RhoGTPases – Function and Regulation Thomas Wieland Institut 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 = TXA 2 -Analog Y = Rho-Kinase Inhibitor C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinum Inhibition 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 botulinum Inhibition of RhoA-C Endothelial cells Thrombin 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 DHPH 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 direct pull down assay Rac1 Cdc42 Rho GEF GTP Pak1-GST Rhotekin-GST p63RhoGEF control p63RhoGEF-DH Tiam-1 Dbl-DH RhoA-GTP Rac1-GTP Cdc42-GTP total RhoA total Rac1 total Cdc42 Direct detection of RhoGTPase activation

relative luciferase expression control RhoGEF C3T C3T indirect luciferase reporter assay Rac1 pSRE firefly luciferase renilla luciferase SRF const. Cdc42 Rho GEF Detection of RhoGTPase activation II

Detection of RhoGTPase activation III FRET

G i PCRG q PCR  GiGi GqGq PM G 12 PCR G  12/13 p63RhoGEF LARG PI3K TIAM-1 RhoA Rac1 VEGF-R NO cGMP p164, Grinch, Gef10 Focus AG Wieland

p63RhoGEF DHPH 580 aa

H2O Cardiomyocytes Non-Cardiomyocytes H2Ol Cardiomyocytes Non-Cardiomyocytes p63RhoGEF Control Heart Brain Placenta Lung Liver Sc. muscle Kidney Pancreas 9,5 7,5 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 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 )

p63RhoGEF control p63RhoGEF-DH Tiam-1 Dbl-DH RhoA-GTP Rac1-GTP Cdc42-GTP total RhoA total Rac1 total Cdc42 relative luciferase expression controlp63RhoGEF C3T Specificity of p63RhoGEF

Upstream regulatory mechanism (1) Lutz et al. J Biol Chem. 280: (2005 )

Upstream regulatory mechanism (2) relative luciferase expression p63 Basal G  q QLG  11 QL Basal p63 G  12 QL G  13 QL Control p63 G  qRC G  qRC+p63 G  12QL+p63 G  12QL G  11QL G  11QL+p63 G  13QL+p63 G  13QL RhoA-GTP total RhoA

p63RhoGEF WB: anti-c-myc WB: anti-G  q/11 IP: anti-c-myc Lysate Control G  11 QL G  q RC Control IP: anti-EE WB: anti-c-myc WB: anti-G  q/11 p63 - EE-G  q QL Physical interaction of p63RhoGEF with G  q in the cell

p Courtesy of John J. Tresmer Physical interaction of p63RhoGEF with G  q in vitro

DHPH p63RhoGEF 580 RhoA GTPGDP G q PCR G  q/11 Physiological relevance of p63RhoGEF mediated RhoA activation Ad p63RhoGEF + Endothelin 1 TRITC-Phalloidin RhoA-GTP total RhoA control p63RhoGEF control p63RhoGEF + 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 G  q/11 proteins. p63RhoGEF interacts directly with active G  q/11 proteins. p63RhoGEF enhances the Endothelin 1 and Phenylephrine induced RhoA activation in cardiomyocytes. b p63RhoGEF - Summary

H 1 -R M 3 -R His G  q/11 p63RhoGEF LARG RhoA G  12/13 PLC/PKC TXA 2 -R U-46619Carb Signaling cascades involving p63RhoGEF and LARG

Larg Control anti-Flag p63 Control anti-c-myc DH PH DH PH RGS PDZ RhoA-GTP total RhoA Control Larg p63 p63RhoGEF (p63) Dominant expression in brain and heart LARG Widespread expression Basal activity of p63RhoGEF and LARG

G 12/13 PCR G q/11 PCR GPCR induced RhoA activation mediated by p63RhoGEF and LARG

Conclusion: Larg needs an active state receptor to induce RhoA activation, whereas p63RhoGEF needs only traces of G  q protein for its activation. The LARG induced Rho activation requires activated GPCRs

G  q/i 1-28aa G  i aa G  q relative Luciferase p Larg M 3 -R GqGq G  q/i The Larg activation ist dependent on the N-terminal part of G  q proteins GqGq G  q/i Conclusion: p63RhoGEF and LARG apparently hold divergent binding sites for G  q

RGS3

RGS proteins contribute to the regulation of G  - gated K + -channels RGS proteins contribute to the regulation of G  - gated K + -channels ACh, 1  M Control 500 nA 25 s Activation of GIRK by M 2 -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 heart RGS3 RGS domain G  binding 70kDa 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