Differential Contribution of GTPase Activation and Effector Antagonism to the Inhibitory Effect of RGS Proteins on Gq-mediated Signaling in Vivo

Abstract

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for ␣ subunits of heterotrimeric G proteins (G␣), thereby accelerating G protein inactivation. RGS proteins can also block G␣-mediated signal production by competing with downstream effectors for G␣ binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on G␣ q -mediated phospholipase C␤ (PLC␤) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M 3 receptors, which couple to endogenous G q protein and mediate a stimulatory effect of carbachol on PLC␤, or constitutively active G␣ q * , which is inert to GTP hydrolysis and activates PLC␤ independent of receptor activation. In M 3 -expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, G␣ q * -induced PLC␤ activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M 3 , G␣ q / G␣ q * , PLC␤, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.Many extracellular stimuli elicit intracellular responses by activating seven-transmembrane receptors that are coupled to heterotrimeric G proteins comprising ␣ and ␤␥ subunits (1, 2). The duration of the response of a cell to external signals is largely determined by the activation/inactivation cycle of G proteins. Activated receptors trigger GTP-for-GDP exchange on G␣ subunits, thus dissociating G␣ from G␤ ␥ and subsequent activation of downstream effectors (such as enzymes and ion channels). The duration of G protein activation is limited by GTPase activity intrinsic to G␣ subunits that catalyzes the conversion of active GTP-bound G␣ into inactive GDP-bound G␣, which in turn can reassociate with G␤␥ and receptors.RGS proteins belong to a family of more than 20 proteins with a conserved RGS core domain of ϳ120 amino acids that is necessary and sufficient for binding to G␣ subunits (3). They are divided into several subfamilies based on their structural similarities, gene organization, and function. RGS proteins act as regulators of G protein signaling by limiting the signals generated by G protein-coupled receptors. They markedly increase the rate at which G␣ subunits hydrolyze GTP to GDP, a property that defines them as GTPase-activating proteins (or GAPs) 1 (4). Hastening of G␣ inactivation facilitates the reass...

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

“…5C). These findings are consistent with a recent report demonstrating that RGS2 blocks muscarinic receptor mediated inositol lipid signaling by both GTPase stimulation and effector antagonism, whereas RGS16 inhibitory effects were due primarily to its GTPase activity (30). Together, these results indicate that the selectivity of RGS2 toward a particular G protein signaling pathway is dictated by its interactions with the receptor, and that, once bound to the receptor, RGS2 relies upon its effector antagonist activity to block receptor and G q/11 ␣ coupling to PLC␤.…”

“…RGS proteins can negatively regulate G␣ signaling by serving both as GAPs and/or effector antagonists (6,7), and a recent report shows differential contributions of GTPase activation and effector antagonism to explain the inhibitory actions of RGS2 and RGS16 on receptor and G q/11 ␣ signaling (30). Specifically, RGS2 appeared to inhibit the stimulation of phospholipase C␤ activity by acting both as a GAP and as an effector antagonist for G q ␣, whereas RGS16 appeared to act as a GAP but not as an effector antagonist.…”

Differential effects of RGS proteins on Gαq and Gα11 activity

“…In contrast, and in accordance with results seen in COS-7 cells [48], RGS2 and RGS3 reduced signalling from both Gα q QL and Gα 11…”

“…This suggests that they do not rely entirely on GAP activity and other mechanisms, such as effector antagonism, are sufficient to mediate their inhibitory effects. This difference in the mode of action of RGS2 and RGS3 to other members of the R4 subfamily has also been observed in mammalian cells [48,49].…”

The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

“…The mechanism by which RGS proteins inhibit signaling pathways is termed effector antagonism and is found in different situations. In this respect, direct binding of RGS2, RGS3, RGS4, and GAIP to phospholipase Cb1 interferes with activated Gaq subunits (Hepler et al, 1997;Anger et al, 2004). Also, RGS2 and RGS 3 interact with adenylyl cyclases reducing cAMP production (Chatterjee et al, 1997;Sinnarajah et al, 2001).…”