RhoGTPases – Function and Regulation RhoGTPases – Function and Regulation

Thomas WielandInstitut für Pharmakologie und Toxikologie

Fakultät für Klinische Medizin Mannheim

Ruprecht-Karls-

Universität

Heidelberg

Physiological function of Rho GTPases

Regulation of the actin cytoskeleton

Actin Vinculin Actin Vinculin

U = TXA2-Analog

Y = Rho-Kinase Inhibitor

C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinumInhibition of RhoA-C

Shape change of platelets

Klages et al. J. Cell Biol. 144 (4) 1999

Keratinocytes

Regulation of cell-cell contacts

C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinumInhibition of RhoA-C

Endothelial cellsThrombin is a strong activator of RhoA

Cadherine

Regulation of proliferation

Neointima formation after vascular injury of a carotid artery

Y27632 = Rho Kinase Inhibitor

Shibata et al. Circulation 103(2) 2001

Cardiac specific overexpression of RhoA in the heart

Posttranslational modification of RhoGTPases

Statins

Beneficial pleiotropic effects of statins

Activation cycle of RhoGTPases

Rho-specific guanine nucleotide exchange factors

DH PH

Catalytic activity~200 aa

Membrane localization ?

Variable specifity for RhoGTPases

Multidomain proteins

Variable size (~ 60 to 800kd)

Variable expression patterns (ubiquitiniously to specific)

Model of PH domain assisted GEF activity

RhoGEFs: The Dbl family

Regulation of RhoGEFs - Autoinhibition

Regulation of RhoGEFs – Protein-Protein-Interaction

Cellular Localization of RhoGEFs

b

Experimental approaches

directpull down assay

Rac1Cdc42 Rho

GEF

GTP GTP GTP

Pak1-GST Rhotekin-GSTp6

3Rho

GEF

cont

rol

p63R

hoG

EF-D

H

Tiam

-1D

bl-D

H

RhoA-GTP

Rac1-GTP

Cdc42-GTP

total RhoA

total Rac1

total Cdc42

Direct detection of RhoGTPase activation

rela

tive

luci

fera

se e

xpre

ssio

n control RhoGEF

0

1

2

3

4

5

+C3T

C3T

indirectluciferase reporter assay

Rac1

pSREfirefly luciferase

renilla luciferase

SRF

const.

Cdc42

Rho

GEF

Detection of RhoGTPase activation II

Detection of RhoGTPase activation IIIFRET

GiPCR GqPCR

Gi Gq

PMG12PCR

G12/13

p63RhoGEFp63RhoGEF LARGLARGPI3K

TIAM-1TIAM-1

RhoARhoARac1Rac1

VEGF-R

NO

cGMP

p164, Grinch, Gef10 p164, Grinch, Gef10

Focus AG Wieland

p63RhoGEF

DH PH

580 aa

H2O

Car

diom

yocy

tes

Non

-Car

diom

yocy

tes

H2Ol

Car

diom

yocy

tes

Non

-Car

diom

yocy

tes

p63RhoGEF Control

Hea

rtB

rain

Place

nta

Lung

Live

rSc.

mus

cle

Kid

ney

Pancr

eas

9,57,5

4,4

2,4

1,3

kb

Northern Blot:mRNA from different human

tissues

RT-PCR:Total RNA from isolated

primary rat cells

Expression of p63RhoGEFExpression of p63RhoGEF

p63RhoGEF induces stress fiber formation

anti-c-myc

20 µm

TRITC-Phalloidin

20 µm

stress fiber

Lutz et al. Naunyn Schmiedebergs Arch Pharmacol. 369:540-6 (2004 )

p63R

hoG

EF

cont

rol

p63R

hoG

EF-D

H

Tiam

-1D

bl-D

H

RhoA-GTP

Rac1-GTP

Cdc42-GTP

total RhoA

total Rac1

total Cdc42

rela

tive

luci

fera

se e

xpre

ssio

n

control p63RhoGEF

0

1

2

3

4

5

+C3T

Specificity of p63RhoGEFSpecificity of p63RhoGEF

Upstream regulatory mechanism (1)Upstream regulatory mechanism (1)

Control M3p63 p63 + M3

0

20

40

60

80

100 Basal

Carbachol

n=8

rela

tive

luc

ifer

ase

exp

res

sio

n

Lutz et al. J Biol Chem. 280:11134-9 (2005 )

Upstream regulatory mechanism (2)Upstream regulatory mechanism (2)re

lati

ve l

uci

fera

seex

pre

ssio

n

0

50

100

150

200

+p63

Basal GqQL G11QL Basal0

50

100

150

+p63

G12QL G13QL

M3-Ach-Receptor

Gq/11 G12/13

Contro

lp6

3

Gq

RCGq

RC+p63

G1

2QL+

p63

G1

2QL

G1

1QL

G1

1QL+

p63

G1

3QL+

p63

G1

3QL

RhoA-GTP

total RhoA

p63

RhoG

EF

p63R

hoG

EF

p63R

hoG

EF

WB:anti-c-myc

WB:anti-Gq/11

anti-Gq/11

IP:anti-c-myc

Lysate

Con

trol

G11QLGqRC

Con

trol

Con

trol

IP:anti-EE

WB:anti-c-myc

WB:anti-Gq/11

p63-

EE-GqQL

Physical interaction of p63RhoGEF with GPhysical interaction of p63RhoGEF with Gq q in the cell in the cell

p63 149-580

p63 149-580

Courtesy of John J. Tresmer

Physical interaction of p63RhoGEF with GPhysical interaction of p63RhoGEF with Gqq in vitroin vitro

DH PH

p63RhoGEF580

RhoA RhoA

GTP GDP

GqPCR

Gq/11

Physiological relevance of Physiological relevance of p63RhoGEF mediated RhoA activationp63RhoGEF mediated RhoA activation

Ad p63RhoGEF+ Endothelin 1

TRITC-Phalloidin

RhoA-GTP

total RhoA

cont

rol

p63R

hoG

EFco

ntro

lp6

3Rho

GEF

+ Endothelin 1

p63RhoGEF

Neonatal Cardiomyocytes

• p63RhoGEF is mainly expressed in heart and brain tissue.

• p63RhoGEF activates RhoA, but not Rac1 or Cdc42

• p63RhoGEF is activated by Gq/11 proteins.

• p63RhoGEF interacts directly with active Gq/11 proteins.

• p63RhoGEF enhances the Endothelin 1 and Phenylephrine induced RhoA activation in cardiomyocytes.

b

p63RhoGEF - Summaryp63RhoGEF - Summary

H1-R M3-R

His

Gq/11

p63RhoGEFLARG

RhoA

G12/13 PLC/PKC

TXA2-R

U-46619

Carb

Signaling cascades involving p63RhoGEF and LARG

Larg

Control

anti-Flag

p63

Control

anti-c-myc

DH PH588

1543

DH PHRGSPDZ

RhoA-GTP

total RhoA

Control

Larg

p63

C

p63

Larg

0

5

10

15

rela

tive

Lu

cife

rase

p63RhoGEF (p63) Dominant expression in brain and heart

LARGWidespread expression

Basal activity of p63RhoGEF and LARG

Control

TXA2-R

TXA2-R+L

ARG

TXA2-R+p

630

25

50

75BasalU-46619 (200nM)

rela

tive

Lu

cife

rase

G12/13PCR Gq/11PCR

Control

M3-

R

M3-

R+LARG

M3-

R+p63

0

25

50

75

100

125BasalCarbachol (1mM)

rela

tive

Lu

cife

rase

GPCR induced RhoA activation mediated by p63RhoGEF and LARG

0 5 10 15 20 250

5

10

15

20

25

30

35Largp63RhoGEF

ng Gq

rela

tive

Lu

cife

rase

Control p63RhoGEF LARG0

25

50

75

100none+GqRC+G12QL

rela

tive

Lu

cife

rase

Conclusion: Larg needs an active state receptor to induce RhoA activation, whereas p63RhoGEF needs only traces of Gq protein for its activation.

The LARG induced Rho activation requires activated GPCRs

Gq/i

1-28aa Gi-3

29-353aa Gq

0

10

20

30

40

50

60

70re

lati

ve L

uci

fera

se

p63 - + - - - + + - - - + + - -

Larg - - + - - - - + + - - - + +

M3-R - - - + - - + - + - - + - +

Gq - - - - + + + + + - - - - -

Gq/i - - - - - - - - - + + + + +

The Larg activation ist dependent on the N-terminal part of Gq proteins

Gq Gq/i

Conclusion: p63RhoGEF and LARG apparently hold divergent binding sites for Gq

RGS3

RGS proteins contribute to the regulation of GRGS proteins contribute to the regulation of G--gated Kgated K+ + -channels-channels

ACh, 1 MControl

500 nA

25 s

Activation of GIRK by M2-AChR in Xenopus oocytes

Doupnik et al. 1997, Proc Natl Acad Sci 94:10461

Regulation by RGS3

+ RGS3

500 nA

25 s

ACh, 1 M

RGS3 - A highly abundant protein in human heartRGS3 - A highly abundant protein in human heart

RGS3 RGS domain

1 394 519509

G binding70kDa

• RGS3 mRNA is upregulated in human HF Owen et al. 2001, Eur Heart J 22:1015

• RGS3 can be induced in NRCM by treatment with bFGF Zhang et al. 1998, J Mol Cell Cardiol 30:269

• RGS3 inhibits G-stimulated PLC activity and PI3K/Akt signaling G scavenger Shi et al. 2001, J Biol Chem 276:24293