Characterization and Comparison of RGS2 and RGS4 as GTPase-Activating Proteins for m2 Muscarinic Receptor-Stimulated G_i

Abstract

RGS2 and RGS4 were studied for their effects as GTPase activating proteins (GAPs) on receptor-activated G i in a novel steady-state assay using membranes from Sf9 cells quadruply infected with baculoviruses encoding the m2 muscarinic receptor, G ␣i2 , G ␤1 , and G ␥2 . In the presence of the muscarinic agonist carbachol, regulator of G protein signaling 2 (RGS2) and RGS4 each produced up to a 10-fold increase in agonistdependent GTPase activity. The observed K m for GTP in this system was increased in the presence of RGS4. NaCl and KCl inhibited the GAP activities of both RGS2 and RGS4, although they had no effect on GTPase activity in the absence of RGS proteins. MgCl 2 had a complex effect on GTPase activity, with optimal RGS2 and RGS4 GAP activities occurring, respectively, at high micromolar and low millimolar concentrations of free Mg 2ϩ . The concentration dependence of RGS GAP activity was assessed, and RGS4 was found to be more potent than RGS2 by up to an order of magnitude. This direct observation confirms a similar difference in potency found when these two RGS proteins were compared for their ability to inhibit signaling downstream of G i (Heximer et al., 1999). RGS2 yielded Hill coefficients greater than 2.0, suggesting that it may bind in a positively cooperative manner to oligomeric structures containing more than one G protein. Furthermore, RGS4 yielded a bell-shaped dose-dependence under low magnesium (0.5 mM) conditions, which is also consistent with the idea of RGS cooperativity.Heterotrimeric G proteins are activated by seven transmembrane spanning receptors, which promote the dissociation of GDP and thereby allow intracellular GTP to bind. G proteins are deactivated by the hydrolysis of GTP to GDP and inorganic phosphate, and the rate of that reaction can be increased up to 2 orders of magnitude by GTPase activating proteins (GAPs) (Wilkie and Ross, 2000). Some effectors act as GAPs toward the G proteins that activate them; for example, phospholipase C␤ (Chidiac and Ross, 1999) and p115Rho-GEF (Kozasa et al., 1998), respectively, increase the rates of GTP hydrolysis by G q and G 12 . However, most GAPs for heterotrimeric G proteins have no known effector function and belong to the regulators of G protein signaling (RGS) protein family. Most RGS proteins are GAPs for members of the G i family (Hepler, 1999), and a subset of those also act as GAPs for G q (Ingi et al., 1998;Scheschonka et al., 2000).The most direct way to measure RGS GAP activities is via increases in the rates at which their targeted G proteins hydrolyze GTP. With isolated G ␣ subunits, GTP turnover is limited by the dissociation of GDP, and therefore the rate of GTP hydrolysis needs to be measured under pre-steady-state conditions using G protein prebound to GTP (Wang et al., 1998a). Alternatively, GDP dissociation is not rate-limiting in the presence of activated receptors, and RGS GAP effects therefore can also be observed under steady-state conditions with receptor-coupled G proteins in the presence of agonist (Wa...

RGS2 Binds Directly and Selectively to the M1 Muscarinic Acetylcholine Receptor Third Intracellular Loop to Modulate Gq/11α Signaling

“…Thus, it seems reasonable that RGS2 would not be recruited by receptors that activate G i/o pathways. However, RGS4 is an efficient GAP in vitro for both G q ␣ and G i ␣ (6) and has been shown to modulate M2-mediated signaling in many cases (25,34,36), yet we did not detect RGS4 binding to the M2i3 or M4i3 loop. One explanation for this discrepancy is that the relative expression levels of receptor, RGS and G␣␤␥ in these assay systems may allow the RGS-G␣ coupling to predominate such that RGS can inhibit G␣ despite a lack of RGS-M2 interaction.…”

“…The baculovirus encoding M1-G 11 ␣ fusion protein was kindly provided by Dr. Tatsuya Haga (Gakushuin University, Tokyo, Japan) and baculoviruses encoding G 1 ␤ and G 2 ␥ were a gift from Dr. Terry Hebert (Montreal Heart Institute, Montreal, Quebec, Canada) (25). Following a 48-h infection at 27°C, membranes were prepared essentially as described (25).…”

“…Steady state GTPase assays were adapted from a previously described method (25). Briefly, membranes (6 g of protein/assay) from Sf9 cells expressing M1 mAChR-G 11 ␣ fusion protein plus G 1 ␤ and G 2 ␥ were preincubated with drugs and purified RGS proteins on ice for 1 h, at which time nucleotides were added to yield 40-l reaction mixtures containing 20 mM HEPES, pH 7.5, 1 mM EDTA, 2 mM MgCl 2 , 10 mM NaCl, 1 mM DTT, 0.1 mM PMSF, 10 g/ml leupeptin, 1 g/ml aprotinin, 0.3 M GTP, 1 mM ATP, 0.1 mM App(NH)p, and 1 ϫ 10 6 cpm/assay [ 32 P]GTP.…”

“…The mix was centrifuged (2000 ϫ g) and [ 32 P] i in the resulting supernatant determined by liquid scintillation counting. The nonspecific signal was estimated as described (25). Membrane GTPase signal was estimated by adding 1 mM unlabeled GTP to the above assay mix and this value subtracted off the total cpm.…”

Up-regulation of Endogenous RGS2 Mediates Cross-desensitization between Gs and Gq Signaling in Osteoblasts

“…RGS2 is unique in preferring G q over G i , with a relatively low affinity for G i as compared with other RGS proteins (17).…”