Publikationsserver der Universitätsbibliothek Marburg

Titel:Regulation of Rho-activating proteins by heterotrimeric G proteins: Sensitivity of Gα RhoGEF interaction is determined by dissociation kinetics
Autor:Bodmann, Eva-Lisa
Weitere Beteiligte: Bünemann, Moritz (Prof.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0474
URN: urn:nbn:de:hebis:04-z2014-04749
DOI: https://doi.org/10.17192/z2014.0474
DDC: Biowissenschaften, Biologie
Titel(trans.):Die Regulation Rho-aktivierender Proteine durch heterotrimere G-Proteine: Die Sensitivität der Gα-RhoGEF-Interaktion wird durch die Dissoziationskinetik reguliert
Publikationsdatum:2015-06-25
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
p63RhoGEF, Fluoreszenz-Resonanz-Energie-Transfer, Gαq, Förster resonance energy transfer, Gα13, Regulator of G protein signaling 2, leukemia-associated RhoGEF, p63RhoGEF, G-Protein gekoppelte Rezeptoren, Gαq, Gα13, Leukämie assoziiertes RhoGEF

Summary:
Activation of RhoGTPases downstream of G protein coupled receptors is important for many physiological functions, such as blood pressure regulation. The subfamilies Rho, Rac and Cdc42 are the best understood RhoGTPases and the present study focused on signaling towards the Rho subfamily member RhoA. In its active state RhoA regulates the cytoskeleton by its influence on actin dynamics, activates important signal transducers such as Rho-associated coiled-coil kinase, which phosphorylates and thereby inactivates myosin light chain phosphatase and induces gene transcription via serum response factor. Most RhoGTPases cycle between a GDP-bound inactive and a GTP-bound active state. The exchange of GTP for GDP and therefore activation is mediated through Rho guanine nucleotide exchange factors (RhoGEFs). In the case of RhoA the largest family of RhoGEFs is functionally and structurally characterized by a DH domain adjunct to a PH domain. The DH domain holds GEF activity and the PH domain has mainly regulatory functions. Some of these RhoGEFs can be activated by Gαq/11 and/or Gα12/13 and the present work focused on their regulation: Downstream of Gα13 RH-RhoGEFs are activated. This group of RhoGEFs shares a regulator of G protein signaling homology domain (RH) in addition to the DH-PH domain, which is also present in the Gαq-activated p63RhoGEF. Knock-out of the RH-RhoGEF leukemia-associated RhoGEF (LARG) protects against salt-induced hypertension in mice and the acute response of vascular smooth muscle cells to angiotensin II treatment is mediated mainly by p63RhoGEF. For both proteins several other physiological functions have been described. Nevertheless, little had been known about why RhoGEFs are activated downstream of two Gα subfamilies and the temporal as well as spatial dynamics of their receptor-mediated activation. Therefore we developed FRET-based assays monitoring RhoGEF activation in living cells for the first time. The Förster resonance energy transfer (FRET) occurs between two fluorophores - in the present study fused to the proteins of interest - with a distance of less than 10nm. Thus an increase in FRET upon stimulation with the agonist reflects convergence of the proteins of interest. Changes in FRET were recorded in single, living cells with a high-speed CCD-camera. The interaction between LARG and Gα13 was monitored in cells transfected with Gα13-mTur2 and YFP-LARG. The stimulation of thromboxane A2 receptor induced a robust increase in FRET. Surprisingly, as shown by the slow decrease in FRET between LARG and Gα13, the interaction of LARG and Gα13 dissociated very slowly (estimated t1/2>5min) compared to the Gα13 inactivation (t1/2=17.50s). This observation was also reflected in the kinetics of LARG translocation to the plasma membrane. Thus LARG and Gα13 interact rapidly upon activation of Gα13, but either LARG inhibits Gα13 inactivation or stays in a complex with Gα13 after inactivation of the same. In our opinion the prolonged interaction is most likely the reason for the almost 100-fold higher sensitivity towards stimulation with a thromboxane agonist of the Gα13 LARG interaction compared with the Gα13 activation. The p63RhoGEF activation was studied by monitoring the interaction of Gαq-CFP and Venus-p63RhoGEF. A robust increase in FRET was observed upon stimulation of Gαq coupled receptors. In contrast to the LARG Gα13 interaction, the p63RhoGEF Gαq interaction mirrored closely the Gαq activation as well as inactivation. In addition also the sensitivity of p63RhoGEF Gαq interaction and Gαq activation was in the same range (EC50 of 500nM histamine). Both observations were also true in a trimeric complex of p63RhoGEF and Gαq with the regulator of G protein signaling RGS2. RGS2 was previously shown to accelerate Gαq inactivation in vitro and consequently we observed an accelerated dissociation of p63RhoGEF and Gαq in the presence of RGS2. Additionally, we could monitor an increase in FRET between p63RhoGEF and RGS2, which is the first evidence for such a trimeric complex in living cells. Thus our data strongly support the concept of a functional activation-dependent p63RhoGEF Gαq RGS2 complex. In this complex RGS2 inhibits downstream signaling. This could be an explanation for severe hypertension, which has been observed in RGS2 knock-out mice (Tang et al., 2003). In summary, LARG as well as p63RhoGEF are both activated upon stimulation of G protein coupled receptors. Nevertheless LARG´s sensitivity towards receptor activation and duration of signaling seems to be remarkably higher and longer than p63RhoGEF´s. The inactivation of p63RhoGEF is further accelerated by RGS2, which also decreases downstream signaling.

Zusammenfassung:
Die Aktivierung der RhoGTPasen durch G-Protein gekoppelte Rezeptoren ist wichtig für viele physiologische Funktionen wie etwa die Blutdruckregulation. Am besten sind die Unterfamilien Rho, Rac und Cdc42 der RhoGTPasen verstanden. In der vorliegenden Arbeit liegt der Fokus auf Signaltransduktions-Mechanismen, welche RhoA aktivieren. Aktives RhoA reguliert zum Beispiel das Zytoskelett durch seinen Einfluss auf die Aktin-Dynamik und reguliert die Rho-Kinase ROCK, welche die Myosin-Leichtketten-Phosphatase durch Phosphorylierung inaktiviert. RhoA induziert weiterhin mittels des Serum responsiven Faktors Gentranskription. Die meisten RhoGTPasen wechseln zwischen einem GDP-gebunden inaktiven und einem GTP-gebunden aktiven Zustand hin und her. Der Austausch von GDP durch GTP und somit die Aktivierung wird durch Rho Guanin Austausch Faktoren (RhoGEFs) vermittelt. Die größte Familie der RhoA-aktivierenden RhoGEFs ist funktionell und strukturell durch eine DH Domäne und eine direkt anschließenden PH Domäne charakterisiert. Die DH Domäne stellt die GEF Aktivität bereit, die PH Domäne hat vor allem regulatorische Funktionen. Manche dieser RhoGEFs werden durch Gαq/11 und/oder Gα12/13 aktiviert. Die vorliegende Arbeit beschäftigt sich mit der Regulation dieser RhoGEFs. Unterhalb von Gα13 werden RH RhoGEFs aktiviert. Alle RH-RhoGEFs besitzen eine Regulator of G protein signaling (RGS) homologe (RH) Domäne zusätzlich zur DH-PH Domäne, welche auch das Gαq-aktivierte p63RhoGEF besitzt. Nach genetischer Depletion des RH-RhoGEFs LARG sind Mäuse gegen Salz-induzierten Bluthochdruck geschützt und die akute Reaktion auf Behandlung mit Angiotensin II fehlt den p63RhoGEF defizienten Mäusen. Trotzdem war bisher unklar, warum RhoGEFs durch zwei Gα-Unterfamilien aktiviert werden. Auch über die zeitliche und räumliche Dynamik der Rezeptor-vermittelten Aktivierung war wenig bekannt. Deshalb entwickelten wir FRET-basierte Messmethoden, welche es ermöglichen, die RhoGEF Aktivierung zum ersten Mal in lebenden Zellen zu beobachten. FRET findet zwischen zwei Fluorophoren statt - in dieser Studie sind die Fluorophore an interessierende Proteine fusioniert –, welche einen Abstand von höchsten 10nm besitzen. Daher reflektiert ein Anstieg im FRET nach Stimulation mit dem Agonisten die Annäherung der interessierenden Proteine. In einzelnen, lebenden Zellen wurden Änderungen in FRET mit einer Hochgeschwindigkeitskamera detektiert. Die Interaktion zwischen LARG und Gα13 wurde in Zellen beobachtet, welche mit Gα13-mTur2 und YFP-LARG transfiziert waren. Die Stimulation des Thromboxan A2 Rezeptors führte zu einem robusten Anstieg des FRET Signals. Wie sich in der langsamen Abnahme des FRET Signals zwischen LARG und Gα13 zeigte, dissoziierten LARG und Gα13 deutlich langsamer (t1/2>5min) als Gα13 inaktivierte (t1/2=17,50s). Dies konnte auch in der Kinetik der LARG Translokation zur Plasmamembrane beobachtet werden. Somit verursacht die Aktivierung des Gα13, dessen schnelle Interaktion mit LARG. Bemerkenswerterweise inhibiert LARG entweder die Gα13´s Inaktivierung oder es bindet an Gα13 nach dessen Inaktivierung. Wir gehen davon aus, dass die verlängerte Interaktion die Ursache für die fast 100fach erhöhte Sensitivität der LARG-Gα13-Interaktion für die Stimulation mit einem Thromboxan Agonisten im Vergleich zur Gα13-Aktivierung ist. Die Aktivierung von p63RhoGEF wurde durch Beobachtung der Interaktion von Gαq-CFP und Venus-p63RhoGEF untersucht. Ein robuster Anstieg im FRET wurde nach Stimulation von Gαq-gekoppelten Rezeptoren detektiert. Im Gegensatz zur LARG-Gα13-Interaktion reflektiert die p63RhoGEF-Gαq-Interaktion zeitlich sehr genau sowohl die Gαq-Aktivierung als auch seine Inaktivierung. Außerdem zeigen die p63RhoGEF-Gαq-Interaktion und die Gαq-Aktivierung eine ähnliche Sensitivität (EC50 von 500nM Histamin). Beide Beobachtungen wurden auch in einem trimären Komplex aus p63RhoGEF, Gαq und RGS2 bestätigt. In der Vergangenheit wurde eine beschleunigte Gαq-Inaktivierung durch RGS2 in vitro beschrieben und auch wir beobachteten eine beschleunigte Dissoziation von p63RhoGEF und Gαq in Anwesenheit von RGS2. Zusätzlich konnten wir einen Anstieg im FRET zwischen p63RhoGEF und RGS2 beobachten. Diese Beobachtung war der erste Beweis für diesen trimären Komplex in lebenden Zellen und unterstützt das Konzept eines funktionalen, aktivierungsabhängigen p63RhoGEFGαq-RGS2-Komplexes. In diesem Komplex inhibiert RGS2 die nachgeordnete Signalweiterleitung. Zusammenfassend werden sowohl LARG als auch p63RhoGEF durch die Stimulation der G-Protein gekoppelter Rezeptoren aktiviert. Trotzdem ist LARGs Sensitivität für die Rezeptoraktivierung deutlich höher und die Dauer der Signalweiterleitung länger als es für p63RhoGEF gezeigt wurde. Die Inaktivierung von p63RhoGEF wird zudem durch RGS2 weiter beschleunigt. Dies hat eine Abnahme der nachgeordneten Signalweiterleitung zur Folge.

Bibliographie / References

  1. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–5.
  2. Neer, E.J., Schmidt, C.J., Nambudripad, R., and Smith, T.F. (1994). The ancient regulatory-protein family of WD-repeat proteins. Nature 371: 297–300.
  3. Langmead, C.J., and Christopoulos, A. (2014). Functional and structural perspectives on allosteric modulation of GPCRs. Curr. Opin. Cell Biol. 27C: 94–101.
  4. Gu, J.L., Müller, S., Mancino, V., Offermanns, S., and Simon, M.I. (2002). Interaction of G alpha(12) with G alpha(13) and G alpha(q) signaling pathways. Proc. Natl. Acad. Sci. U. S. A. 99: 9352–7.
  5. Mao, J., Yuan, H., Xie, W., Simon, M.I., and Wu, D. (1998). Specific involvement of G proteins in regulation of serum response factor-mediated gene transcription by different receptors. J. Biol. Chem. 273: 27118–23.
  6. Simon, M.I., Strathmann, M.P., and Gautam, N. (1991). Diversity of G proteins in signal transduction. Science 252: 802–8.
  7. Zhang, P., and Mende, U. (2011). Regulators of G-protein signaling in the heart and their potential as therapeutic targets. Circ. Res. 109: 320–33.
  8. Momotani, K., Artamonov, M. V, Utepbergenov, D., Derewenda, U., Derewenda, Z.S., and Somlyo, A. V (2011). p63RhoGEF couples Gα(q/11)-mediated signaling to Ca2+ sensitization of vascular smooth muscle contractility. Circ. Res. 109: 993–1002.
  9. Basile, J.R., Barac, A., Zhu, T., Guan, K., and Gutkind, J.S. (2004). Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res. 64: 5212–24.
  10. Dohlman, H.G., Song, J., Apanovitch, D.M., DiBello, P.R., and Gillen, K.M. (1998). Regulation of G protein signalling in yeast. Semin. Cell Dev. Biol. 9: 135–41.
  11. Hiley, E., McMullan, R., and Nurrish, S.J. (2006). The Galpha12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans. EMBO J. 25: 5884–95.
  12. Xu, N., Voyno-Yasenetskaya, T., and Gutkind, J.S. (1994). Potent transforming activity of the G13 alpha subunit defines a novel family of oncogenes. Biochem. Biophys. Res. Commun. 201: 603– 9.
  13. McCudden, C.R., Hains, M.D., Kimple, R.J., Siderovski, D.P., and Willard, F.S. (2005). G-protein signaling: back to the future. Cell. Mol. Life Sci. 62: 551–77.
  14. Berstein, G., Blank, J.L., Jhon, D.Y., Exton, J.H., Rhee, S.G., and Ross, E.M. (1992). Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell 70: 411–8.
  15. Moreira, I.S. (2014). Structural features of the G-protein/GPCR interactions. Biochim. Biophys. Acta 1840: 16–33.
  16. Yeung, W.W.S., and Wong, Y.H. (2009). The RhoA-specific guanine nucleotide exchange factor p63RhoGEF binds to activated Galpha(16) and inhibits the canonical phospholipase Cbeta pathway. Cell. Signal. 21: 1317–25.
  17. Galpha q allosterically activates and relieves autoinhibition of p63RhoGEF. Cell. Signal. 22: 1114–23.
  18. Chow, C.R., Suzuki, N., Kawamura, T., Hamakubo, T., and Kozasa, T. (2013). Modification of p115RhoGEF Ser(330) regulates its RhoGEF activity. Cell. Signal. 25: 2085–92.
  19. Sánchez-Fernández, G., Cabezudo, S., García-Hoz, C., Benincá, C., Aragay, A.M., Mayor, F., et al. (2014). Gαq signalling: the new and the old. Cell. Signal. 26: 833–48.
  20. Hendriks-Balk, M.C., Peters, S.L.M., Michel, M.C., and Alewijnse, A.E. (2008). Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins. Eur. J. Pharmacol. 585: 278–91.
  21. Hayashi, A., Hiatari, R., Tsuji, T., Ohashi, K., and Mizuno, K. (2013). p63RhoGEF-mediated formation of a single polarized lamellipodium is required for chemotactic migration in breast carcinoma cells. FEBS Lett. 587: 698–705.
  22. Zinovyeva, M., Sveshnikova, E., Visser, J., and Belyavsky, A. (2004). Molecular cloning, sequence and expression pattern analysis of the mouse orthologue of the leukemia-associated guanine nucleotide exchange factor. Gene 337: 181–8.
  23. Vaqué, J.P., Dorsam, R.T., Feng, X., Iglesias-Bartolome, R., Forsthoefel, D.J., Chen, Q., et al. (2013). A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors. Mol. Cell 49: 94–108.
  24. Marinissen, M.J., and Gutkind, J.S. (2005). Scaffold proteins dictate Rho GTPase-signaling specificity. Trends Biochem. Sci. 30: 423–6.
  25. Hao, J., Michalek, C., Zhang, W., Zhu, M., Xu, X., and Mende, U. (2006). Regulation of cardiomyocyte signaling by RGS proteins: differential selectivity towards G proteins and susceptibility to regulation. J. Mol. Cell. Cardiol. 41: 51–61.
  26. Zhou, J., Moroi, K., Nishiyama, M., Usui, H., Seki, N., Ishida, J., et al. (2001). Characterization of RGS5 in regulation of G protein-coupled receptor signaling. Life Sci. 68: 1457–69.
  27. Sprang, S.R., Chen, Z., and Du, X. (2007). Structural basis of effector regulation and signal termination in heterotrimeric Galpha proteins. Adv. Protein Chem. 74: 1–65.
  28. Jones, T.L.Z. (2004). Role of palmitoylation in RGS protein function. Methods Enzymol. 389: 33–55.
  29. Barrett, K., Leptin, M., and Settleman, J. (1997). The Rho GTPase and a putative RhoGEF mediate a signaling pathway for the cell shape changes in Drosophila gastrulation. Cell 91: 905–15.
  30. Dascal, N. (1997). Signalling via the G protein-activated K+ channels. Cell. Signal. 9: 551–73.
  31. Zhang, H., Wang, L., Kao, S., Whitehead, I.P., Hart, M.J., Liu, B., et al. (1999). Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF. Curr. Biol. 9: 1271–4.
  32. Bhattacharya, S., and Vaidehi, N. (2010). Computational mapping of the conformational transitions in agonist selective pathways of a G-protein coupled receptor. J. Am. Chem. Soc. 132: 5205–14.
  33. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477: 611–5.
  34. Qin, K., Dong, C., Wu, G., and Lambert, N. a (2011). Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers. Nat. Chem. Biol. 7: 740–7.
  35. Schoner, W. (2008). Salt abuse: the path to hypertension. Nat. Med. 14: 16–7.
  36. Rossman, K.L., Der, C.J., and Sondek, J. (2005). GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6: 167–80.
  37. Hamazaki, Y., Kojima, H., Mano, H., Nagata, Y., Todokoro, K., Abe, T., et al. (1998). Tec is involved in G protein-coupled receptor-and integrin-mediated signalings in human blood platelets. Oncogene 16: 2773–9.
  38. Milde, M., Rinne, A., Wunder, F., Engelhardt, S., and Bünemann, M. (2013). Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi- mediated inhibition of cAMP production. Biochem. J. 454: 515–23.
  39. Kuner, R., Swiercz, J.M., Zywietz, A., Tappe, A., and Offermanns, S. (2002). Characterization of the expression of PDZ-RhoGEF, LARG and Galpha12/Galpha13 proteins in the murine nervous system. Eur. J. Neurosci. 16: 2333–41.
  40. Zeng, W., Xu, X., Popov, S., Mukhopadhyay, S., Chidiac, P., Swistok, J., et al. (1998). The N-terminal domain of RGS4 confers receptor-selective inhibition of G protein signaling. J. Biol. Chem. 273: 34687–90.
  41. Heximer, S.P., Srinivasa, S.P., Bernstein, L.S., Bernard, J.L., Linder, M.E., Hepler, J.R., et al. (1999). G protein selectivity is a determinant of RGS2 function. J. Biol. Chem. 274: 34253–9.
  42. Waheed, A.A., and Jones, T.L.Z. (2002). Hsp90 interactions and acylation target the G protein Galpha 12 but not Galpha 13 to lipid rafts. J. Biol. Chem. 277: 32409–12.
  43. Bhattacharyya, R., and Wedegaertner, P.B. (2000). Galpha 13 requires palmitoylation for plasma membrane localization, Rho-dependent signaling, and promotion of p115-RhoGEF membrane binding. J. Biol. Chem. 275: 14992–9.
  44. Sagi, S.A., Seasholtz, T.M., Kobiashvili, M., Wilson, B.A., Toksoz, D., and Brown, J.H. (2001). Physical and functional interactions of Galphaq with Rho and its exchange factors. J. Biol. Chem. 276: 15445–52.
  45. Shi, C.S., Lee, S.B., Sinnarajah, S., Dessauer, C.W., Rhee, S.G., and Kehrl, J.H. (2001). Regulator of G-protein signaling 3 (RGS3) inhibits Gbeta1gamma 2-induced inositol phosphate production, mitogen-activated protein kinase activation, and Akt activation. J. Biol. Chem. 276: 24293–300.
  46. Wells, C.D., Liu, M.-Y., Jackson, M., Gutowski, S., Sternweis, P.M., Rothstein, J.D., et al. (2002). Mechanisms for reversible regulation between G13 and Rho exchange factors. J. Biol. Chem. 277: 1174–81.
  47. Jaiswal, M., Gremer, L., Dvorsky, R., Haeusler, L.C., Cirstea, I.C., Uhlenbrock, K., et al. (2011). Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G- protein signaling (RGS)-containing Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). J. Biol. Chem. 286: 18202–12.
  48. Brandt, D., Gimona, M., Hillmann, M., Haller, H., and Mischak, H. (2002). Protein kinase C induces actin reorganization via a Src-and Rho-dependent pathway. J. Biol. Chem. 277: 20903–10.
  49. Chikumi, H., Vázquez-Prado, J., Servitja, J.-M., Miyazaki, H., and Gutkind, J.S. (2002b). Potent activation of RhoA by Galpha q and Gq-coupled receptors. J. Biol. Chem. 277: 27130–4.
  50. Salim, S., Sinnarajah, S., Kehrl, J.H., and Dessauer, C.W. (2003). Identification of RGS2 and type V adenylyl cyclase interaction sites. J. Biol. Chem. 278: 15842–9.
  51. Chen, Z., Singer, W.D., Wells, C.D., Sprang, S.R., and Sternweis, P.C. (2003). Mapping the Galpha13 binding interface of the rgRGS domain of p115RhoGEF. J. Biol. Chem. 278: 9912–9.
  52. Papoucheva, E., Dumuis, A., Sebben, M., Richter, D.W., and Ponimaskin, E.G. (2004). The 5- hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein. J. Biol. Chem. 279: 3280–91.
  53. Bernstein, L.S., Ramineni, S., Hague, C., Cladman, W., Chidiac, P., Levey, A.I., et al. (2004). RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. J. Biol. Chem. 279: 21248–56.
  54. Kristelly, R., Gao, G., and Tesmer, J.J.G. (2004). Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J. Biol. Chem. 279: 47352–62.
  55. Oleksy, A., Opalinski, Ł., Derewenda, U., Derewenda, Z.S., and Otlewski, J. (2006). The molecular basis of RhoA specificity in the guanine nucleotide exchange factor PDZ-RhoGEF. J. Biol. Chem. 281: 32891–7.
  56. RhoB deficiency in thymic medullary epithelium leads to early thymic atrophy. Int. Immunol. 23: 593–600.
  57. Kozasa, T., Hajicek, N., Chow, C.R., and Suzuki, N. (2011). Signalling mechanisms of RhoGTPase regulation by the heterotrimeric G proteins G12 and G13. J. Biochem. 150: 357–69.
  58. Calò, L.A., Davis, P.A., Pagnin, E., Dal Maso, L., Maiolino, G., Seccia, T.M., et al. (2014). Increased level of p63RhoGEF and RhoA/Rho kinase activity in hypertensive patients. J. Hypertens. 32: 331–8.
  59. Hilgers, R.H.P., Todd, J., and Webb, R.C. (2007). Increased PDZ-RhoGEF/RhoA/Rho kinase signaling in small mesenteric arteries of angiotensin II-induced hypertensive rats. J. Hypertens. 25: 1687–97.
  60. Schmidt, A., and Hall, A. (2002). Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev. 16: 1587–609.
  61. Hakem, A., Sanchez-Sweatman, O., You-Ten, A., Duncan, G., Wakeham, A., Khokha, R., et al. (2005). RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis. Genes Dev. 19: 1974–9.
  62. Kitzing, T.M., Sahadevan, A.S., Brandt, D.T., Knieling, H., Hannemann, S., Fackler, O.T., et al. (2007). Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion. Genes Dev. 21: 1478–83.
  63. Thumkeo, D., Shimizu, Y., Sakamoto, S., Yamada, S., and Narumiya, S. (2005). ROCK-I and ROCK- II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo. Genes Cells 10: 825–34.
  64. Itoh, M., Nagatomo, K., Kubo, Y., and Saitoh, O. (2006). Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation. J. Neurochem. 99: 1505–16.
  65. Jin, L., Ying, Z., Hilgers, R.H.P., Yin, J., Zhao, X., Imig, J.D., et al. (2006). Increased RhoA/Rho- kinase signaling mediates spontaneous tone in aorta from angiotensin II-induced hypertensive rats. J. Pharmacol. Exp. Ther. 318: 288–95.
  66. Sun, X., Kaltenbronn, K.M., Steinberg, T.H., and Blumer, K.J. (2005). RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling. Mol. Pharmacol. 67: 631–9.
  67. Bodenstein, J., Sunahara, R.K., and Neubig, R.R. (2007). N-terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol. Pharmacol. 71: 1040–50.
  68. Gu, S., Tirgari, S., and Heximer, S.P. (2008). The RGS2 gene product from a candidate hypertension allele shows decreased plasma membrane association and inhibition of Gq. Mol. Pharmacol. 73: 1037–43.
  69. Hoffmann, C., Nuber, S., Zabel, U., Ziegler, N., Winkler, C., Hein, P., et al. (2012). Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells. Mol. Pharmacol. 82: 236–45.
  70. Cladman, W., and Chidiac, P. (2002). Characterization and comparison of RGS2 and RGS4 as GTPase-activating proteins for m2 muscarinic receptor-stimulated G(i). Mol. Pharmacol. 62: 654–9.
  71. Lohse, M.J., Nuber, S., and Hoffmann, C. (2012). Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol. Rev. 64: 299–336.
  72. Hollinger, S., and Hepler, J.R. (2002). Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol. Rev. 54: 527–59.
  73. Reiter, E., Ahn, S., Shukla, A.K., and Lefkowitz, R.J. (2012). Molecular mechanism of β-arrestin- biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52: 179–97.
  74. Cario-Toumaniantz, C., Ferland-McCollough, D., Chadeuf, G., Toumaniantz, G., Rodriguez, M., Galizzi, J.-P., et al. (2012). RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension. Am. J. Physiol. Cell Physiol. 302: C1394–404.
  75. Satoh, K., Fukumoto, Y., and Shimokawa, H. (2011). Rho-kinase: important new therapeutic target in cardiovascular diseases. Am. J. Physiol. Heart Circ. Physiol. 301: H287–96.
  76. Tang, X., Jin, R., Qu, G., Wang, X., Li, Z., Yuan, Z., et al. (2013). GPR116, an adhesion G-protein- coupled receptor, promotes breast cancer metastasis via the Gαq-p63RhoGEF-Rho GTPase pathway. Cancer Res. 73: 6206–18.
  77. Polymorphisms and haplotypes of the regulator of G protein signaling-2 gene in normotensives and hypertensives. Hypertension 47: 415–20.
  78. Calò, L.A., Davis, P.A., and Pessina, A.C. (2011). Does p63RhoGEF, a new key mediator of angiotensin II signalling, play a role in blood pressure regulation and cardiovascular remodelling in humans? J. Renin. Angiotensin. Aldosterone. Syst. 12: 634–6.
  79. Calò, L.A., Pagnin, E., Davis, P.A., Sartori, M., Ceolotto, G., Pessina, A.C., et al. (2004). Increased expression of regulator of G protein signaling-2 (RGS-2) in Bartter's/Gitelman's syndrome. A role in the control of vascular tone and implication for hypertension. J. Clin. Endocrinol. Metab. 89: 4153–7.
  80. Wennerberg, K., and Der, C.J. (2004). Rho-family GTPases: it's not only Rac and Rho (and I like it).
  81. Nethe, M., and Hordijk, P.L. (2010). The role of ubiquitylation and degradation in RhoGTPase signalling. J. Cell Sci. 123: 4011–8.
  82. Kohara, K., Tabara, Y., Nakura, J., Imai, Y., Ohkubo, T., Hata, A., et al. (2008). Identification of hypertension-susceptibility genes and pathways by a systemic multiple candidate gene approach: the millennium genome project for hypertension. Hypertens. Res. 31: 203–12.
  83. Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP- kinase cascade via the Gα(q/11)-p63RhoGEF-RhoA axis. PLoS Pathog. 9: e1003385.
  84. References Braman, J., Papworth, C., and Greener, A. (1996). Site-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol. Biol. 57: 31–44.
  85. Bianconi, E., Piovesan, A., Facchin, F., Beraudi, A., Casadei, R., Frabetti, F., et al. (2013). An estimation of the number of cells in the human body. Ann. Hum. Biol. 40: 463–71.
  86. Carter, A.M., Gutowski, S., and Sternweis, P.C. (2014). Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs). J. Biol. Chem. 289: 19737–46.
  87. Beadling, C., Druey, K.M., Richter, G., Kehrl, J.H., and Smith, K.A. (1999). Regulators of G protein signaling exhibit distinct patterns of gene expression and target G protein specificity in human lymphocytes. J. Immunol. 162: 2677–82.
  88. RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane. Dev. Cell 7: 855–69.
  89. McNair, K., Spike, R., Guilding, C., Prendergast, G.C., Stone, T.W., Cobb, S.R., et al. (2010). A role for RhoB in synaptic plasticity and the regulation of neuronal morphology. J. Neurosci. 30: 3508– 17.
  90. Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue. J. Biol. Chem. 278: 19301–8.
  91. Goedhart, J., Unen, J. van, Adjobo-Hermans, M.J.W., and Gadella, T.W.J. (2013). Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location. Sci. Rep. 3: 2284.
  92. Jares-Erijman, E., and Jovin, T.M. (2003). FRET imaging. Nat. Biotechnol. 21: 1387–95.
  93. Bos, J.L., Rehmann, H., and Wittinghofer, A. (2007). GEFs and GAPs: critical elements in the control of small G proteins. Cell 129: 865–77.
  94. Herzog, D., Loetscher, P., Hengel, J. van, Knüsel, S., Brakebusch, C., Taylor, V., et al. (2011). The small GTPase RhoA is required to maintain spinal cord neuroepithelium organization and the neural stem cell pool. J. Neurosci. 31: 5120–30.
  95. Cappello, S., Böhringer, C.R.J., Bergami, M., Conzelmann, K.-K., Ghanem, A., Tomassy, G.S., et al. (2012). A radial glia-specific role of RhoA in double cortex formation. Neuron 73: 911–24.
  96. Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis. Blood 119: 1054–63.
  97. Souchet, M., Portales-Casamar, E., Mazurais, D., Schmidt, S., Léger, I., Javré, J.-L., et al. (2002). Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere. J. Cell Sci. 115: 629–40.
  98. Semplicini, A., Lenzini, L., Sartori, M., Papparella, I., Calò, L.A., Pagnin, E., et al. (2006). Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II. J. Hypertens. 24: 1115–24.
  99. Zhou, J.Y., Toth, P.T., and Miller, R.J. (2003). Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3. J. Pharmacol. Exp. Ther. 305: 460–6.
  100. Lutz, S., Freichel-Blomquist, A., Rümenapp, U., Schmidt, M., Jakobs, K.H., and Wieland, T. (2004). p63RhoGEF and GEFT are Rho-specific guanine nucleotide exchange factors encoded by the same gene. Naunyn. Schmiedebergs. Arch. Pharmacol. 369: 540–6.
  101. References Hein, P., and Bünemann, M. (2009). Coupling mode of receptors and G proteins. Naunyn. Schmiedebergs. Arch. Pharmacol. 379: 435–43.
  102. Galpha(12/13) mediates alpha(1)-adrenergic receptor-induced cardiac hypertrophy. Circ. Res. 91: 961–9.
  103. Suzuki, N., Tsumoto, K., Hajicek, N., Daigo, K., Tokita, R., Minami, S., et al. (2009b). Activation of leukemia-associated RhoGEF by Galpha13 with significant conformational rearrangements in the interface. J. Biol. Chem. 284: 5000–9.
  104. Park, D., Jhon, D.Y., Lee, C.W., Lee, K.H., and Rhee, S.G. (1993). Activation of phospholipase C isozymes by G protein beta gamma subunits. J. Biol. Chem. 268: 4573–6.
  105. Hoffmann, C., Gaietta, G., Bünemann, M., Adams, S.R., Oberdorff-Maass, S., Behr, B., et al. (2005). A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat. Methods 2: 171–6.
  106. Maier-Peuschel, M., Frölich, N., Dees, C., Hommers, L.G., Hoffmann, C., Nikolaev, V.O., et al. (2010). A fluorescence resonance energy transfer-based M2 muscarinic receptor sensor reveals rapid kinetics of allosteric modulation. J. Biol. Chem. 285: 8793–800.
  107. Itoh, K., Yoshioka, K., Akedo, H., Uehata, M., Ishizaki, T., and Narumiya, S. (1999). An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat. Med. 5: 221–5.
  108. Kreutz, B., Yau, D.M., Nance, M.R., Tanabe, S., Tesmer, J.J.G., and Kozasa, T. (2006). A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins. Biochemistry 45: 167–74.
  109. Heximer, S.P. (2013). A " new twist " on RGS protein selectivity. Structure 21: 319–20.
  110. Li, Y., Hashim, S., and Anand-Srivastava, M.B. (2005). Angiotensin II-evoked enhanced expression of RGS2 attenuates Gi-mediated adenylyl cyclase signaling in A10 cells. Cardiovasc. Res. 66: 503–11.
  111. Madaule, P., Furuyashiki, T., Reid, T., Ishizaki, T., Watanabe, G., Morii, N., et al. (1995). A novel partner for the GTP-bound forms of rho and rac. FEBS Lett. 377: 243–8.
  112. Madaule, P., and Axel, R. (1985). A novel ras-related gene family. Cell 41: 31–40.
  113. Leung, T., Manser, E., Tan, L., and Lim, L. (1995). A novel serine/threonine kinase binding the Ras- related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270: 29051–4.
  114. Barnes, W.G., Reiter, E., Violin, J.D., Ren, X.-R., Milligan, G., and Lefkowitz, R.J. (2005). beta- Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation. J. Biol. Chem. 280: 8041–50.
  115. Krasel, C., Bünemann, M., Lorenz, K., and Lohse, M.J. (2005). Beta-arrestin binding to the beta2- adrenergic receptor requires both receptor phosphorylation and receptor activation. J. Biol. Chem. 280: 9528–35.
  116. Kelly, P., Casey, P.J., and Meigs, T.E. (2007). Biologic functions of the G12 subfamily of heterotrimeric g proteins: growth, migration, and metastasis. Biochemistry 46: 6677–87.
  117. Pi, M., Spurney, R.F., Tu, Q., Hinson, T., and Quarles, L.D. (2002). Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor. Endocrinology 143: 3830–8.
  118. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., et al. (1997). Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–4.
  119. Hubbard, K.B., and Hepler, J.R. (2006). Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins. Cell. Signal. 18: 135–50.
  120. Zhou, X., and Zheng, Y. (2013). Cell type-specific signaling function of RhoA GTPase: lessons from mouse gene targeting. J. Biol. Chem. 288: 36179–88.
  121. Irannejad, R., Tomshine, J.C., Tomshine, J.R., Chevalier, M., Mahoney, J.P., Steyaert, J., et al. (2013). Conformational biosensors reveal GPCR signalling from endosomes. Nature 495: 1–8.
  122. Kobilka, B.K., and Deupi, X. (2007). Conformational complexity of G-protein-coupled receptors. Trends Pharmacol. Sci. 28: 397–406.
  123. Moepps, B., Tulone, C., Kern, C., Minisini, R., Michels, G., Vatter, P., et al. (2008). Constitutive serum response factor activation by the viral chemokine receptor homologue pUS28 is differentially regulated by Galpha(q/11) and Galpha(16). Cell. Signal. 20: 1528–37.
  124. Soisson, S.M., Nimnual, A.S., Uy, M., Bar-Sagi, D., and Kuriyan, J. (1998). Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein. Cell 95: 259–68.
  125. Caruthers, M.H., Beaucage, S.L., Becker, C., Efcavitch, J.W., Fisher, E.F., Galluppi, G., et al. (1983). Deoxyoligonucleotide synthesis via the phosphoramidite method. Gene Amplif. Anal. 3: 1–26.
  126. Ren, X.D., and Schwartz, M.A. (2000). Determination of GTP loading on Rho. Methods Enzymol. 325: 264–72.
  127. Miyawaki, A. (2011). Development of probes for cellular functions using fluorescent proteins and fluorescence resonance energy transfer. Annu. Rev. Biochem. 80: 357–73.
  128. Day, P.W., Carman, C. V., Sterne-Marr, R., Benovic, J.L., and Wedegaertner, P.B. (2003). Differential interaction of GRK2 with members of the G alpha q family. Biochemistry 42: 9176–84.
  129. Bondar, A., and Lazar, J. (2014). Dissociated GαGTP and Gβγ protein subunits are the major activated form of heterotrimeric Gi/o proteins. J. Biol. Chem. 289: 1271–81.
  130. Bodmann, E.-L., Rinne, A., Brandt, D., Lutz, S., Wieland, T., Grosse, R., et al. (2014). Dynamics of Gαq-protein-p63RhoGEF interaction and its regulation by RGS2. Biochem. J. 458: 131–40.
  131. Heim, R., and Tsien, R.Y. (1996). Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6: 178–82.
  132. Wilkie, T.M., and Yokoyama, S. (1994). Evolution of the G protein alpha subunit multigene family. Soc. Gen. Physiol. Ser. 49: 249–70.
  133. Wilkie, T.M., Gilbert, D.J., Olsen, A.S., Chen, X.N., Amatruda, T.T., Korenberg, J.R., et al. (1992). Evolution of the mammalian G protein alpha subunit multigene family. Nat. Genet. 1: 85–91.
  134. Ziegler, N., Bätz, J., Zabel, U., Lohse, M.J., and Hoffmann, C. (2011). FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors. Bioorg. Med. Chem. 19: 1048–54.
  135. Lyon, A.M., Dutta, S., Boguth, C.A., Skiniotis, G., and Tesmer, J.J.G. (2013). Full-length Gα(q)- phospholipase C-β3 structure reveals interfaces of the C-terminal coiled-coil domain. Nat. Struct. Mol. Biol. 20: 355–62.
  136. Togashi, H., Nagata, K., Takagishi, M., Saitoh, N., and Inagaki, M. (2000). Functions of a rho-specific guanine nucleotide exchange factor in neurite retraction. Possible role of a proline-rich motif of KIAA0380 in localization. J. Biol. Chem. 275: 29570–8.
  137. Takefuji, M., Wirth, A., Lukasova, M., Takefuji, S., Boettger, T., Braun, T., et al. (2012). G(13)- mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure. Circulation 126: 1972–82.
  138. Meigs, T.E., Fedor-Chaiken, M., Kaplan, D.D., Brackenbury, R., and Casey, P.J. (2002). Galpha12 and Galpha13 negatively regulate the adhesive functions of cadherin. J. Biol. Chem. 277: 24594– 600.
  139. Galpha12/Galpha13 deficiency causes localized overmigration of neurons in the developing cerebral and cerebellar cortices. Mol. Cell. Biol. 28: 1480–8.
  140. Rojas, R.J., Yohe, M.E., Gershburg, S., Kawano, T., Kozasa, T., and Sondek, J. (2007). Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology- associated pleckstrin homology domain. J. Biol. Chem. 282: 29201–10.
  141. Sadja, R., Alagem, N., and Reuveny, E. (2003). Gating of GIRK channels: details of an intricate, membrane-delimited signaling complex. Neuron 39: 9–12.
  142. Lefkowitz, R.J. (1998). G Protein-coupled Receptors: III. NEW ROLES FOR RECEPTOR KINASES AND -ARRESTINS IN RECEPTOR SIGNALING AND DESENSITIZATION. J. Biol. Chem. 273: 18677–80.
  143. Hein, P., Rochais, F., Hoffmann, C., Dorsch, S., Nikolaev, V.O., Engelhardt, S., et al. (2006). Gs activation is time-limiting in initiating receptor-mediated signaling. J. Biol. Chem. 281: 33345– 51.
  144. Ross, E.M., and Wilkie, T.M. (2000). GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69: 795– 827.
  145. Calebiro, D., Nikolaev, V.O., and Lohse, M.J. (2010). Imaging of persistent cAMP signaling by internalized G protein-coupled receptors. J. Mol. Endocrinol. 45: 1–8.
  146. Ying, Z., Jin, L., Dorrance, A.M., and Webb, R.C. (2004). Increaseed expression of mRNA for regulator of G protein signaling domain-containing Rho guanine nucleotide exchange factors in aorta from stroke-prone spontaneously hypertensive rats. Am. J. Hypertens. 17: 981–5.
  147. Meigs, T.E., Fields, T.A., McKee, D.D., and Casey, P.J. (2001). Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release. Proc. Natl. Acad. Sci. U. S. A. 98: 519–24.
  148. Camps, M., Carozzi, A., Schnabel, P., Scheer, A., Parker, P.J., and Gierschik, P. (1992). Isozyme- selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature 360: 684–6.
  149. Waldo, G.L., Ricks, T.K., Hicks, S.N., Cheever, M.L., Kawano, T., Tsuboi, K., et al. (2010). Kinetic scaffolding mediated by a phospholipase C-beta and Gq signaling complex. Science 330: 974–80.
  150. Wedegaertner, P.B., Wilson, P.T., and Bourne, H.R. (1995). Lipid modifications of trimeric G proteins. J. Biol. Chem. 270: 503–6.
  151. Vilardaga, J.-P., Bünemann, M., Krasel, C., Castro, M., and Lohse, M.J. (2003). Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21: 807–12.
  152. Swenson-Fields, K.I., Sandquist, J.C., Rossol-Allison, J., Blat, I.C., Wennerberg, K., Burridge, K., et al. (2008). MLK3 limits activated Galphaq signaling to Rho by binding to p63RhoGEF. Mol. Cell 32: 43–56.
  153. Zamponi, G.W., and Snutch, T.P. (1998). Modulation of voltage-dependent calcium channels by G proteins. Curr. Opin. Neurobiol. 8: 351–6.
  154. Sambrook, J., and Russel, D.W. (2001). Molecular Cloning (Cold Spring Harbor Laboratory Press).
  155. Hirose, M., Ishizaki, T., Watanabe, N., Uehata, M., Kranenburg, O., Moolenaar, W.H., et al. (1998). Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J. Cell Biol. 141: 1625–36.
  156. Ben-Chaim, Y., Chanda, B., Dascal, N., Bezanilla, F., Parnas, I., and Parnas, H. (2006). Movement of " gating charge " is coupled to ligand binding in a G-protein-coupled receptor. Nature 444: 106–9.
  157. Kahsai, A.W., Xiao, K., Rajagopal, S., Ahn, S., Shukla, A.K., Sun, J., et al. (2011). Multiple ligand- specific conformations of the β2-adrenergic receptor. Nat. Chem. Biol. 7: 692–700.
  158. Liu, X., Wang, H., Eberstadt, M., Schnuchel, A., Olejniczak, E.T., Meadows, R.P., et al. (1998). NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor Trio. Cell 95: 269–77.
  159. Degtyarev, M.Y., Spiegel, A.M., and Jones, T.L. (1994). Palmitoylation of a G protein alpha i subunit requires membrane localization not myristoylation. J. Biol. Chem. 269: 30898–903.
  160. Dror, R.O., Pan, A.C., Arlow, D.H., Borhani, D.W., Maragakis, P., Shan, Y., et al. (2011b). Pathway References Girkontaite, I., Missy, K., Sakk, V., Harenberg, A., Tedford, K., Pötzel, T., et al. (2001). Lsc is required for marginal zone B cells, regulation of lymphocyte motility and immune responses. Nat. Immunol. 2: 855–62.
  161. Thumkeo, D., Watanabe, S., and Narumiya, S. (2013). Physiological roles of Rho and Rho effectors in mammals. Eur. J. Cell Biol. 92: 303–15.
  162. Swiercz, J.M., Kuner, R., Behrens, J., and Offermanns, S. (2002). Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology. Neuron 35: 51–63.
  163. Welch, H.C.E., Coadwell, W.J., Ellson, C.D., Ferguson, G.J., Andrews, S.R., Erdjument-Bromage, H., et al. (2002). P-Rex1, a PtdIns(3,4,5)P3-and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac. Cell 108: 809–21.
  164. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., and Cormier, M.J. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111: 229–33.
  165. Mellor, H., Flynn, P., Nobes, C.D., Hall, A., and Parker, P.J. (1998). PRK1 is targeted to endosomes by the small GTPase, RhoB. J. Biol. Chem. 273: 4811–4.
  166. Protein kinase A-mediated phosphorylation of the Galpha13 switch I region alters the Galphabetagamma13-G protein-coupled receptor complex and inhibits Rho activation. J. Biol. Chem. 278: 124–30.
  167. Kozasa, T., and Gilman, A.G. (1996). Protein kinase C phosphorylates G12 alpha and inhibits its interaction with G beta gamma. J. Biol. Chem. 271: 12562–7.
  168. Cunningham, M.L., Waldo, G.L., Hollinger, S., Hepler, J.R., and Harden, T.K. (2001). Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling. J. Biol. Chem. 276: 5438–44.
  169. Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., et al. (1996). Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho. Science 271: 645–8.
  170. G12/G13 Family G Proteins Regulate Marginal Zone B Cell Maturation, Migration, and Polarization. J. Immunol. 177: 2985–93.
  171. Singer, W.D., Miller, R.T., and Sternweis, P.C. (1994). Purification and characterization of the alpha subunit of G13. J. Biol. Chem. 269: 19796–802.
  172. Kozasa, T., and Gilman, A.G. (1995). Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of alpha 12 and inhibition of adenylyl cyclase by alpha z. J. Biol. Chem. 270: 1734–41.
  173. Liu, G., and Voyno-Yasenetskaya, T.A. (2005). Radixin stimulates Rac1 and Ca2+/calmodulin- dependent kinase, CaMKII: cross-talk with Galpha13 signaling. J. Biol. Chem. 280: 39042–9.
  174. Pagnin, E., Semplicini, A., Sartori, M., Pessina, A.C., and Calò, L.A. (2005). Reduced mRNA and protein content of rho guanine nucleotide exchange factor (RhoGEF) in Bartter's and Gitelman's syndromes: relevance for the pathophysiology of hypertension. Am. J. Hypertens. 18: 1200–5.
  175. Zamponi, G.W., and Currie, K.P.M. (2013). Regulation of Ca(V)2 calcium channels by G protein coupled receptors. Biochim. Biophys. Acta 1828: 1629–43.
  176. Chikumi, H., Fukuhara, S., and Gutkind, J.S. (2002a). Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. J. Biol. Chem. 277: 12463–73.
  177. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245–8.
  178. Rhee, S.G. (2001). Regulation of phosphoinositide-specific phospholipase C. Annu. Rev. Biochem. 70: 281–312.
  179. Zalcman, G., Closson, V., Linarès-Cruz, G., Lerebours, F., Honoré, N., Tavitian, A., et al. (1995). Regulation of Ras-related RhoB protein expression during the cell cycle. Oncogene 10: 1935–45.
  180. Puetz, S., Lubomirov, L.T., and Pfitzer, G. (2009). Regulation of smooth muscle contraction by small GTPases. Physiology (Bethesda). 24: 342–56.
  181. Tang, K.M., Wang, G., Lu, P., Karas, R.H., Aronovitz, M., Heximer, S.P., et al. (2003). Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nat. Med. 9: 1506–12.
  182. Li, J., Adams, L.D., Wang, X., Pabon, L., Schwartz, S.M., Sane, D.C., et al. (2004). Regulator of G protein signaling 5 marks peripheral arterial smooth muscle cells and is downregulated in atherosclerotic plaque. J. Vasc. Surg. 40: 519–28.
  183. Hepler, J.R., Berman, D.M., Gilman, A.G., and Kozasa, T. (1997). RGS4 and GAIP are GTPase- activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio- GTP-Gq alpha. Proc. Natl. Acad. Sci. U. S. A. 94: 428–32.
  184. Burridge, K., Wennerberg, K., Hill, C., and Carolina, N. (2004). Rho and Rac Take Center Stage Review. 116: 167–79.
  185. Ohashi, K., Nagata, K., Maekawa, M., Ishizaki, T., Narumiya, S., and Mizuno, K. (2000). Rho- associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J. Biol. Chem. 275: 3577–82.
  186. Liu, A.X., Rane, N., Liu, J.P., and Prendergast, G.C. (2001). RhoB is dispensable for mouse development, but it modifies susceptibility to tumor formation as well as cell adhesion and growth factor signaling in transformed cells. Mol. Cell. Biol. 21: 6906–12.
  187. Sah, V.P., Hoshijima, M., Chien, K.R., and Brown, J.H. (1996). Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes. Dissociation of Ras and Rho pathways. J. Biol. Chem. 271: 31185–90.
  188. Nobes, C.D., and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81: 53–62.
  189. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J. Biol. Chem. 271: 13556–60.
  190. Carman, C. V., Parent, J.L., Day, P.W., Pronin, A.N., Sternweis, P.M., Wedegaertner, P.B., et al. (1999). Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. J. Biol. Chem. 274: 34483–92.
  191. Selective uncoupling of G alpha 12 from Rho-mediated signaling. J. Biol. Chem. 280: 18049–55.
  192. Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A., Iwamatsu, A., et al. (1999). Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285: 895–8.
  193. Hamm, H.E., Deretic, D., Arendt, A., Hargrave, P.A., Koenig, B., and Hofmann, K.P. (1988). Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. Science 241: 832–5.
  194. Li, Y., Sternweis, P.M., Charnecki, S., Smith, T.F., Gilman, A.G., Neer, E.J., et al. (1998). Sites for Galpha binding on the G protein beta subunit overlap with sites for regulation of phospholipase Cbeta and adenylyl cyclase. J. Biol. Chem. 273: 16265–72.
  195. Tesmer, V.M., Kawano, T., Shankaranarayanan, A., Kozasa, T., and Tesmer, J.J.G. (2005). Snapshot of activated G proteins at the membrane: the Galphaq-GRK2-Gbetagamma complex. Science 310: 1686–90.
  196. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1: 263–73.
  197. Medlin, M.D., Staus, D.P., Dubash, A.D., Taylor, J.M., and Mack, C.P. (2010). Sphingosine 1- phosphate receptor 2 signals through leukemia-associated RhoGEF (LARG), to promote smooth muscle cell differentiation. Arterioscler. Thromb. Vasc. Biol. 30: 1779–86.
  198. Lyon, A.M., Taylor, V.G., and Tesmer, J.J.G. (2014). Strike a pose: Gαq complexes at the membrane. Trends Pharmacol. Sci. 35: 23–30.
  199. Davis, T.L., Bonacci, T.M., Sprang, S.R., and Smrcka, A. V. (2005). Structural and molecular characterization of a preferred protein interaction surface on G protein beta gamma subunits. Biochemistry 44: 10593–604.
  200. Snyder, J.T., Worthylake, D.K., Rossman, K.L., Betts, L., Pruitt, W.M., Siderovski, D.P., et al. (2002). Structural basis for the selective activation of Rho GTPases by Dbl exchange factors. Nat. Struct. Biol. 9: 468–75.
  201. Westfield, G.H., Rasmussen, S.G.F., Su, M., Dutta, S., DeVree, B.T., Chung, K.Y., et al. (2011). Structural flexibility of the G alpha s alpha-helical domain in the beta2-adrenoceptor Gs complex. Proc. Natl. Acad. Sci. U. S. A. 108: 16086–91.
  202. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh, T. (1991). Structure and function of signal- transducing GTP-binding proteins. Annu. Rev. Biochem. 60: 349–400.
  203. Rebecchi, M.J., and Pentyala, S.N. (2000). Structure, function, and control of phosphoinositide- specific phospholipase C. Physiol. Rev. 80: 1291–335.
  204. Lutz, S., Shankaranarayanan, A., Coco, C., Ridilla, M., Nance, M.R., Vettel, C., et al. (2007). Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs. Science 318: 1923–7.
  205. Tesmer, J.J., Berman, D.M., Gilman, A.G., and Sprang, S.R. (1997). Structure of RGS4 bound to AlF4-activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 89: 251– 61.
  206. Chen, Z., Singer, W.D., Sternweis, P.C., and Sprang, S.R. (2005). Structure of the p115RhoGEF rgRGS domain-Galpha13/i1 chimera complex suggests convergent evolution of a GTPase activator. Nat. Struct. Mol. Biol. 12: 191–7.
  207. Goss, D.J., Parkhurst, L.J., Mehta, H.B., Woodley, C.L., and Wahba, A.J. (1984). Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation. J. Biol. Chem. 259: 7374–7.
  208. Watkins, J.L., Kim, H., Markwardt, M.L., Chen, L., Fromme, R., Rizzo, M.A., et al. (2013). The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein. Acta Crystallogr. D. Biol. Crystallogr. 69: 767–73.
  209. Logothetis, D.E., Kurachi, Y., Galper, J., Neer, E.J., and Clapham, D.E. (1987). The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325: 321– 6.
  210. Nygaard, R., Zou, Y., Dror, R.O., Mildorf, T.J., Arlow, D.H., Manglik, A., et al. (2013). The dynamic process of β(2)-adrenergic receptor activation. Cell 152: 532–42.
  211. Oka, Y., Saraiva, L.R., Kwan, Y.Y., and Korsching, S.I. (2009). The fifth class of Galpha proteins. Proc. Natl. Acad. Sci. U. S. A. 106: 1484–9.
  212. Berman, D.M., Kozasa, T., and Gilman, A.G. (1996a). The GTPase-activating Protein RGS4 Stabilizes the Transition State for Nucleotide Hydrolysis. J. Biol. Chem. 271: 27209–27212.
  213. Lutz, S., Freichel-Blomquist, A., Yang, Y., Rümenapp, U., Jakobs, K.H., Schmidt, M., et al. (2005). The guanine nucleotide exchange factor p63RhoGEF, a specific link between Gq/11-coupled receptor signaling and RhoA. J. Biol. Chem. 280: 11134–9.
  214. Hamm, H.E. (1998). The many faces of G protein signaling. J. Biol. Chem. 273: 669–72.
  215. Preininger, A.M., Eps, N. van, Yu, N.-J., Medkova, M., Hubbell, W.L., and Hamm, H.E. (2003). The myristoylated amino terminus of Galpha(i)(1) plays a critical role in the structure and function of Galpha(i)(1) subunits in solution. Biochemistry 42: 7931–41.
  216. Hill, C.S., Wynne, J., and Treisman, R. (1995). The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159–70.
  217. Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., and Hall, A. (1992). The small GTP- binding protein rac regulates growth factor-induced membrane ruffling. Cell 70: 401–10.
  218. Ridley, A.J., and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–99.
  219. Kobilka, B.K. (2013). The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 52: 6380–8.
  220. Williams, S.L., Lutz, S., Charlie, N.K., Vettel, C., Ailion, M., Coco, C., et al. (2007). Trio's Rho- specific GEF domain is the missing Galpha q effector in C. elegans. Genes Dev. 21: 2731–46.
  221. Tang, W.J., and Gilman, A.G. (1991). Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science 254: 1500–3.
  222. Momotani, K., and Somlyo, A. V (2012). p63RhoGEF: a new switch for G(q)-mediated activation of smooth muscle. Trends Cardiovasc. Med. 22: 122–7.
  223. Ma, P., and Zemmel, R. (2002). Value of novelty? Nat. Rev. Drug Discov. 1: 571–2.
  224. Offermanns, S., Mancino, V., Revel, J.P., and Simon, M.I. (1997). Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency. Science 275: 533–6.
  225. Hughes, T.E., Zhang, H., Logothetis, D.E., and Berlot, C.H. (2001). Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or . J. Biol. Chem. 276: 4227–35.
  226. Wheeler, A.P., and Ridley, A.J. (2004). Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp. Cell Res. 301: 43–9.
  227. Vilardaga, J.-P., Bünemann, M., Feinstein, T.N., Lambert, N., Nikolaev, V.O., Engelhardt, S., et al. (2009). GPCR and G proteins: drug efficacy and activation in live cells. Mol. Endocrinol. 23: 590–9.
  228. Dorsch, S., Klotz, K., Engelhardt, S., Lohse, M.J., and Bünemann, M. (2009). Analysis of receptor oligomerization by FRAP microscopy. Nat. Methods 6: 225–30.
  229. Grant, S.L., Lassègue, B., Griendling, K.K., Ushio-Fukai, M., Lyons, P.R., and Alexander, R.W. (2000). Specific regulation of RGS2 messenger RNA by angiotensin II in cultured vascular smooth muscle cells. Mol. Pharmacol. 57: 460–7.
  230. Nakamura, S., Kreutz, B., Tanabe, S., Suzuki, N., and Kozasa, T. (2004). Critical role of lysine 204 in switch I region of Galpha13 for regulation of p115RhoGEF and leukemia-associated RhoGEF. Mol. Pharmacol. 66: 1029–34.
  231. Grabocka, E., and Wedegaertner, P.B. (2007). Disruption of oligomerization induces nucleocytoplasmic shuttling of leukemia-associated Rho Guanine-nucleotide exchange factor. Mol. Pharmacol. 72: 993–1002.
  232. Birnboim, H.C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513.
  233. Meyer, B.H., Freuler, F., Guerini, D., and Siehler, S. (2008). Reversible translocation of p115- RhoGEF by G(12/13)-coupled receptors. J. Cell. Biochem. 104: 1660–70.
  234. Guilluy, C., Brégeon, J., Toumaniantz, G., Rolli-Derkinderen, M., Retailleau, K., Loufrani, L., et al. (2010). The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nat. Med. 16: 183–90.
  235. Rujkijyanont, P., Beyene, J., Wei, K., Khan, F., and Dror, Y. (2007). Leukaemia-related gene expression in bone marrow cells from patients with the preleukaemic disorder Shwachman- Diamond syndrome. Br. J. Haematol. 137: 537–44.
  236. Pollinger, T. (2012). Spatiotemporale Organisation der Interaktion von G q Protein-Untereinheiten und der Phospholipase Cβ3. Universität Würzburg.
  237. Khan, S.M., Sleno, R., Gora, S., Zylbergold, P., Laverdure, J.-P., Labbé, J.-C., et al. (2013). The expanding roles of Gβγ subunits in G protein-coupled receptor signaling and drug action. Pharmacol. Rev. 65: 545–77.
  238. Ruiz-Velasco, V., and Ikeda, S.R. (2003). A splice variant of the G protein beta 3-subunit implicated in disease states does not modulate ion channels. Physiol. Genomics 13: 85–95.
  239. Coleman, M.L., Sahai, E.A., Yeo, M., Bosch, M., Dewar, A., and Olson, M.F. (2001). Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat. Cell Biol. 3: 339–45.
  240. Sahai, E., and Marshall, C.J. (2002). ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat. Cell Biol. 4: 408–15.
  241. Cavin, S., Maric, D., and Diviani, D. (2014). A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts. Biochim. Biophys. Acta 1843: 335–45.
  242. Jaffe, A.B., and Hall, A. (2005). Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21: 247–69.
  243. Li, H., Choe, N.H., Wright, D.T., and Adler, K.B. (1995). Histamine provokes turnover of inositol phospholipids in guinea pig and human airway epithelial cells via an H1-receptor/G protein- dependent mechanism. Am. J. Respir. Cell Mol. Biol. 12: 416–24.
  244. Zheng, M., Cierpicki, T., Momotani, K., Artamonov, M. V, Derewenda, U., Bushweller, J.H., et al. (2009). On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF. BMC Struct. Biol. 9: 36.
  245. Wuertz, C.M., Lorincz, A., Vettel, C., Thomas, M.A., Wieland, T., and Lutz, S. (2010). p63RhoGEF-a key mediator of angiotensin II-dependent signaling and processes in vascular smooth muscle cells. FASEB J. 24: 4865–76.
  246. Schmidt, C.J., Thomas, T.C., Levine, M.A., and Neer, E.J. (1992). Specificity of G protein beta and gamma subunit interactions. J. Biol. Chem. 267: 13807–10.
  247. Bünemann, M., Bücheler, M.M., Philipp, M., Lohse, M.J., and Hein, L. (2001). Activation and deactivation kinetics of alpha 2A-and alpha 2C-adrenergic receptor-activated G protein-activated inwardly rectifying K+ channel currents. J. Biol. Chem. 276: 47512–7.
  248. Ponimaskin, E.G., Profirovic, J., Vaiskunaite, R., Richter, D.W., and Voyno-Yasenetskaya, T.A. (2002). 5-Hydroxytryptamine 4(a) receptor is coupled to the Galpha subunit of heterotrimeric G13 protein. J. Biol. Chem. 277: 20812–9.
  249. Vogt, S., Grosse, R., Schultz, G., and Offermanns, S. (2003). Receptor-dependent RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement of Gq/G11. J. Biol. Chem. 278: 28743–9.
  250. Illenberger, D., Walliser, C., Nurnberg, B., Diaz Lorente, M., and Gierschik, P. (2003). Specificity and structural requirements of phospholipase C-beta stimulation by Rho GTPases versus G protein beta gamma dimers. J. Biol. Chem. 278: 3006–14.
  251. Skowronek, K.R., Guo, F., Zheng, Y., and Nassar, N. (2004). The C-terminal basic tail of RhoG assists the guanine nucleotide exchange factor trio in binding to phospholipids. J. Biol. Chem. 279: 37895–907.
  252. Kelly, P., Stemmle, L.N., Madden, J.F., Fields, T.A., Daaka, Y., and Casey, P.J. (2006b). A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion. J. Biol. Chem. 281: 26483–90.
  253. Shankaranarayanan, A., Thal, D.M., Tesmer, V.M., Roman, D.L., Neubig, R.R., Kozasa, T., et al. (2008). Assembly of high order G alpha q-effector complexes with RGS proteins. J. Biol. Chem. 283: 34923–34.
  254. Han, J., Huang, N., Kim, D., and Kehrl, J.H. (2006). RGS1 and RGS13 mRNA silencing in a human B lymphoma line enhances responsiveness to chemoattractants and impairs desensitization. J. Leukoc. Biol. 79: 1357–68.
  255. Katayama, K., Leslie, J.R., Lang, R.A., Zheng, Y., and Yoshida, Y. (2012). Left-right locomotor circuitry depends on RhoA-driven organization of the neuroepithelium in the developing spinal cord. J. Neurosci. 32: 10396–407.
  256. Goulimari, P., Knieling, H., Engel, U., and Grosse, R. (2008). LARG and mDia1 link Galpha12/13 to cell polarity and microtubule dynamics. Mol. Biol. Cell 19: 30–40.
  257. Sondek, J., Bohm, A., Lambright, D.G., Hamm, H.E., and Sigler, P.B. (1996). Crystal structure of a G- protein beta gamma dimer at 2.1A resolution. Nature 379: 369–74.
  258. Worthylake, D.K., Rossman, K.L., and Sondek, J. (2000). Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 408: 682–8.
  259. Rosenbaum, D.M., Rasmussen, S.G.F., and Kobilka, B.K. (2009). The structure and function of G- protein-coupled receptors. Nature 459: 356–63.
  260. Rasmussen, S.G.F., DeVree, B.T., Zou, Y., Kruse, A.C., Chung, K.Y., Kobilka, T.S., et al. (2011). Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477: 549–55.
  261. Wirth, A., Benyó, Z., Lukasova, M., Leutgeb, B., Wettschureck, N., Gorbey, S., et al. (2008). G12- G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat. Med. 14: 64–8.
  262. Moers, A., Nieswandt, B., Massberg, S., Wettschureck, N., Grüner, S., Konrad, I., et al. (2003). G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat. Med. 9: 1418–22.
  263. Vigil, D., Cherfils, J., Rossman, K.L., and Der, C.J. (2010). Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy? Nat. Rev. Cancer 10: 842–57.
  264. Rask-Andersen, M., Almén, M.S., and Schiöth, H.B. (2011). Trends in the exploitation of novel drug targets. Nat. Rev. Drug Discov. 10: 579–90.
  265. Oldham, W.M., and Hamm, H.E. (2008). Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9: 60–71.
  266. Chikumi, H., Barac, A., Behbahani, B., Gao, Y., Teramoto, H., Zheng, Y., et al. (2004). Homo-and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential. Oncogene 23: 233–40.
  267. Cook, D.R., Rossman, K.L., and Der, C.J. (2014). Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease. Oncogene 33: 4021–35.
  268. Barberan, S., McNair, K., Iqbal, K., Smith, N.C., Prendergast, G.C., Stone, T.W., et al. (2011). Altered apoptotic responses in neurons lacking RhoB GTPase. Eur. J. Neurosci. 34: 1737–46.
  269. Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka, A., et al. (1997). p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J. 16: 3044–56.
  270. Offermanns, S., Zhao, L.P., Gohla, A., Sarosi, I., Simon, M.I., and Wilkie, T.M. (1998). Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice. EMBO J. 17: 4304–12.
  271. Ren, X.D., Kiosses, W.B., and Schwartz, M.A. (1999). Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18: 578–85.
  272. Pogozheva, I.D., Lomize, A.L., and Mosberg, H.I. (1997). The transmembrane 7-alpha-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints. Biophys. J. 72: 1963–85.
  273. Lee, M.J., Tasaki, T., Moroi, K., An, J.Y., Kimura, S., Davydov, I. V., et al. (2005). RGS4 and RGS5 are in vivo substrates of the N-end rule pathway. Proc. Natl. Acad. Sci. U. S. A. 102: 15030–5.
  274. References Terrillon, S., and Bouvier, M. (2004). Roles of G-protein-coupled receptor dimerization. EMBO Rep. 5: 30–4.
  275. Berney, C., and Danuser, G. (2003). FRET or no FRET: a quantitative comparison. Biophys. J. 84: 3992–4010.
  276. Hein, P., Frank, M., Hoffmann, C., Lohse, M.J., and Bünemann, M. (2005). Dynamics of receptor/G protein coupling in living cells. EMBO J. 24: 4106–14.
  277. Kelly, P., Moeller, B.J., Juneja, J., Booden, M.A., Der, C.J., Daaka, Y., et al. (2006a). The G12 family of heterotrimeric G proteins promotes breast cancer invasion and metastasis. Proc. Natl. Acad. Sci. U. S. A. 103: 8173–8.
  278. Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J. Clin. Invest. 111: 445–52.
  279. Kourlas, P.J., Strout, M.P., Becknell, B., Veronese, M.L., Croce, C.M., Theil, K.S., et al. (2000). Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: evidence for its fusion with MLL in acute myeloid leukemia. Proc. Natl. Acad. Sci. U. S. A. 97: 2145–50.
  280. Thumkeo, D., Keel, J., Ishizaki, T., Hirose, M., Nonomura, K., Oshima, H., et al. (2003). Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death. Mol. Cell. Biol. 23: 5043–55.
  281. Digby, G.J., Lober, R.M., Sethi, P.R., and Lambert, N.A. (2006). Some G protein heterotrimers physically dissociate in living cells. Proc. Natl. Acad. Sci. U. S. A. 103: 17789–94.
  282. Oliveira-dos-Santos, A.J., Matsumoto, G., Snow, B.E., Bai, D., Houston, F.P., Whishaw, I.Q., et al. (2000). Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc. Natl. Acad.
  283. Chhatriwala, M.K., Betts, L., Worthylake, D.K., and Sondek, J. (2007). The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation. J. Mol. Biol. 368: 1307–20.
  284. Bustelo, X.R., Sauzeau, V., and Berenjeno, I.M. (2007). GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29: 356–70.
  285. Kishi, K., Sasaki, T., Kuroda, S., Itoh, T., and Takai, Y. (1993). Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J. Cell Biol. 120: 1187–95.
  286. Klages, B., Brandt, U., Simon, M.I., Schultz, G., and Offermanns, S. (1999). Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J. Cell Biol. 144: 745–54.
  287. Taya, S., Inagaki, N., Sengiku, H., Makino, H., Iwamatsu, A., Urakawa, I., et al. (2001). Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF. J. Cell Biol. 155: 809–20.
  288. Bansal, G., Druey, K.M., and Xie, Z. (2007). R4 RGS proteins: regulation of G-protein signaling and beyond. Pharmacol. Ther. 116: 473–95.
  289. Violin, J.D., Zhang, J., Tsien, R.Y., and Newton, A.C. (2003). A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161: 899–909.
  290. Sebbagh, M., Hamelin, J., Bertoglio, J., Solary, E., and Bréard, J. (2005). Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner. J. Exp. Med. 201: 465–71.
  291. Li, Y., Hanf, R., Otero, A.S., Fischmeister, R., and Szabo, G. (1994). Differential effects of pertussis toxin on the muscarinic regulation of Ca2+ and K+ currents in frog cardiac myocytes. J. Gen. Physiol. 104: 941–59.
  292. Mintert, E., Bösche, L.I., Rinne, A., Timpert, M., Kienitz, M.-C., Pott, L., et al. (2007). Generation of a constitutive Na+-dependent inward-rectifier current in rat adult atrial myocytes by overexpression of Kir3.4. J. Physiol. 585: 3–13.
  293. Qin, K., Sethi, P.R., and Lambert, N. a (2008). Abundance and stability of complexes containing inactive G protein-coupled receptors and G proteins. FASEB J. 22: 2920–7.
  294. Siehler, S. (2009). Regulation of RhoGEF proteins by G12/13-coupled receptors. Br. J. Pharmacol. 158: 41–9.
  295. Tuteja, N. (2009). Signaling through G protein coupled receptors. Plant Signal. Behav. 4: 942–7.
  296. Suzuki, N., Hajicek, N., and Kozasa, T. (2009a). Regulation and physiological functions of G12/13- mediated signaling pathways. Neurosignals. 17: 55–70.
  297. Ong, D.C.T., Ho, Y.M., Rudduck, C., Chin, K., Kuo, W.-L., Lie, D.K.H., et al. (2009). LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast and colorectal cancer. Oncogene 28: 4189–200.
  298. Chung, C.T., Niemela, S.L., and Miller, R.H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U. S. A. 86: 2172–5.
  299. Heasman, S.J., Carlin, L.M., Cox, S., Ng, T., and Ridley, A.J. (2010). Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration. J. Cell Biol. 190: 553–63.
  300. Jackson, B., Peyrollier, K., Pedersen, E., Basse, A., Karlsson, R., Wang, Z., et al. (2011). RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes. Mol. Biol. Cell 22: 593–605.
  301. Olson, E.N., and Nordheim, A. (2010). Linking actin dynamics and gene transcription to drive cellular motile functions. Nat. Rev. Mol. Cell Biol. 11: 353–65.
  302. Bünemann, M., Frank, M., and Lohse, M.J. (2003). Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. U. S. A. 100: 16077–82.
  303. Melendez, J., Stengel, K., Zhou, X., Chauhan, B.K., Debidda, M., Andreassen, P., et al. (2011). RhoA GTPase is dispensable for actomyosin regulation but is essential for mitosis in primary mouse embryonic fibroblasts. J. Biol. Chem. 286: 15132–7.
  304. Katayama, K., Melendez, J., Baumann, J.M., Leslie, J.R., Chauhan, B.K., Nemkul, N., et al. (2011). Loss of RhoA in neural progenitor cells causes the disruption of adherens junctions and hyperproliferation. Proc. Natl. Acad. Sci. U. S. A. 108: 7607–12.
  305. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat. Cell Biol. 13: 724–9.
  306. Raamsdonk, C.D. van, Griewank, K.G., Crosby, M.B., Garrido, M.C., Vemula, S., Wiesner, T., et al. (2010). Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363: 2191–9.
  307. Xiang, S.Y., Vanhoutte, D., Re, D.P. del, Purcell, N.H., Ling, H., Banerjee, I., et al. (2011). RhoA protects the mouse heart against ischemia/reperfusion injury. J. Clin. Invest. 121: 3269–76.
  308. Vega, F.M., Fruhwirth, G., Ng, T., and Ridley, A.J. (2011). RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. J. Cell Biol. 193: 655–65.
  309. An autoinhibitory helix in the C-terminal region of phospholipase C-β mediates Gαq activation. Nat. Struct. Mol. Biol. 18: 999–1005.
  310. Chauhan, B.K., Lou, M., Zheng, Y., and Lang, R.A. (2011). Balanced Rac1 and RhoA activities regulate cell shape and drive invagination morphogenesis in epithelia. Proc. Natl. Acad. Sci. U. S. A. 108: 18289–94.
  311. Dror, R.O., Arlow, D.H., Maragakis, P., Mildorf, T.J., Pan, A.C., Xu, H., et al. (2011a). Activation mechanism of the β2-adrenergic receptor. Proc. Natl. Acad. Sci. U. S. A. 108: 18684–9.
  312. Zhang, S., Zhou, X., Lang, R.A., and Guo, F. (2012). RhoA of the Rho family small GTPases is essential for B lymphocyte development. PLoS One 7: e33773.
  313. Orchard, R.C., and Alto, N.M. (2012). Mimicking GEFs: a common theme for bacterial pathogens. Cell. Microbiol. 14: 10–8.
  314. Kach, J., Sethakorn, N., and Dulin, N.O. (2012). A finer tuning of G-protein signaling through regulated control of RGS proteins. Am. J. Physiol. Heart Circ. Physiol. 303: H19–35.
  315. Chen, Z., Guo, L., Hadas, J., Gutowski, S., Sprang, S.R., and Sternweis, P.C. (2012). Activation of p115-RhoGEF requires direct association of Gα13 and the Dbl homology domain. J. Biol. Chem. 287: 25490–500.
  316. Rinne, A., Birk, A., and Bünemann, M. (2013). Voltage regulates adrenergic receptor function. Proc. Natl. Acad. Sci. U. S. A. 110: 1536–41.
  317. Takefuji, M., Krüger, M., Sivaraj, K.K., Kaibuchi, K., Offermanns, S., and Wettschureck, N. (2013). RhoGEF12 controls cardiac remodeling by integrating G protein-and integrin-dependent signaling cascades. J. Exp. Med. 210: 665–73.
  318. Medina, F., Carter, A.M., Dada, O., Gutowski, S., Hadas, J., Chen, Z., et al. (2013). Activated RhoA is a positive feedback regulator of the Lbc family of Rho guanine nucleotide exchange factor proteins. J. Biol. Chem. 288: 11325–33.
  319. Morgan-Fisher, M., Wewer, U.M., and Yoneda, A. (2013). Regulation of ROCK activity in cancer. J. Histochem. Cytochem. 61: 185–98.
  320. Mikelis, C.M., Palmby, T.R., Simaan, M., Li, W., Szabo, R., Lyons, R., et al. (2013). PDZ-RhoGEF and LARG are essential for embryonic development and provide a link between thrombin and LPA receptors and Rho activation. J. Biol. Chem. 288: 12232–43.
  321. Nance, M.R., Kreutz, B., Tesmer, V.M., Sterne-Marr, R., Kozasa, T., and Tesmer, J.J.G. (2013). Structural and functional analysis of the regulator of G protein signaling 2-gαq complex. Structure 21: 438–48.
  322. Shirley, M.D., Tang, H., Gallione, C.J., Baugher, J.D., Frelin, L.P., Cohen, B., et al. (2013). Sturge- Weber syndrome and port-wine stains caused by somatic mutation in GNAQ. N. Engl. J. Med. 368: 1971–9.
  323. Banerjee, J., and Wedegaertner, P.B. (2004). Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton. Mol. Biol. Cell 15: 1760–75.
  324. Chung, K.Y. (2013). Structural Aspects of GPCR-G Protein Coupling. Toxicol. Res. 29: 149–55.
  325. Ying, Z., Giachini, F.R.C., Tostes, R.C., and Webb, R.C. (2009). PYK2/PDZ-RhoGEF links Ca2+ signaling to RhoA. Arterioscler. Thromb. Vasc. Biol. 29: 1657–63.
  326. Sun, C., Liu, C., Li, S., Li, H., Wang, Y., Xie, Y., et al. (2014). Overexpression of GEFT, a Rho family guanine nucleotide exchange factor, predicts poor prognosis in patients with rhabdomyosarcoma. Int. J. Clin. Exp. Pathol. 7: 1606–15.
  327. Katritch, V., Fenalti, G., Abola, E.E., Roth, B.L., Cherezov, V., and Stevens, R.C. (2014). Allosteric sodium in class A GPCR signaling. Trends Biochem. Sci. 39: 233–44.
  328. Towbin, H., Staehelin, T., and Gordon, J. (1992). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24: 145–9.
  329. Boussif, O., Lezoualc'h, F., Zanta, M. a, Mergny, M.D., Scherman, D., Demeneix, B., et al. (1995). A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 92: 7297–301.
  330. Loirand, G., and Pacaud, P. (2014). Involvement of Rho GTPases and their regulators in the pathogenesis of hypertension. Small GTPases 5: e28846.
  331. Offermanns, S., Laugwitz, K.L., Spicher, K., and Schultz, G. (1994). G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc. Natl. Acad. Sci. U. S. A. 91: 504–8.
  332. Lang, P., Gesbert, F., Delespine-Carmagnat, M., Stancou, R., Pouchelet, M., and Bertoglio, J. (1996). Protein kinase A phosphorylation of RhoA mediates the morphological and functional effects of cyclic AMP in cytotoxic lymphocytes. EMBO J. 15: 510–9.
  333. Xu, N., Bradley, L., Ambdukar, I., and Gutkind, J.S. (1993). A mutant alpha subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells. Proc. Natl. Acad. Sci. U. S. A. 90: 6741–5.
  334. Strathmann, M.P., and Simon, M.I. (1991). G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits. Proc. Natl. Acad. Sci. U. S. A. 88: 5582–6.
  335. Wilkie, T.M., Scherle, P.A., Strathmann, M.P., Slepak, V.Z., and Simon, M.I. (1991). Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines. Proc. Natl. Acad. Sci. U. S. A. 88: 10049–53.
  336. Rossman, K.L., and Campbell, S.L. (2000). Bacterial expressed DH and DH/PH domains. Methods Enzymol. 325: 25–38.
  337. Suzuki, N., Nakamura, S., Mano, H., and Kozasa, T. (2003). Galpha 12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF. Proc. Natl. Acad. Sci. U. S. A. 100: 733– 8.
  338. Sanger, F., Nicklen, S., and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 74: 5463–7.
  339. Wall, M.A., Coleman, D.E., Lee, E., Iñiguez-Lluhi, J.A., Posner, B.A., Gilman, A.G., et al. (1995). The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell 83: 1047–58.
  340. Pedersen, E., and Brakebusch, C. (2012). Rho GTPase function in development: how in vivo models change our view. Exp. Cell Res. 318: 1779–87.
  341. Booden, M.A., Siderovski, D.P., and Der, C.J. (2002). Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA. Mol. Cell. Biol. 22: 4053–61.
  342. Xiang, S., Kim, E.Y., Connelly, J.J., Nassar, N., Kirsch, J., Winking, J., et al. (2006). The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor. J. Mol. Biol. 359: 35–46.
  343. Berman, D.M., Wilkie, T.M., and Gilman, A.G. (1996b). GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86: 445–52.
  344. Wieland, T., and Mittmann, C. (2003). Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system. Pharmacol. Ther. 97: 95–115.
  345. Riobo, N.A., and Manning, D.R. (2005). Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol. Sci. 26: 146–54.
  346. Worzfeld, T., Wettschureck, N., and Offermanns, S. (2008). G(12)/G(13)-mediated signalling in mammalian physiology and disease. Trends Pharmacol. Sci. 29: 582–9.
  347. Bellanger, J.-M., Estrach, S., Schmidt, S., Briançon-Marjollet, A., Zugasti, O., Fromont, S., et al. (2003). Different regulation of the Trio Dbl-Homology domains by their associated PH domains. Biol. Cell 95: 625–34.
  348. Karlsson, R., Pedersen, E.D., Wang, Z., and Brakebusch, C. (2009). Rho GTPase function in tumorigenesis. Biochim. Biophys. Acta 1796: 91–8.
  349. Pfreimer, M., Vatter, P., Langer, T., Wieland, T., Gierschik, P., and Moepps, B. (2011). LARG links histamine-H1-receptor-activated Gq to Rho-GTPase-dependent signaling pathways. Cell. Signal. 24: 652–63.
  350. Sahai, E., Ishizaki, T., Narumiya, S., and Treisman, R. (1999). Transformation mediated by RhoA requires activity of ROCK kinases. Curr. Biol. 9: 136–45.
  351. Chen, Z., Singer, W.D., Danesh, S.M., Sternweis, P.C., and Sprang, S.R. (2008). Recognition of the activated states of Galpha13 by the rgRGS domain of PDZRhoGEF. Structure 16: 1532–43.
  352. Mossessova, E., Corpina, R.A., and Goldberg, J. (2003). Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism. Mol. Cell 12: 1403–11.
  353. Kehrl, J.H., and Sinnarajah, S. (2002). RGS2: a multifunctional regulator of G-protein signaling. Int. J. Biochem. Cell Biol. 34: 432–8.
  354. Hart, M.J., Jiang, X., Kozasa, T., Roscoe, W., Singer, W.D., Gilman, A.G., et al. (1998). Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science 280: 2112–4.
  355. Schmidt, S., and Debant, A. (2014). Function and regulation of the Rho guanine nucleotide exchange factor Trio. Small GTPases 5: e29769.
  356. Kempf, A., Tews, B., Arzt, M.E., Weinmann, O., Obermair, F.J., Pernet, V., et al. (2014). The sphingolipid receptor S1PR2 is a receptor for Nogo-a repressing synaptic plasticity. PLoS Biol. 12: e1001763.
  357. Lessey-Morillon, E.C., Osborne, L.D., Monaghan-Benson, E., Guilluy, C., O'Brien, E.T., Superfine, R., et al. (2014). The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1- dependent mechanotransduction in endothelial cells to stimulate transendothelial migration. J. Immunol. 192: 3390–8.
  358. Chiu, W.-C., Juang, J.-M., Chang, S.-N., Wu, C.-K., Tsai, C.-T., Tseng, C., et al. (2012). Differential baseline expression and angiotensin II-stimulation of leukemia-associated RhoGEF in vascular smooth muscle cells of spontaneously hypertensive rats. Int. J. Nanomedicine 7: 5929–39.
  359. Siderovski, D.P., and Willard, F.S. (2005). The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits. Int. J. Biol. Sci. 1: 51–66.
  360. Ridley, A.J. (2011). Life at the leading edge. Cell 145: 1012–22.
  361. Ridley, A.J. (2006). Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 16: 522–9.
  362. Ridley, A.J. (2013). RhoA, RhoB and RhoC have different roles in cancer cell migration. J. Microsc. 251: 242–9.
  363. Vega, F.M., and Ridley, A.J. (2007). SnapShot: Rho family GTPases. Cell 129: 1430.


* Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten