RGS3 Is a GTPase-Activating Protein for Giαand Gqαand a Potent Inhibitor of Signaling by GTPase-Deficient Forms of Gqαand G11α

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Regulator of G-protein signaling 3 (RGS3) enhances the intrinsic rate at which G␣ i and G␣ q hydrolyze GTP to GDP, thereby limiting the duration in which GTP-G␣ i and GTP-G␣ q can activate effectors. Since GDP-G␣ subunits rapidly combine with free G␤␥ subunits to reform inactive heterotrimeric G-proteins, RGS3 and other RGS proteins may also reduce the amount of G␤␥ subunits available for effector interactions. Although RGS6, RGS7, and RGS11 bind G␤ 5 in the absence of a G␥ subunit, RGS proteins are not known to directly influence G␤␥ signaling. Here we show that RGS3 binds G␤ 1 ␥ 2 subunits and limits their ability to trigger the production of inositol phosphates and the activation of Akt and mitogen-activated protein kinase. Co-expression of RGS3 with G␤ 1 ␥ 2 inhibits G␤ 1 ␥ 2 -induced inositol phosphate production and Akt activation in COS-7 cells and mitogen-activated protein kinase activation in HEK 293 cells. The inhibition of G␤ 1 ␥ 2 signaling does not require an intact RGS domain but depends upon two regions in RGS3 located between acids 313 and 390 and between 391 and 458. Several other RGS proteins do not affect G␤ 1 ␥ 2 signaling in these assays. Consistent with the in vivo results, RGS3 inhibits G␤␥-mediated activation of phospholipase C␤ in vitro. Thus, RGS3 may limit G␤␥ signaling not only by virtue of its GTPase-activating protein activity for G␣ subunits, but also by directly interfering with the activation of effectors.Heterotrimeric G-proteins link seven transmembrane receptors to downstream signaling pathways. Receptor activation triggers the exchange of GTP for GDP by the G␣ subunit of the heterotrimeric G-protein, causing a conformational change in the G␣ subunit, which facilitates its dissociation from the receptor and G␤␥ subunits. GTP-bound G␣ and free G␤␥ subunits then bind and activate downstream effectors. However, G␣ subunits possess an intrinsic GTPase activity, which returns G␣ to its GDP bound state and thereby limits the duration of G␣ signaling. Because GDP-G␣ possesses a high affinity for G␤␥ subunits, the heterotrimeric G-protein rapidly reforms, effectively ending G␤␥-mediated signaling as well (reviewed in Refs. 1 and 2).Cells possess another important mechanism that curtails the duration in which a G␣ subunit remains GTP bound. Members of a family of proteins termed regulators of G-protein signaling (RGS) 1 dramatically accelerate the intrinsic rate that certain G␣ subunits hydrolyze GTP, a property that identifies them as GTPase-activating proteins (GAPs). The mammalian RGS proteins have a 120-amino acid region, RGS domain, or RGS box, which binds G␣ i and G␣ q subfamily members in a transition state of the GTP hydrolysis reaction, thereby lowering the free energy of the reaction (reviewed in Refs. 3 and 4). In addition, Rho guanine nucleotide exchange factors have a divergent RGS domain, which accelerates the intrinsic GTPase activity of G␣ 12 and G␣ 13 (5, 6). RGS proteins with GAP activity for members of the G␣ s subfamily remain enigmatic.The mammalian RGS proteins ca...

Section: Rgs3 Impairs the Generation Of Inositolmentioning

confidence: 99%

mentioning

confidence: 99%

Regulator of G-protein Signaling 3 (RGS3) Inhibits Gβ1γ2-induced Inositol Phosphate Production, Mitogen-activated Protein Kinase Activation, and Akt Activation

Shi

Lee

Sinnarajah

et al. 2001

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“…To further separate responses dependent on intracellular S1P from those that are receptor-mediated, we specifically blocked signaling of the heterotrimeric G proteins that S1PRs couple to and signal through. Regulators of G protein signaling, such as RGS3, function as GTPase-activating proteins for G␣ i and G␣ q subunits of heterotrimeric G proteins, resulting in their inactivation (44). It was previously shown that transfection of HEK 293 cells with RGS3 completely prevented signaling mediated by activation of S1PRs with exogenous S1P (45).…”

Section: Spp-1 Expression Does Not Abrogate Egf-induced Tyrosine Phosmentioning

confidence: 99%

Role of Sphingosine-1-phosphate Phosphatase 1 in Epidermal Growth Factor-induced Chemotaxis

Stunff

Mikami

Giussani

et al. 2004

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF-or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate G␣ i suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.The bioactive sphingolipid, sphingosine-1-phosphate (S1P), 1 is a ligand for a family of five specific G protein-coupled receptors (S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) (1) that are coupled to various G proteins. These receptors regulate diverse cellular processes and have been implicated in cytoskeletal rearrangement and regulation of cell movement (2, 3). The founding member of this receptor family, S1P 1 , couples to a G i pathway (4), and its gene disruption in mice demonstrated an essential function for vascular maturation (5). More recently, several studies have established that S1P 1 is also essential for lymphocyte recirculation and that it regulates egress from peripheral lymphoid organs (6, 7). In addition to its extracellular functions, S1P acts intracellularly to regulate cell survival (8), yet its direct targets have not yet been elucidated.As with other potent lipid mediators, levels of S1P are low and tightly regulated in a spatial-temporal manner. Sphingosine kinase (SphK) catalyzes the synthesis of S1P (2), which can be irreversibly degraded by S1P lyase (9 -11) and reversibly conver...

“…Whereas all GRKs have putative amino-terminal RH domains, G␣ interaction has only been observed for GRK2 and GRK3. Unlike other G␣ q -binding RGS proteins such as RGS2 (37), RGS3 (38), RGS4 (22), and RGS18 (39), the GRK2 RH domain does not stimulate the GTPase activity of G␣ q in a single turnover GAP assay and only weakly stimulates GTPase when G␣ q is reconstituted with M1 muscarinic receptor and assayed in an agonist-induced steady-state GTPase assay (34). Because the GRK2 RH domain inhibits G␣ q -stimulated PLC␤ activity both in vivo and in vitro yet lacks significant GAP activity in vitro, it has been postulated that GRK2 RGS acts by sequestration of G␣ q .…”

mentioning

confidence: 99%

G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

Sterne‐Marr

Tesmer

Day

et al. 2003

113

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G␣ q/ll and inhibiting G␣ q stimulation of the effector phospholipase C␤. The binding site for G␣ q/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the G␣ q/ll binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G␣ q/ll . These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G␣ q/ll binding defects impair recruitment to the plasma membrane by activated G␣ q and regulation of G␣ q -stimulated phospholipase C␤ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G␣ binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices ␣3 and ␣4, ␣5 and ␣6, and ␣7 and ␣8. The adenomatous polyposis coli (APC) binding site of axin involves residues on ␣ helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G␣ q/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its ␣5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.