Specific Involvement of G Proteins in Regulation of Serum Response Factor-mediated Gene Transcription by Different Receptors

Abstract

Regulation of serum response factor (SRF)-mediated gene transcription by G protein subunits and G proteincoupled receptors was investigated in transfected NIH3T3 cells and in a cell line that was derived from mice lacking G␣ q and G␣ 11 . We found that the constitutively active forms of the ␣ subunits of the G q and G 12 class of G proteins, including G␣ q , G␣ 11 , G␣ 14 , G␣ 16 , G␣ 12 , and G␣ 13 , can activate SRF in NIH3T3 cells. We also found that the type 1 muscarinic receptor (m1R) and ␣ 1 -adrenergic receptor (AR)-mediated SRF activation is exclusively dependent on G␣ q/11 , while the receptors for thrombin, lysophosphatidic acid (LPA), thromboxane A2, and endothelin can activate SRF in the absence of G␣ q/11 . Moreover, RGS12 but not RGS2, RGS4, or Axin was able to inhibit G␣ 12 and G␣ 13 -mediated SRF activation. And RGS12, but not other RGS proteins, blocked thrombin-and LPA-mediated SRF activation in the G␣ q/11 -deficient cells. Therefore, the thrombin, LPA, thromboxane A2, and endothelin receptors may be able to couple to G␣ 12/13 . On the contrary, receptors including ␤ 2 -and ␣ 2 -ARs, m2R, the dopamine receptors type 1 and 2, angiotensin receptors types 1 and 2, and interleukin-8 receptor could not activate SRF in the presence or absence of G␣ q/11 , suggesting that these receptors cannot couple to endogenous G proteins of the G 12 or G q classes.Hormones, neurotransmitters, and many other biologically active molecules, such as lysophosphatidic acid (LPA), 1 thrombin, catecholamines, endothelin, etc., transduce their signals through heterotrimeric G proteins (1, 2). Molecular cloning has revealed at least four classes of G protein ␣ subunits: G␣ s , G␣ i , G␣ q , and G␣ 12 (3). The G␣ s subunits and G␣ i subunits regulate adenylyl cyclase activities, while the G␣ q subunits regulate phospholipase C activities. However, the function of the G␣ 12 class of G proteins, which includes G␣ 12 and G␣ 13 , remains to be elucidated. Activated forms of G␣ 12 and G␣ 13 , when transfected into fibroblast cells, were shown to induce transformation phenotypes (4 -6), suggesting that this class of G proteins may be involved in cell growth regulation. Moreover, G␣ 12 and G␣ 13 were shown to induce formation of stress fibers in fibroblast cells through small G protein RhoA (7). This observation was supported by the report that G␣ 12 activated serum response factor (SRF) through RhoA (8). The in vivo function of G␣ 13 was also investigated using the gene-targeting technique in mice. Mice lacking G␣ 13 are embryonic lethal apparently due to the failure to develop vasculature structures, indicating that G␣ 13 may be involved in the function of endothelial cells (9). In the same study, thrombin-mediated chemotaxis of fibroblasts lacking G␣ 13 was blocked, indicating that the thrombin receptor couples to G␣ 13 . This is consistent with the observation that thrombin as well as a thromboxane A2 receptor agonist could stimulate the binding of a photo-affinity GTP analog to G␣ 13 (10). However, there were contradictory r...

RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho?

“…This research was made possible by the development of embryonic ®broblast cell lines derived from knocked out animals lacking each class of G protein a subunits (see review by O ermanns, 1999). For example, m1-muscarinic and a 1 -adrenergic receptors failed to induce SRF-dependent gene expression in cells lacking G q and G 11 , and this response was restored by the expression of wild type G q (Mao et al, 1998b). Similarly, the stimulation of stress ®ber formation by m1-muscarinic and metabotropic glutamate-1a receptors was not observed in G q -and G 11 -de®cient ®broblasts, whereas LPA, bradykinin B2 and serotonin 2C receptors failed to induce stress ®ber formation in G 13 -de®cient ®broblasts (Gohla et al, 1999).…”

“…Interestingly, SRE activation in response to thrombin and LPA was attenuated by the expression of dominant negative molecules for RGS-RhoGEFs, suggesting that these RhoGEFs may mediate some of the biological responses elicited by these and other G 12 -coupled receptors (Fukuhara et al, 1999;Mao et al, 1998b). In this regard, p115-RhoGEF, PDZ-RhoGEF and LARG are themselves focus-forming when overexpressed in NIH3T3 cells (Figure 2), and a recent report indicates that the transformation of these cells induced by G2A, an oncogenic GPCR, can be suppressed by co-expression of the RGS domain of Lsc, the murine homolog of p115 RhoGEF (Zohn et al, 2000).…”

Functional consequences of Gα13 mutations that disrupt interaction with p115RhoGEF

“…Activated G proteins a q/11 , a 12 , and a 13 have been shown to stimulate SRE-mediated gene transcription through RhoA-dependent activation of the serum response factor (SRF) (Mao et al, 1998b). The ability of constitutively active a 13 mutants to activate Rhodependent signaling was determined using the SREmediated luciferase gene transcription assay, where HEK293 cells were transiently transfected with the constitutively active a 13 or mutants and a reporter plasmid that expresses the luciferase gene under the control of SRE.…”

“…a 12 and a 13 can activate the Na þ /H þ exchanger (Voyno-Yasenetskaya et al, 1994a), the c-Jun NH2-terminal kinase (Prasad et al, 1995), extracellular-signal-regulated kinases (ERK) (VoynoYasenetskaya et al, 1996), tyrosine kinases (Mao et al, 1998a;Shi et al, 2000), serum response element (SRE)-mediated gene transcription (Mao et al, 1998b), stress fiber and focal adhesion formation (Buhl et al, 1995;Gohla et al, 1999), neurite retraction (Katoh et al, 1998;Kranenburg et al, 1999), and apoptosis (Althoefer et al, 1997;Berestetskaya et al, 1998). Several of these pathways are mediated by the monomeric Rho GTPase, a member of the Ras superfamily of GTPases.…”

Signaling mechanisms for regulation of chemotaxis

“…We recently added PAK1 to the list by demonstrating the interaction between Gβγ and PAK1, which is involved in chemoattractant-mediated activation of Cdc42 and PAK1 [10]. Chemoattractants can also couple to G16 [11] and probably G12/13 [12], which have been shown to activate small GTPase RhoA via a guanine nucleotide exchange factor (GEF) 115 [13][14][15]. In the past ten years, many of the chemoattractant signaling pathways were comprehensively characterizing using a combination of biochemical, molecular and cell biological, and transgenic approaches.…”