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.

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.     

Socius, a novel binding partner of Galpha12/13, promotes the Galpha12-induced RhoA activation

Heterotrimeric G proteins act as a molecular switch that conveys signals from G protein-coupled receptors in the cell membrane to intracellular downstream effectors. The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions through their specific downstream effectors. On the other hand, regulatory systems of the activity of Galpha(12) and Galpha(13) have not been fully clear. Here, we show that Socius, a previously identified Rho family small GTPase Rnd1 interacting protein, binds directly to Galpha(12) and Galpha(13) through its NH(2)-terminal region. Socius increased the amounts of GTP-bound active form of Galpha(12) in 293T cells. Furthermore, Socius promotes the Galpha(12)-induced RhoA activation in 293T cells. These results demonstrate that Socius is a novel activator of the Galpha(12) family.     

Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.

The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

Physical and functional interactions of Galphaq with Rho and its exchange factors

Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.     

Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.     

Activation of RhoA by association of Galpha(13) with Dbl

RhoA is a small G protein that is implicated in the regulation of the actin cytoskeleton, gene expression, and cell cycle progression. It is activated by many agonists whose receptors are linked to heterotrimeric G proteins, but the mechanisms are incompletely understood. In this study, we show that the constitutively active alpha-subunit of the heterotrimeric G protein G(13) associated with the Rho family guanine nucleotide exchange factor Dbl in NIH 3T3 cells and that this resulted in activation of RhoA. This activation was not seen with wild-type Galpha(13) or if Dbl and active Galpha(13) were expressed separately and mixed. In contrast, coexpression of constitutively active Galpha(q) with Dbl did not lead to their association and caused a weak activation of RhoA that was no greater than that observed with wild-type Galpha(q). These findings illustrate that activated Galpha(13) and Dbl can associate in vivo and that this leads to Rho activation.     

Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides

Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.     

Gbetagamma signaling and Ca2+ mobilization co-operate synergistically in a Sos and Rac-dependent manner in the activation of JNK by Gq-coupled receptors

The mechanism by which G(q)-coupled receptors stimulate the c-Jun N-terminal kinase (JNK) activity has not been fully delineated. Here, we showed that stimulation of endogenous G(q)-coupled receptors in human hepatocarcinoma HepG2 cells resulted in an Src family kinase- and Ca(2+)-dependent JNK activation. Cos-7 cells transfected with HA-tagged JNK and various G(q)-coupled receptors also exhibited similar characteristics and provided further evidence for the involvement of Gbetagamma, an upstream intermediate for Src family kinases. The Ca(2+) and Gbetagamma signals operate in a high degree of independence. Transient expression of Gbetagamma subunits and elevation of cytoplasmic Ca(2+) level by thapsigargin activated JNK in a synergistic fashion. JNK activities triggered by G(q)-coupled receptors, Gbetagamma and thapsigargin were all suppressed by dominant negative (DN) mutants of Son of sevenless (Sos) and Rac. We propose that the co-operative effect between Gbetagamma-mediated signaling and the increased intracellular Ca(2+) level represents a robust mechanism for the stimulation of JNK by G(q)-coupled receptors.     

Prostaglandin receptors: advances in the study of EP3 receptor signaling

Prostaglandin (PG) E(2) produces a broad range of physiological and pharmacological actions in diverse tissues through specific receptors on plasma membranes for maintenance of local homeostasis in the body. PGE receptors are divided into four subtypes, EP1, EP2, EP3, and EP4, which have been identified and cloned. These EP receptors are members of the G-protein coupled receptor family. Among these subtypes, the EP3 receptor is unique in its ability to couple to multiple G proteins. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via G(i) activation, and in Ca(2+)-mobilization through G(beta)(gamma) from G(i). Along with G(i) activation, the EP3 receptor can stimulate cAMP production via G(s) activation. Recent evidence indicates that the EP3 receptor can augment G(s)-coupled receptor-stimulated adenylyl cyclase activity, and can also be coupled to the G(13) protein, resulting in activation of the small G protein Rho followed by morphological changes in neuronal cells. This article focuses on recent studies on the novel pathways of EP3 receptor signaling.     

Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection

Sphingosine-1-phosphate (S1P) induces an initial Ca(2+)-dependent contraction followed by a sustained Ca(2+)-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P(1) and S1P(2) receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P(1) or S1P(2) receptors. Phospholipase C (PLC)-beta activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P(1) receptors abolished S1P-stimulated activation of Galpha(i3) and partially inhibited activation of Galpha(i1), whereas silencing of S1P(2) receptors abolished activation of Galpha(q), Galpha(13), and Galpha(i2) and partially inhibited activation of Galpha(i1). Silencing of S1P(2) but not S1P(1) receptors suppressed S1P-stimulated PLC-beta and Rho kinase activities, implying that both signaling pathways were mediated by S1P(2) receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P(1) receptor agonist SEW2871 did not stimulate PLC-beta or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P(1) receptors so as to enable chemical inactivation of S1P(2) receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P(2) receptors. Thus S1P(1) and S1P(2) receptors are coupled to distinct complements of G proteins. Only S1P(2) receptors activate PLC-beta and Rho kinase and mediate initial and sustained contraction.     

Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain

The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).     

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to 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 Galpha12 family, Galpha12 and Galpha13, 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 Galpha12 and Galpha13. 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 Galpha12 family which, in turn, activate an exchange factor acting on Rho.