A Novel PDZ Domain Containing Guanine Nucleotide Exchange Factor Links Heterotrimeric G Proteins to Rho
1999; Elsevier BV; Volume: 274; Issue: 9 Linguagem: Inglês
10.1074/jbc.274.9.5868
ISSN1083-351X
AutoresShigetomo Fukuhara, Cristina Murga, Muriel Zohar, Tadashi Igishi, J. Silvio Gutkind,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoSmall GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Gα12 family, Gα12 and Gα13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Gα12 and Gα13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Gα12 family which, in turn, activate an exchange factor acting on Rho. Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Gα12 family, Gα12 and Gα13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Gα12 and Gα13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Gα12 family which, in turn, activate an exchange factor acting on Rho. guanine nucleotide exchange factor Lsc homology domain mitogen-activated protein kinase c-Jun amino-terminal kinase serum response element serum response factor GTPase-activating protein Dbl homology domain pleckstrin-homology domain Src homology regulators of G protein signaling chloramphenicol acetyltransferase The Ras superfamily of GTPases comprises approximately 50 members that can be divided into several families, Ras, Rho, Sar, Rab, Arf, and Ran, based on their primary sequence as well as on their cellular activities (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2660) Google Scholar, 2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar, 3Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar). Whereas the Rab, Arf, and Sar groups participate in the transport of proteins and vesicles among different intracellular compartments, the Ran proteins function in nuclear transport, and Ras plays a central role in cell proliferation and differentiation (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar,4Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (764) Google Scholar). In the case of Ras, recent studies have revealed how it works at the molecular level. This small GTP-binding protein exchanges GDP for GTP upon activation of Ras-specific guanine nucleotide exchange factors (GEFs)1 (5Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (340) Google Scholar), and in the GTP-bound state, Ras physically associates with Raf (6Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (85) Google Scholar) thereby recruiting this serine threonine kinase to the plasma membrane. This causes the activation of Raf and initiates the activity of a sequential cascade of kinases leading to the stimulation of mitogen-activated protein kinases (MAPKs), p42MAPK and p44MAPK, also known as extracellular signal-regulated kinases-2 and -1, respectively, which, in turn, control the activity of nuclear transcription factors that are critical for cell growth (7Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar, 8Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3188) Google Scholar).The Rho family of GTP-binding proteins, which consists of the Rho, Rac, and Cdc42 subfamilies, has been shown to regulate several aspects of cytoskeleton function (4Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (764) Google Scholar). For example, Rho participates in the formation of actin stress fibers and mediates the redistribution of cytoskeletal components (4Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (764) Google Scholar, 9Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar). Rac is involved in the regulation of lamellipodia (pleat-shaped protrusions at the cell periphery) and membrane ruffling (10Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar); and Cdc42 regulates the formation of thin finger-like cytoplasmic extensions known as filopodia (11Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-92Crossref PubMed Scopus (690) Google Scholar). These proteins play an important role in the regulation of cell morphology, cell aggregation, tissue polarity, cytokinesis, cell motility, and also in smooth muscle contraction (12Hotchin N.A. Hall A. Cancer Surv. 1996; 27: 311-322PubMed Google Scholar, 13Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (328) Google Scholar, 14Narumiya S. J. Biochem. (Tokyo). 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar). However, recent evidence suggests that Rho proteins are also integral components of signaling pathways leading to transcriptional control. For example, Rac and Cdc42 regulate the activity of the c-Jun amino-terminal kinase (JNK) thereby affecting the transcriptional activity of c-Jun (15Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar), and Rho has been recently shown to induce expression from the serum responsive element (SRE) through the transcriptional activation of the serum response factor (SRF) (16Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar).The functional activity of small GTP-binding proteins of the Ras superfamily is tightly regulated in vivo by proteins that control their GDP/GTP bound state. Whereas GEFs promote the exchange of GDP for GTP thus activating Ras-like proteins (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar), GTPase-activating proteins increase the low intrinsic rate of GTP hydrolysis of small GTPases (reviewed in Ref. 3Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar) and are negative modulators. The mechanisms of activation of GEFs for Ras by cell surface receptors have been intensely investigated. For example, the biochemical route connecting the epidermal growth factor-receptor tyrosine kinase to Ras has been recently identified (5Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 17Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4222) Google Scholar), and includes the phosphorylation of the receptor itself on tyrosine residues, thus creating docking sites for adapter molecules such as Grb2 and Crk. These adapter molecules, in turn, help recruit to the membrane Sos, a Ras-GEF, thereby inducing the exchange of GDP for GTP on Ras. In contrast, the signaling pathway regulating the activity of GEFs for Rho family members is still poorly understood.Many GEFs for Rho, Rac, and Cdc42, including dbl,ost, lfc, lbc, vav,ect2, tim, and net (reviewed in Refs.18Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar and 19Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar) were discovered by virtue of their ability to transform NIH 3T3 cells when overexpressed or when activated by truncations. All these proteins share a 250-amino acid stretch of significant sequence similarity with Dbl, termed Dbl homology (DH) domain, adjacent to a pleckstrin-homology (PH) domain (18Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar, 19Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar). The DH domain was shown to be responsible for nucleotide exchange activity toward GTPases of the Rho family (20Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar, 21Hart M.J. Eva A. Evans T. Aaronson S.A. Cerione R.A. Nature. 1991; 354: 311-314Crossref PubMed Scopus (335) Google Scholar). Of interest, one of these GEFs for Rho-like proteins, the protein product of the vav proto-oncogene, proto-Vav, also exhibits a Src homology (SH) 2 domain flanked by two SH3 domains (22Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (302) Google Scholar, 23Bustelo X.R. Barbacid M. Science. 1992; 256: 1196-1199Crossref PubMed Scopus (168) Google Scholar), and we have recently shown that tyrosine phosphorylation of proto-Vav by hematopoietic specific tyrosine kinases can activate its GEF activity for Rac both in vitro and in vivo(24Teramoto H. Salem P. Robbins K.C. Bustelo X.R. Gutkind J.S. J. Biol. Chem. 1997; 272: 10751-10755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 25Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (678) Google Scholar). However, no other GEF for Rho-like proteins has been found to be regulated by tyrosine phosphorylation, nor to contain a phosphotyrosine-binding domain such as an SH2 or PTB domains. Furthermore, the vast majority of the known GEFs for small GTP-binding proteins of the Rho family are expressed in a very restricted tissue-specific manner (18Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar, 19Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2083) Google Scholar), and their mechanism of activation is still largely unknown.In this study, we explored the existence of novel GEFs for Rho-like proteins possessing structural domains that might suggest a role in signal transduction. Here, we report the identification of a novel, ubiquitously expressed GEF for Rho-like proteins containing a PDZ domain. This protein, termed PDZ-RhoGEF, was found to activate biochemical pathways specific for Rho, in a Rho-dependent manner. Interestingly, PDZ-RhoGEF was found to be closely related to the Drosophila DRhoGEF2, and recent genetic analysis suggests that DRhoGEF2 acts downstream of the concertinagene, a Drosophila Gα12 homolog. Here, we found that PDZ-RhoGEF physically associates in vivo with activated α subunits of heterotrimeric G proteins of the G12 family, Gα12 and Gα13. Association was found to occur through a novel structural domain, termed Lsc homology (LH) domain, located between the PDZ and the DH domain, and also present in the NH2-terminal, regulatory domain of the lscproto-oncogene product and its human homolog, p115-RhoGEF. This LH domain is distantly related to the G protein-binding region of a family of proteins known as regulators of G proteinsignaling (RGSs) (26Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (98) Google Scholar). Together, our present findings suggest the existence of a novel pathway by which the large family of G protein-coupled receptors communicates to Rho through the activation of G12/G13 and the physical association between Gα12 or Gα13 with LH containing GEFs for Rho, thereby stimulating Rho-dependent pathways.DISCUSSIONAlthough small GTP-binding proteins of the Rho family play a critical role in a variety of cellular functions, including the organization of the actin cytoskeleton and the activity of biochemical routes regulating gene expression and cell growth, how these GTPases are activated by cell surface receptors is still largely unknown. Thus, we investigated whether novel exchange factors for Rho-related GTPases might exist, exhibiting functional domains suggestive of a role in signal transduction. For this, we took advantage of the observation that all known GEFs for Rho proteins exhibit a DH domain, a 250-amino acid stretch of significant sequence similarity to Dbl, a transforming protein that was originally isolated from a diffuse B cell lymphoma (46Srivastava S.K. Wheelock R.H. Aaronson S.A. Eva A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8868-8872Crossref PubMed Scopus (33) Google Scholar). Using a consensus sequence for DH domains of Rho-exchange factors (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1752) Google Scholar) to search DNA data bases, we identified a yet uncharacterized molecule exhibiting a putative DH domain. Detailed analysis of its primary sequence revealed that this molecule contained additional areas of similarity with known modular domains, including a PH domain, a PDZ domain, a proline-rich region, and an area of homology to p115-RhoGEF, DRhoGEF2 and Lsc, that was not found in any other GEF described so far, and that was termed LH domain. As many of these protein regions are likely candidates to participate in signal transmission, we decided to investigate further this novel putative exchange factor.When the DH domain of this new molecule was compared with those of other DH containing proteins, we found that it was highly related to that of two Rho-specific GEFs, p115-RhoGEF (30Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and Lsc (31Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and to a recently described Drosophila Rho GEF, DRhoGEF2 (33Hacker U. Perrimon N. Genes Dev. 1998; 12: 274-284Crossref PubMed Scopus (250) Google Scholar). However, PDZ-RhoGEF was more distantly related to the DH domain of exchange factors activating Rac1 and/or Cdc42 such as Tiam1 (47Habets G.G. van der Kammen R.A. Stam J.C. Michiels F. Collard J.G. Oncogene. 1995; 10: 1371-1376PubMed Google Scholar), Vav (48Bustelo X.R. Crit. Rev. Oncog. 1996; 7: 65-88Crossref PubMed Scopus (64) Google Scholar), Ost (49Horii Y. Beeler J.F. Sakaguchi K. Tachibana M. Miki T. EMBO J. 1994; 13: 4776-4786Crossref PubMed Scopus (185) Google Scholar), and Dbl (46Srivastava S.K. Wheelock R.H. Aaronson S.A. Eva A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8868-8872Crossref PubMed Scopus (33) Google Scholar), and to those acting on Ras, including Sos (50Bonfini L. Karlovich C.A. Dasgupta C. Banerjee U. Science. 1992; 255: 603-606Crossref PubMed Scopus (198) Google Scholar) and Ras-GRF (51Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar). Consistent with this observation, expression of epitope-tagged forms of this novel GEF did not elevate the activity of co-transfected HA-tagged forms of MAPK and JNK, but potently stimulated the transcriptional activity of SRF, as judged by the use of a reporter plasmid under the control of a mutated SRE (16Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar). Furthermore, experiments with the use of botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, and an inactivation resistant form of Rho, RhoI41, indicated that the enhanced expression from the SRE-driven plasmid was dependent on the availability of a functional Rho. In addition, we have recently observed that expression of this molecule in Madin-Darby canine kidney cells potently induces the formation of actin stress fibers, to an extent comparable to that caused by expressing activated forms of Rho. 2M. Zohar and J. S. Gutkind, unpublished data. Thus, taken together, the primary sequence similarity with Rho GEFs and these biochemical profiles strongly suggest that this novel DH-containing molecule, that was designated PDZ-RhoGEF, can stimulate in vivoRho-specific pathways.In addition to the DH domain, a PH domain is present in all GEFs for Rho-related proteins described so far, located adjacent to the carboxyl end of the DH domain (18Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). PH domains are found in a wide variety of signaling molecules (52Musacchio A. Gibson T. Rice P. Thompson J. Saraste M. Trends Biochem. Sci. 1993; 18: 343-348Abstract Full Text PDF PubMed Scopus (485) Google Scholar) and have been implicated in both protein-protein and protein-lipid interactions (53Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Although a DH domain is necessary and sufficient for the exchange activity on Rho proteins in vitro (20Hart M.J. Eva A. Zangrilli D. Aaronson S.A. Evans T. Cerione R.A. Zheng Y. J. Biol. Chem. 1994; 269: 62-65Abstract Full Text PDF PubMed Google Scholar), the integrity of the PH domain is required for the activity in vivo of this family of exchange factors (31Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) most likely by facilitating membrane translocation (31Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Consistent with those observations, deletion of the DH domain, PH domain, or DH/PH domains abolished the biochemical activity of PDZ-RhoGEF. Similarly, PDZ-RhoGEF readily induced the appearance of foci of transformation when expressed in NIH 3T3 cells, and this activity required the presence of an intact DH and PH domain (data not shown), supporting a critical role for the DH and PH domains for the functional activity of PDZ-RhoGEF.The most striking feature of this novel exchange factor was the presence of a PDZ domain, a protein-protein interaction domain originally identified as an area of homology between the product of theDrosophila dlg tumor suppresor gene and the synaptic protein PSD-95 (54Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1001) Google Scholar), currently found in more than 60 distinct gene products (35Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). These domains can either bind specific recognition sequences such as the (S/T)XV motif at the carboxyl termini of certain proteins, or they can form hetero- or homodimers, suggesting that this modular protein-binding domain can participate in the formation of macromolecular complexes (35Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Thus, the PDZ domain was expected to contribute to PDZ-RhoGEF function. Surprisingly, however, when this domain was deleted, we did not observe any demonstrable effect on the ability of PDZ-RhoGEF to induce Rho-dependent pathways. Similarly, deletion of the PDZ domain was shown not to affect the biological activities of the Rac1 exchange factor Tiam1 (55Michiels F. Stam J.C. Hordijk P.L. van der Kammen R.A. Ruuls-Van Stalle L. Feltkamp C.A. Collard J.G. J. Cell Biol. 1997; 137: 387-398Crossref PubMed Scopus (210) Google Scholar). Although, based on these results, we cannot rule out the possibility that the PDZ domain facilitates the interaction of PDZ-RhoGEF and other exchange factors with yet to be identified signaling molecules (see below), we therefore decided to focus our efforts on other putative functional domains. One such interesting domain is a stretch of 197 amino acids located upstream from the DH domain, that was termed LH domain forLsc homology domain, and that is also found in Lsc, p115-RhoGEF, and DRhoGEF2 but not in any other GEF. This suggests that the LH domain represents a distinctive feature of this subgroup of exchange factors, which might bear functional relevance. Indeed, deletion of the LH domain enhanced the ability of PDZ-RhoGEF to stimulate SRE, to an extent comparable to that of a truncation mutant lacking the entire NH2-terminal regulatory region. Similarly, we found that NH2-terminal truncated or LH-deleted forms of PDZ-RhoGEF exhibit enhanced focus forming activity in NIH 3T3 cells when compared with the wild-type form. 3S. Fukuhara and J. S. Gutkind, unpublished results. Thus, PDZ-RhoGEF, like other GEFs, appears to be negatively regulated by inhibitory sequences within the non-catalytic region (18Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar), and this inhibitory function most likely resides in the LH domain.Because of the possibility that the LH domain might have a regulatory function, we searched data bases for molecules displaying sequences related to the LH domain. Surprisingly, we found that the catalytic region of a recently described GTPase-activating proteins for heterotrimeric G proteins, RGS14, exhibited a limited sequence similarity to the LH domain. RGSs were initially identified as homologues of Sst2 proteins, which are negative regulators of pheromone signaling in yeast (56Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). This protein family, currently with 19 members, shares a 120-amino acid core, termed GH domain, which is essential for accelerating the rate of GTP hydrolysis on Gα proteins (56Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). RGS14, together with RGS12, represent novel members of this family, characterized for being substantially larger (∼60 and ∼140 kDa, respectively) than the majority of the other known RGSs (∼25 kDa) (26Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (98) Google Scholar). This data suggested that the LH domain might confer to PDZ-RhoGEF the ability to interact with G protein α subunits. Indeed, we found that PDZ-RhoGEF could form stable complexes in vivospecifically with members of the G12 family of G protein α subunits, Gα12 and Gα13, and that this interaction required the presence of an intact LH domain. Taken together, we can conclude that PDZ-RhoGEF can interact physically with a particular subset of Gα proteins, thereby providing a direct link between hetereotrimeric G proteins and small GTP-binding proteins of the Rho family.These findings may have important implications regarding the function of G proteins of the G12 family, Gα12 and Gα13. These ubiquitously expressed G proteins were discovered by M. Simon's group upon amplification of mouse brain cDNA by polymerase chain reaction using degenerated oligonucleotides corresponding to regions highly conserved among G proteins (57Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (200) Google Scholar). Gα12 and Gα13 exhibit 67% amino acid identity with each other, but only 35–44% of amino acid identity to α subunits of other classes, such as Gq,Gi, and Gs (57Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Crossref PubMed Scopus (200) Google Scholar). Furthermore, whereas members of the Gαq family of G proteins activate phosphatidylinositol-specific phospholipases, the Gαsfamily stimulate adenylyl cyclases, and Gαi inhibits adenylyl cyclases and activate certain phosphodiesterases and promote the opening of several ion channels (58Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (522) Google Scholar, 59Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 60Birnbaumer L. Birnbaumer M. J. Recept. Signal Transduct. Res. 1995; 15: 213-252Crossref PubMed Scopus (94) Google Scholar), members of the Gα12 family of GTPases appear not to affect any of these second messenger-generating systems (60Birnbaumer L. Birnbaumer M. J. Recept. Signal Transduct. Res. 1995; 15: 213-252Crossref PubMed Scopus (94) Google Scholar). In this regard, the finding that concertina (cta), a Drosophilagene involved in embryogenesis (41Parks S. Wieschaus E. Cell. 1991; 64: 447-458Abstract Full Text PDF PubMed Scopus (244) Google Scholar), and that Gα12 and Gα13 can behave as remarkably potent oncogenes (29Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar, 42Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1994; 201: 603-609Crossref PubMed Scopus (86) Google Scholar), provided early indications that this G protein class might be involved in growth regulation, albeit through poorly defined mechanisms. Intense investigation in many laboratories has recently generated a wealth of information on how Gα12 and Gα13 may act (see Ref. 61Dhanasekaran N. Dermott J.M. Cell Signal. 1996; 8: 235-245Crossref PubMed Scopus (127) Google Scholar, for a recent review). In particular, one such study (62Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) demonstrated that activated Gα12 and Gα13, but not Gαi2 and Gαq or different combinations of β and γ subunits, mimicked the effect of activated RhoA on stress fibers and focal adhesion assembly, and we have recently provided evidence that Gα12 stimulates nuclear responses and cellular transformation through Rho (27Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar). Furthermore, several studies have now provided evidence that members of the G12family of G proteins link many G protein-coupled receptors, including receptors for lysophosphatidic acid, thrombin, thromboxane A2, and acetylcholine to the activation of Rho-dependent pathways and, in many cases, cell growth control (27Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar, 63Aragay A.M. Collins L.R. Post G.R. Watson A.J. Feramisco J.R. Brown J.H. Simon M.I. J. Biol. Chem. 1995; 270: 20073-20077Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 64Collins L.R. Ricketts W.A. Olefsky J.M. Brown J.H. Oncogene. 1997; 15: 595-600Crossref PubMed Scopus (29) Google Scholar, 65Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google
Referência(s)