The α-Helical Domain of Gαt Determines Specific Interaction with Regulator of G Protein Signaling 9

Abstract

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein ␣ subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the G i subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the ␣ subunit of transducin, G␣ t . Here we report another unique property of RGS9: high specificity for G␣ t . The core (RGS) domain of RGS9 (RGS9) stimulates G␣ t GTPase activity by 10-fold and G␣ i1 GTPase activity by only 2-fold at a concentration of 10 M. Using chimeric G␣ t /G␣ i1 subunits we demonstrated that the ␣-helical domain of G␣ t imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated G␣ subunits as measured by a change in fluorescence of a mutant G␣ t (Chi6b) selectively labeled at Cys-210. K d values for RGS9 complexes with G␣ t and G␣ i1 calculated from the direct binding and competition experiments were 185 nM and 2 M, respectively. The ␥ subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of P␥ to increase the affinity of RGS9 for G␣ t . A distinct, nonoverlapping pattern of RGS and P␥ interaction with G␣ t suggests a unique mechanism of effector-mediated GAP function of the RGS9.The ␣ subunits of heterotrimeric G proteins function as molecular switches that determine active and inactive states of signaling pathways. The crystal structures of G␣ t 1 and G␣ i1 in their activated, inactive, and transition state forms have revealed the nature of the molecular switches (switches I, II, and III), which are local conformational changes in the regions around the nucleotide binding pocket depending on whether GTP or GDP is bound (1-4). G protein ␣ subunits are activated by seven helical membrane receptors that catalyze the exchange of GDP for GTP by decreasing the affinity of GDP for the ␣ subunit. The lifetime of the active state of an ␣ subunit is defined by the rate of intrinsic GTPase activity that converts GTP to GDP. Therefore, termination of the signal response is dependent on G␣ GTPase activity. Purified G␣ subunits typically display slow (ϳ 4/min) GTP hydrolysis that often cannot account for the deactivation rates of G protein-controlled processes, for example phototransduction (5) and ion channel regulation (6).A large family of regulatory proteins that modulate the inactivation rate of G␣ subunits by accelerating their intrinsic GTPase activity has recently been identified (7,8). These proteins, known as regulators of G protein signaling (RGS), are encoded by at least 19 genes and have been identified in mammalian tissues based on homology to the diagnostic RGS core domain of ϳ 120 amino acid residues. It is not yet certain that all RGS proteins are GAPs (GTPase-accelerati...

Action of Pasteurella multocida Toxin Depends on the Helical Domain of Gαq

“…For example, it was shown by crystal structure analysis that the G o LOCO motif (also called G protein regulatory motif), which is found in some GTPase-activating proteins of the RGS (regulator of G protein signaling) family (e.g. RGS12 and RGS14), binds to the all-helical domain of G proteins (22,23). Also AGS proteins (activators of G protein signaling), which function as guanine nucleotide dissociation inhibitors, possess a G o LOCO domain (24).…”

A regulator of G protein signaling interaction surface linked to effector specificity

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The Cytoplasmic Membrane-proximal Domain of the HtrII Transducer Interacts with the E-F Loop of Photoactivated Natronomonas pharaonis Sensory Rhodopsin II

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