Characterization of RGS5 in regulation of G protein-coupled receptor signaling

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (DeltaN-RGS5). RGS5 bound to G alpha (i1), Ga alpha (i3), G alpha (0), G alpha (0) and G alpha (q) but not to G alpha (s) and G alpha (13) in the presence of GDP/AlF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha (i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT(1a) receptors (AT(1a)-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGSS was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGSS induced about 20-25 % reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT(1a) receptors showed that RGSS inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT(1a) receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT(1a)-293T cells, DeltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and DeltaN-RGS5 revealed that the N terminal (1-33) of RGSS plays a role in targeting this protein to membranes, and that the N terminal region of RGSS is not essential for exerting activities. (C) 2001 Elsevier Science Inc. All rights reserved.