CFTR in the plasma membrane may be regulated by insertion and retrieval

mechanisms and that these processes may provide a mechanism to augment PKA activation in

regulating CFTR in the plasma membrane.

Studies investigating the traffic of CFTR argue that it is internalized exclusively through

the clathrin-mediated endocytic pathway (12,13). While inhibition of clathrin coated vesicle

formation inhibits the removal of CFTR from the cell surface of native epithelial cells, disruption

of alternative pathways, such as caveolae, does not affect the internalization of CFTR (12).

Furthermore, both immunological and electrophysiological techniques show that CFTR can be

found in clathrin coated vesicles in both native epithelial cells and in cultured cells (11).

Proteins that target to clathrin coated vesicles are recruited to these structures by interacting with

clathrin adaptor complexes. Association of AP complexes with membrane proteins is mediated

by the presence of sorting motifs in the cytoplasmic tails of such proteins (for review see ref 20).

We have previously demonstrated that CFTR interacts with the endocytic adaptor complex, AP-

2, and that this interaction is mediated through the carboxyl-tail of CFTR (33). Analysis of the

carboxyl tails of several species of CFTR shows the presence of a highly conserved tyrosine-


25

based sorting motif at amino acids Y1424DSI (10,33). This conforms to the paradigm of a YXXΦ

sorting motif, where X is any amino acid and Φ is a hydrophobic amino acid. Indeed, mutations

in this sorting motif inhibit the internalization of full-length CFTR, and block the interaction

between the carboxyl-terminus of CFTR and AP-2 complexes in (10,33,35).

The tyrosine based sorting motif YDSI has been shown to be necessary for efficient

endocytosis of CFTR. To evaluate the selectivity of the interaction between this sorting motifs

and clathrin adaptor complexes, we performed surface plasmon resonance analysis using an

immobilized peptide corresponding to the YDSI motif of CFTR and recorded interactions with

purified adaptor complexes AP-2 and Golgi-specific adaptor complexes, AP-1. No interaction

was detected with AP-1 complexes, or clathrin. However, CFTR peptides did interact with AP-2

adaptors, indicating that the sorting motif in the carboxyl-terminus of CFTR specifically interacts

with plasma membrane adaptors, consistent with our previous studies (33). AP-2 and AP-1

complexes are responsible for selecting cargo that traffics through CCV that originate at the

plasma membrane and TGN respectively and recognize similar sorting signals in the cytoplasmic

domains of such proteins. The observation that the tyrosine signal, YDSI, selectively interacts

with AP-2 complexes but not AP-1 complexes indicates that this signal in the carboxyl terminus

of CFTR is important for directing CFTR to endocytic clathrin coated vesicles at the plasma

membrane.

Despite many protein-protein interaction assays, including bead pull-down, surface

plasmon resonance, phage display, interaction overlay and yeast two-hybrid analysis,


26

demonstrating the association between endocytic sorting motifs, from a variety of proteins, and

AP-2 clathrin adaptor complexes, whether such interactions occur in vivo or are physiologically

relevant has not always been established. For example, one study has shown in vitro interactions

between the asialoglycoprotein receptor tail and the β subunit of AP-2 complexes (25).

However, most evidence, including yeast two-hybrid, peptide cross-linking and crystal structure

data suggests that the µ subunit mediates the interaction between adaptor complexes and

tyrosine-based sorting motifs (22,48-50). Having established that CFTR contains a tyrosine-

based sorting motif that interacts with AP-2 complexes, we sought to define the amino acid

requirements for the interaction as well as identify the subunit of AP-2 that recognizes this motif.

Photoreactive peptides containing the YDSI sorting motif of CFTR crosslinked a 50 kDa subunit

of the AP-2 complex which is the apparent molecular weight of µ2. Recapturing experiments

confirmed that this crosslinked species was indeed µ2. This interaction between CFTR peptides

and µ2 was dependent upon the tyrosine motif as mutations at Y1424 and I1427 abolished

crosslinking. Furthermore, peptides containing the tyrosine-based sorting motif of TGN38

(SDYQRL) inhibited the crosslinking of CFTR peptides to µ2, indicating that the sorting motif

of CFTR and TGN38 likely interact at the same or similar sites on µ2.

Mutagenesis studies on µ2 have revealed at least two regions on µ2, D176 and W421,

that are important for interacting with tyrosine-based sorting motifs (40). The crystal structure of

µ2 (residues 158-435) complexed with tyrosine-containing peptides has been resolved and

corroborate mutagenesis studies, demonstrating that D176 and W421 are directly involved in


27

binding the tyrosine motif of TGN38 (50). GST-fusion proteins containing the carboxyl tail of

CFTR (GST-CT) efficiently bound isolated wild type µ2 but mutations at D176A/W421A in µ2

abolished the interaction. These results are consistent with previous data that show D176 and

W421 constitute the internalization signal-binding interface of µ2. Furthermore, while other

subunits have been proposed to mediate the interaction between tyrosine-sorting motifs and AP-2

complexes, these results demonstrate that µ2 exclusively mediates the interaction between YDSI

and AP-2. AP-2 complexes containing mutant µ2 failed to bind to CFTR in pull-down

experiments, confirming that the tyrosine based sorting motif of CFTR solely interacts with the

µ2 subunit of AP-2. Another tyrosine-based sorting motif binding site has been proposed at the

amino terminal domain of µ2 (residues 102-125); however, as the D176A/W421A mutations

blocked the interaction between µ2 and CFTR, it does not appear that these residues contribute

significantly to the interaction with the tyrosine sorting motif of CFTR (51).

Recent studies examining the interaction between the EGF receptor and µ2 have shown

that while these two proteins interact in vitro, mutations that affect their interaction do not result

in the inhibition of EGF internalization, suggesting the possibility of µ2-dependent and µ2-

independent pathways of clathrin mediated endocytosis (40). These results also highlight the

importance of evaluating the functional relevance of protein-protein interactions observed in

vitro. Our results show that CFTR was efficiently internalized from the plasma membrane when

transiently expressed in cells expressing the wild type endogenous µ2, but that the

overexpression of the dominant negative mutant µ2 significantly reduced the efficiency with


28

which CFTR was retrieved from the cell surface. Furthermore, quantitative analysis of CFTR

internalization in the dominant negative mutant µ2-expressing cells support the phenomenon

observed by immunofluorescence, that the D176A/W421A mutation inhibits the endocytosis of

CFTR. While over 60% of CFTR is internalized in 15 minutes in wild type HeLa cells, only

25% of CFTR is removed from the cell surface in cells expressing the dominant negative mutant

µ2. These results strongly correlate with the reduction in endocytosis observed in CFTR

Y1424A mutants (10,35), suggesting that the interaction specifically affected is that between µ2

and the Y1424DSI endocytosis signal. Thus, the interactions between µ2 and CFTR that were

initially demonstrated in vitro correspond to a functional interaction that is necessary for the

selective and efficient mechanism of CFTR endocytosis. Furthermore, this provides the first

characterized example of an endocytosis signal identified for an ion channel.

While the above results clearly demonstrate the functional relevance of AP-2 and CFTR

interactions for CFTR internalization, the data also indicate that a portion of CFTR is still able to

undergo internalization in HeLa cells expressing the dominant negative mutant µ2. This could

argue for additional endocytosis signals in CFTR. Indeed, very recent studies by Hu et al.

indicate that the efficient endocytosis of CFTR requires multiple internalization signals located

in the carboxyl-terminus (34). These authors identified that a phenylalanine, F1413 and a

dileucine signal, L1430L, co-operate with the Y1424DSI signal in the carboxyl-terminus of CFTR

to efficiently drive its internalization from the plasma membrane. These results appear to be at

odds, however, with previous studies by Prince et al. examining the dileucine signal in the


29

carboxyl-terminus of CFTR, as similar studies performed with CFTR/transferrin receptor

chimeras demonstrate that mutagenesis of the dileucine motif L1430L to a dialanine has no effect

on the rate of CFTR internalization (10). Moreover, the only mutation identified which affected

CFTR internalization was the YXXΦ motif in the carboxyl-terminus of CFTR, Y1424A.

Presumably, additional signals could interact with clathrin adaptor proteins much in the same

manner described here for the YXXΦ motif Y1424DSI. For example, AP complexes have been

shown to interact with dileucine signals in vitro much the same as tyrosine-based signals.

However, co-immunoprecipitation studies have yet to show the interaction between AP-2

complexes and dileucine containing proteins in vivo, prompting some groups to conclude that

dileucine signals constitute a low-affinity site for AP-2 interactions(52) (51). While

acknowledging the potential for multiple signals in CFTR that could bind to AP-2, the results

presented here clearly demonstrate that the YXXΦ sorting motif identified in the carboxyl-

terminus of CFTR follows the paradigm of other well-described YXXΦ motifs, such that it

interacts with the defined YXXΦ binding site on µ2, and is important in the efficient endocytosis

of CFTR.

The most clinically important mutation of CFTR, ∆F508 CFTR, results in insufficient

quantities of CFTR at the plasma membrane. A great deal of effort has gone toward rescuing

this mutation in the biosynthetic pathway by chemical chaperones or manipulation of

endogenous chaperones with the goal of increasing the delivery of ∆F508 to the plasma

membrane. However, recent reports suggest that ∆F508 CFTR is removed from the plasma


30

membrane at a rate faster than that of wild type CFTR (53). Therefore, to increase the amount of

CFTR at the plasma membrane it is necessary to address the issue of residence time of CFTR in

the plasma membrane as well as CFTR delivery to the cell surface. Thus, gaining a clearer

understanding of the mechanisms by which CFTR is removed from the plasma membrane and

the molecular interactions involved in this process will aid in identifying strategies to enhance

CFTR expression at the plasma membrane. The results presented here demonstrate that the

interaction between the tyrosine-based sorting motif in the carboxyl terminus of CFTR (YDSI)

and the µ2 subunit of plasma membrane clathrin adaptors AP-2 provide a mechanism for the

selective internalization of CFTR into clathrin coated vesicles.


31

ACKNOWLEDEGMENTS

The authors gratefully acknowledge the technical assistance of Mark R. Silvis. The

authors appreciate the generosity of Dr. Juan Bonafacino for providing the GST-∆µ constructs

and Dr. Alexander Sorkin for providing the HeLa Tet-off cell line and the cDNA for µ2. We

also thank Drs. Michael Marks, Alexander Sorkin and Juan Bonafacino for their helpful

discussion. This work was supported by grants from the NIDDK of the NIH (1P50DK56490)

and the North American Cystic Fibrosis Foundation (BRADBU00G0).


32

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37

TABLE LEGENDS

Table I. Schematic Representation of Photoactivatable Peptides Used for Cross-linking

experiments.

The amino acid sequence of the carboxyl tail of CFTR containing the tyrosine motif, YDSI is

depicted to compare with the synthetic peptides used in this study. Photoactivatable peptides

were generated that contain the CFTR WT endocytic sorting motif *YDSI, mutations in the

sorting motif, *ADSA, and the sorting motif from the carboxyl tail of TGN38, *YQRL. Peptides

were amended with a biotinylated Lysine at the N-terminus of the peptide to aid in biochemical

detection of cross-linked proteins by immunoblot analysis using streptavidin-HRP. BPA denotes

the photoactive site, benzoylphenylalanine.


38

FIGURE LEGENDS

Figure 1. Surface Plasmon Resonance Analysis of CFTR peptide interactions with purified

proteins of the clathrin mediated endocytic pathway.

CFTR peptides containing the Y1424DSI endocytic sorting motif were immobilized onto a sensor

chip via avidin linkage as described under “Materials and Methods”. Samples containing

purified AP-2, AP-1, or clathrin were injected under continuous flow conditions at the

concentration indicated and the bar indicates perfusion of protein containing buffer. The

resonance is given in arbitrary units (RU). Only solutions containing purified AP-2 complexes

bound to the YDSI peptide of CFTR. Perfusion of solutions containing AP-1 complexes or

clathrin did not result in a shift in resonance units much beyond baseline.

Figure 2A. The *YDSI Sorting Motif of CFTR Cross-links to the µµµµ2 Subunit of the AP-2

Complex.

Purified AP-2 and AP-1 complexes were incubated with photoactivatable peptides and subject

to UV crosslinking. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose and

immunoblotted with Streptavidin-HRP. *YDSI containing peptides cross-link to a 50 kDa

subunit of the AP-2 complex, the apparent molecular weight of µ2, Lane 4. Peptides with

mutations in the sorting motif, *ADSA, fail to interact with AP-2, Lane 3. Neither peptide cross-

links to any of the AP-1 subunits, Lanes 1 and 2. 2B Immunoblot of AP-2 complexes and 2C

Coomassie Staining of cross-linking reactions demonstrate that all four subunits of the AP-2 are

present in the reaction.


39

Figure 3. Peptides Containing Tyrosine-Based Sorting Motifs Compete for AP-2 Cross-

linking.

Cross-linking reactions were performed with the addition of 100-fold excess competitor peptides.

Peptides containing mutations in the Tyrosine sorting motif, ADSA, did not compete with wt

peptides for AP-2 cross-linking, Lane 2. Peptides containing the Tyrosine-based sorting motif

from TGN38 did successfully compete with WT peptides for AP-2 cross-linking, Lane 3, as did

an excess of non-biotinylated wt peptide, Lane 4.

Figure 4. Recapturing Experiments confirm that *YDSI only crosslinks µµµµ2.

AP-2 complexes purified from bovine brain were incubated with the cross-linking peptide

*YDSI and UV irradiated. AP-2 complexes were immunoprecipitated with AP-2 specific

antibodies against the α-subunit. AP-2 complexes were fractionated by SDS-PAGE and

crosslinked species were detected by immunoblot with Streptavidin-HRP. *YDSI containing

peptides cross-link to a 50 kDa subunit of the AP-2 complex, the apparent molecular weight of

µ2, Lane 2. In parallel photolabeling reactions, isolated AP-2 complexes were dissociated into

individual subunits by denaturation and boiling followed by immunoprecipitation with antibodies

against the individual AP-2 subunits, α (lane 4), β1/β2 (lane 5), or µ2 (lane 6). Recaptured

subunits were analyzed by SDS-PAGE and immunoblot with streptavidin-HRP. µ2 was the only

subunit of the AP-2 complex that was crosslinked by the *YDSI peptide, lane 6.


40

Figure 5. YDSI specifically cross-links to isolated µ2. µ2. µ2. µ2.

GST, GST-∆µ2 and GST-∆µ1 were incubated with the photoreactive peptides *YDSI and

*ADSA and subject to UV irradiation. Proteins were resolved by SDS-PAGE, transferred to

nitrocellulose and incubated with streptavidin-HRP. Coomassie blue staining of the GST fusion

proteins is shown in lanes 1-3. Lanes 4-6 are the ECL observed with the mutant cross-linking

peptide, *ADSA. Lanes 7-9 demonstrate the cross-linking results obtained with the wt peptide,

*YDSI.

Figure 6. Dominant Negative Mutant µµµµ2 Fails to Interact with CFTR.

Wild type µ2 or dominant negative mutant µ2 (D176A/W421A) was translated in vitro in the

presence of 35S-methionine. GSH-Sepharose immobilized GST-fusion proteins were incubated

with 4% of translation reaction diluted in binding buffer as described in “Material and Methods”.

Bound proteins were resolved by SDS-PAGE and processed for autoradiography. GST fusion

proteins containing the carboxyl-terminus of CFTR (GST-CT) efficiently pulled-down wt µ2 but

not mutant µ2 (lanes 8 and 9 respectively). GST alone did not bind either µ2 protein (lanes 2

and 3). A GST fusion protein containing the equine immunodeficiency virus protein p9 was

used as a positive control for wild type µ2 binding (lane 5) and a GST fusion protein containing

the human immunodeficiency virus type 1 p6 protein which does not interact with µ2 was used

as a negative control (lane 4). An aliquot of the µ2 translation reaction (2%) is shown in lane 1.

Figure 7. Mutant AP-2 Complexes Fail to Interact with the C-terminus of CFTR.


41

GST-fusion proteins containing the C-terminus of CFTR were incubated with cell lysates from

HeLa cells expressing wild-type AP-2 (+Tet) and HeLa cells expressing the dominant negative

AP-2 complexes (-Tet). Bound proteins were resolved by SDS-PAGE, transferred to

nitrocellulose, and immunoblotted with the AP-2 specific antibody, anti-α adaptin. GST-CT

efficiently interacted with WT AP-2 complexes, Lane 3, however, mutant AP-2 complexes failed

to interact with GST-CT, Lane 1. GST alone (Lanes 2 and 4) failed to interact with either AP

complex.

Figure 8. Dominant Negative AP-2 Complexes Inhibit the Internalization of CFTR.

CFTR was transiently expressed in HeLa cells expressing wt AP-2 complexes or in HeLa cells

expressing mutant AP-2 complexes. Cell surface CFTR was labeled with anti-CFTR antibodies

at 4°C followed by incubation at 37°C for fifteen minutes to monitor internalization of CFTR

from the cell surface (Panels B and E). Prior to fixation Rhodamine conjugated Wheat Germ

Agglutinin was used to label the cell surface, (Panels A and B). Panel C shows a merge of panels

A and B, intracellular green staining demonstrates that CFTR endocytosis proceeds in wild type

cells. Panel F, a merge of D and E, shows that CFTR remains at the cells surface, indicated by

the overlap of red (cell surface WGA) and green (CFTR) signals to yield a yellow cell surface

staining pattern.


42

Figure 9. CFTR Remains at the Cell Surface in Dominant Negative Mutant HeLa Cells

The co-localized pixel densities of WGA and CFTR are shown by differential intensity plot

using black/white intensity representation derived from confocal image acquisition of HeLa cells

expressing dominant negative mutant µ2 (Panel A) and wild type cells (Panel B). CFTR and cell

surface WGA showed a higher degree of co-localization in mutant cells (Panel A) compared to

wild type cells (Panel B) indicated by the appearance of significantly more pixels in Panel A

compared to Panel B. The differential intensity maps (lower panels) of the co-localized regions

in mutant cells (Panel A) and wild type cells (Panel B) show that CFTR and cell surface WGA

share greater spectral intensity indicative of a higher degree of co-localization.

Figure 10. Dominant Negative Mutant µµµµ2 Inhibits the Internalization of CFTR. Full-length CFTR was transiently expressed in HeLa cells expressing wild-type µ2 (+ Tet; dark

shading) or HeLa cells expressing dominant negative mutant µ2 (- Tet; light shading). The

surface density of CFTR after 15 minutes of internalization was determined with the radioactive

antibody binding assay as described in Methods. Cell surface CFTR was labeled with anti-

CFTR antibodies at 4°C followed by 15 minutes of internalization at 37°C. The remaining

CFTR antibody at the cell surface was measured by 125I-Protein A. Results are expressed as a

percent of CFTR internalized from the cell surface at 15 minutes relative to the amount of total

cell surface CFTR at time zero. Results are mean ± s.e.m. for three separate experiments.

...CQQFVIEENKVRQYDSIQKLLNERS...

Tyrosine-basedEndocytic Motif

CFTR C-terminus(aa. 1411-1435)

YDSI KVIEENKVRQYDSIQ

CFTR-*YDSI

Biotin

KVIEENK RQYDSIQ

BPA

KVIEENK RQADSAQ

Biotin BPA

CFTR-*ADSA

Table 1 Weixel and Bradbury

Time (sec)

0 100 200 300 400 500 600

20180

20200

20220

20240

20260

20280

20300

20320

0 100 200 300 400 500 600

Res

onan

ceU

nit

s

Clathrin (10 M)�

AP-1 (1.0 M)�

AP-2 (0.1 M)�

Addition

Association Dissociation

Washout

Figure 1 Weixel and Bradbury

Cross-linking

*AD

SA

*AD

SA

*YD

SI

*YD

SI

50

37

25

100

150

MWkDa

�2

� � �

AP-2

Immunoblot

AP-2

Coomassie blue

AP-1 AP-2

��2

�2

�2

A B C

Figure 2 Weixel and Bradbury

*YD

SI

AD

SA

TG

N38

YD

SI

*YDSI +

1 2 3 4

noA

ban

ti-�

116

MWkDa no

Ab

anti-a

anti-

1/2

��

anti-

2�

1 52 3 4 6

precipitation re-precipitation after boiling

�2

97.4

66

45

31

GST-��2

GST-��1

GST +

+

+

+

+

+

+

+

+

GST-��2

Coomassie Blue *ADSA *YDSI

D176A

/W421A

wt

D176A

/W421A

wt�transl

ation

GST-CTP6 P9GST

D176A

/W421A

wt

D176A

/W421A

wt

1 2 3 4 5 6 7 8 9

1 2 3 4

% C

FTR

Inte

rnal

ized

20

30

40

50

60

70

80

wild-type AP-2(+ Tet)

dom-neg AP-2(- Tet)

Fig. 10. Weixel and Bradbury

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