RGS6, RGS7, RGS9, and RGS11 Stimulate GTPase Activity of Gi Family G-proteins with Differential Selectivity and Maximal Activity

Abstract

Regulator of G-protein signaling (RGS) proteins areGTPase activating proteins (GAPs) of heterotrimeric Gproteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein ␣-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a G␥-like (GGL) domain that mediates dimeric interaction with G␤ 5 . G␤ 5 /R7 dimers stimulated steady state GTPase activity of G␣-subunits of the G i family, but not of G␣ q or G␣ 11 , when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the G␤ 5 /R7 proteins revealed differences in potencies and efficacies toward G␣-subunits of the G i family. Although all four G␤ 5 /R7 proteins exhibited similar potencies toward G␣ o , G␤ 5 /RGS9 and G␤ 5 /RGS11 were more potent GAPs of G␣ i1 , G␣ i2 , and G␣ i3 than were G␤ 5 /RGS6 and G␤ 5 /RGS7. The maximal GAP activity exhibited by G␤ 5 /RGS11 was 2-to 4-fold higher than that of G␤ 5 /RGS7 and G␤ 5 /RGS9, with G␤ 5 /RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious G␤ 5 /RGS7 and G␤ 5 /RGS9 inhibited G␤ 5 / RGS11-stimulated GTPase activity of G␣ o . Therefore, R7 family RGS proteins are G i family-selective GAPs with potentially important differences in activities.Heterotrimeric guanine nucleotide-binding proteins (G-proteins) act as molecular switches in multiple GPCR 1 signaling pathways via regulation of specific effector molecules such as phospholipase C and adenylyl cyclase. The biological activity of G-protein ␣-subunits is determined by the identity of the bound guanine nucleotide (GTP or GDP), which in turn is governed by the relative rates of guanine nucleotide exchange and hydrolysis of GTP by the intrinsic GTPase activity of G␣-subunits.These opposing reactions are stimulated by agonist-occupied GPCR and GTPase-activating proteins (GAPs).Although some effector proteins exhibit GAP activity (1-3), the primary regulators of GTPase activity of G␣-subunits are a diverse family of regulator of G-protein signaling (RGS) proteins that act as GAPs for heterotrimeric G-protein ␣-subunits (4 -7). This family is defined by a conserved RGS domain, which markedly increases the rate of GTP hydrolysis by G␣-subunits and terminates effector activation by both G␣-and G␤␥-subunits. More than 30 RGS proteins have been identified and organized into subfamilies based on sequence similarity and domain structure. These families vary in size and complexity, from the R4 family whose structure is largely limited to the RGS domain to the R12 and RhoGEF families whose members are large multifunctional proteins containing several domains (for reviews see Refs. 8 -10).The R7 RGS family is a unique multidomain family, which consists of RGS proteins containing a novel G-␥-like (GGL) domain homologous to the G␥-subunit of heterotrimeric Gproteins (11). This domain, found in the mammalian proteins RGS6, -7, -9, and -11 and the Caenorhabdi...

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

“…First, we show that in the cellular environment RGS9-2/G␤5 and RGS7/G␤5 display a strong preference for G␣o over G␣i. These results are in overall agreement with published in vitro data (26,38). The RGS7 complex showed the greatest selectivity for G␣o and was completely unable to inactivate G␣i in the absence of R7BP.…”

“…Finally, we report that in living cells, RGS7 shows a much more potent activity relative to the RGS9-2 complex. In contrast, previous in vitro observations with purified proteins reported approximately equal catalytic activities of these two proteins (26). This illustrates the importance of considering a native, physiologically relevant for MOR-Gi-RGS9-2-R7BP, 3.7 ϫ 10 Ϫ4 Ϯ 3.7 ϫ 10 Ϫ5 for MOR-Gi-RGS9-2, 3.0 ϫ 10 Ϫ4 Ϯ 5.9 ϫ 10 Ϫ5 for MOR-Gi-RGS7-R7BP, and 1.2 ϫ 10 Ϫ5 Ϯ 6.8 ϫ 10…”

“…Regulate the G␣i/o Subfamily in the absence or presence of R7BP-Members of the R7 RGS family have been shown previously to be selective GAPs for the G␣i/o proteins in the in vitro biochemical assays (26). However, their selectivity was never examined in the physiological context of living cells.…”

Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction

“…Similarly, GRK2 GAP activity is dependent on the presence of a G protein-coupled receptor (26). Indeed, the recent comprehensive study by Hooks et al (27) shows that in the presence of receptors incorporated into lipid vesicles, RGS7 and other G protein ␥-like RGS proteins have activity to G␣ i subunits as well as G␣ o . Thus, the lack of RGS7 GAP activity toward G␣ q could be due to the lack of effector, receptor, or other necessary components that are present in a cell.…”

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

“…RGS9-2 as well as highly homologous RGS6 and RGS7 (RGS6/7) are among the most abundant RGSs in the CNS (Gold et al, 1997;Terzi et al, 2009). They belong to the R7 subfamily that additionally contains RGS9-1 and RGS11 proteins prevalent in the retina (Hooks et al, 2003;Anderson et al, 2009). Members of the R7 family share several domains and form constitutive complexes with the type 5 G protein b subunit (Gb5) (Sondek and Siderovski, 2001;Cheever et al, 2008).…”