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RhoGTPases – Function and Regulation

Ruprecht-Karls- Universität Heidelberg. RhoGTPases – Function and Regulation. Thomas Wieland Institut für Pharmakologie und Toxikologie Fakultät für Klinische Medizin Mannheim. Physiological function of Rho GTPases. Vinculin. Actin. Vinculin. Actin.

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RhoGTPases – Function and Regulation

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  1. Ruprecht-Karls- Universität Heidelberg RhoGTPases – Function and Regulation Thomas Wieland Institut für Pharmakologie und Toxikologie Fakultät für Klinische Medizin Mannheim

  2. Physiological function of Rho GTPases

  3. Vinculin Actin Vinculin Actin Regulation of the actin cytoskeleton

  4. Shape change of platelets U = TXA2-Analog Y = Rho-Kinase Inhibitor C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinum Inhibition of RhoA-C Klages et al. J. Cell Biol. 144 (4) 1999

  5. Regulation of cell-cell contacts Cadherine Keratinocytes C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinum Inhibition of RhoA-C Endothelial cells Thrombin is a strong activator of RhoA

  6. Regulation of proliferation Neointima formation after vascular injury of a carotid artery Y27632 = Rho Kinase Inhibitor Shibata et al. Circulation 103(2) 2001

  7. Cardiac specific overexpression of RhoA in the heart

  8. Posttranslational modification of RhoGTPases

  9. Statins

  10. Beneficial pleiotropic effects of statins

  11. Activation cycle of RhoGTPases

  12. 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)

  13. Model of PH domain assisted GEF activity

  14. RhoGEFs: The Dbl family

  15. Regulation of RhoGEFs - Autoinhibition

  16. Regulation of RhoGEFs – Protein-Protein-Interaction

  17. Cellular Localization of RhoGEFs

  18. b Experimental approaches Direct detection of RhoGTPase activation direct pull down assay p63RhoGEF-DH p63RhoGEF Dbl-DH Tiam-1 control GEF RhoA-GTP total RhoA Rho Rac1 Cdc42 GTP GTP GTP Rac1-GTP total Rac1 Cdc42-GTP total Cdc42 Rhotekin-GST Pak1-GST

  19. indirect luciferase reporter assay 5 +C3T 4 relative luciferase expression GEF 3 2 Rho 1 Rac1 0 C3T control RhoGEF Cdc42 SRF firefly luciferase pSRE renilla luciferase const. Detection of RhoGTPase activation II

  20. Detection of RhoGTPase activation III FRET

  21. bg Focus AG Wieland VEGF-R G12PCR PM GiPCR GqPCR Ga12/13 Gai Gaq NO PI3K p63RhoGEF LARG cGMP p164, Grinch, Gef10 TIAM-1 RhoA Rac1

  22. p63RhoGEF 580 aa DH PH

  23. Expression of p63RhoGEF Sc. muscle Kidney Pancreas kb Placenta Liver Lung Brain Heart 9,5 Northern Blot: mRNA from different human tissues 7,5 4,4 2,4 1,3 Non-Cardiomyocytes Non-Cardiomyocytes Cardiomyocytes Cardiomyocytes H2Ol H2O RT-PCR: Total RNA from isolated primary rat cells Control p63RhoGEF

  24. p63RhoGEF induces stress fiber formation stress fiber 20 µm 20 µm TRITC-Phalloidin anti-c-myc Lutz et al. Naunyn Schmiedebergs Arch Pharmacol. 369:540-6(2004 )

  25. 5 +C3T 4 relative luciferase expression 3 2 1 0 control p63RhoGEF Specificity of p63RhoGEF p63RhoGEF-DH p63RhoGEF Dbl-DH Tiam-1 control RhoA-GTP total RhoA Rac1-GTP total Rac1 Cdc42-GTP total Cdc42

  26. Basal 100 Carbachol 80 60 expression relative luciferase 40 20 n=8 0 Control M3 p63 p63 + M3 Upstream regulatory mechanism (1) Lutz et al. J Biol Chem. 280:11134-9 (2005 )

  27. M3-Ach-Receptor 200 150 +p63 +p63 150 100 relative luciferase expression 100 50 50 Ga12/13 0 0 Basal Ga12QL Ga13QL GaqQL Ga11QL Gaq/11 Basal Ga11QL+p63 Ga13QL+p63 Ga12QL+p63 GaqRC+p63 Ga13QL Ga11QL Ga12QL Control GaqRC p63 RhoA-GTP total RhoA Upstream regulatory mechanism (2)

  28. EE-GaqQL - p63 WB: anti-c-myc IP: anti-EE WB: anti-Gaq/11 Physical interaction of p63RhoGEF with Gaq in the cell p63RhoGEF p63RhoGEF p63RhoGEF Control Control Control WB: anti-c-myc IP: anti-c-myc WB: anti-Gaq/11 Lysate anti-Gaq/11 Ga11QL GaqRC

  29. Physical interaction of p63RhoGEF with Gaqin vitro p63 149-580 p63 149-580 Courtesy of John J. Tresmer

  30. Physiological relevance of p63RhoGEF mediated RhoA activation Ad p63RhoGEF + Endothelin 1 Gaq/11 TRITC-Phalloidin p63RhoGEF 580 DH PH RhoA RhoA GTP GDP GqPCR Neonatal Cardiomyocytes p63RhoGEF control p63RhoGEF control + Endothelin 1 RhoA-GTP total RhoA p63RhoGEF

  31. p63RhoGEF - Summary • p63RhoGEF is mainly expressed in heart and brain tissue. • p63RhoGEF activates RhoA, but not Rac1 or Cdc42 • p63RhoGEF is activated by Gaq/11 proteins. • p63RhoGEF interacts directly with active Gaq/11 proteins. • p63RhoGEFenhances the Endothelin 1 and Phenylephrine induced RhoA activation in cardiomyocytes. • b

  32. PLC/PKC LARG Signaling cascades involving p63RhoGEF and LARG His U-46619 Carb H1-R M3-R TXA2-R Gaq/11 Ga12/13 p63RhoGEF RhoA

  33. Control Larg anti-Flag 15 10 relative Luciferase 5 0 C Larg p63 Basal activity of p63RhoGEF and LARG p63RhoGEF (p63) Dominant expression in brain and heart Control p63 Larg Control p63 RhoA-GTP anti-c-myc total RhoA 588 PH DH LARG Widespread expression 1543 RGS PH PDZ DH

  34. GPCR induced RhoA activation mediated by p63RhoGEF and LARG G12/13PCR Gq/11PCR

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

  36. 70 60 50 40 relative Luciferase 30 20 10 0 The Larg activation ist dependent on the N-terminal part of Gaq proteins Gaq Gaq/i Gaq/i 1-28aa Gai-3 29-353aa Gaq Conclusion: p63RhoGEF and LARG apparently hold divergent binding sites for Gaq

  37. RGS3

  38. Activation of GIRK by M2-AChR in Xenopus oocytes Regulation by RGS3 + RGS3 Control ACh, 1 mM ACh, 1 mM 500 nA 500 nA 25 s 25 s Doupnik et al. 1997, Proc Natl Acad Sci 94:10461 RGS proteins contribute to the regulation of Gbg-gated K+ -channels

  39. 394 509 1 519 RGS3 RGS domain RGS3 - A highly abundant protein in human heart 70kDa Gbg binding • 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 Gbg-stimulated PLC activity and • PI3K/Akt signaling  Gbg scavenger • Shi et al. 2001, J Biol Chem 276:24293

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