Abstract
The R7 subfamily of RGS proteins critically regulates neuronal G protein-signaling pathways that are essential for vision, nociception, motor coordination, and reward processing. A member of the R7 RGS family, RGS11, is a GTPase-accelerating protein specifically expressed in retinal ON-bipolar cells where it forms complexes with the atypical G protein beta subunit, Gbeta(5), and transmembrane protein R9AP. Association with R9AP has been shown to be critical for the proteolytic stability of the complex in the retina. In this study we report that R9AP can in addition stimulate the GTPase-accelerating protein activity of the RGS11 x Gbeta(5) complex at Galpha(o). Single turnover GTPase assays reveal that R9AP co-localizes RGS11 x Gbeta(5) and Galpha(o) on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11 x Gbeta(5). Reconstitution of mGluR6-Galpha(o) signaling in Xenopus oocytes indicates that RGS11 x Gbeta(5)-mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-Galpha(o) signaling by the RGS11 x Gbeta(5) x R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes.
Highlights
Regulators of G protein signaling (RGS)3 are ubiquitous signaling molecules that critically shape cellular responses mediated by G protein-coupled receptor (GPCR) pathways [1]
Because urea treatment removes the G protein transducin (G␣t) and associated GTPase-accelerating protein (GAP) but leaves endogenous R9AP and GPCR rhodopsin intact [20], we used this preparation as an initial reagent to study the effects of R9AP on the RGS111⁄7G5 complex
Previous studies have shown that anchoring purified RGS9-11⁄7G5 by native R9AP contained within the uROS preparation markedly potentiates its GAP activity toward exogenously added G proteins (G␣)t (19 –21)
Summary
Antibodies—The generation of rabbit anti-R9AP (against amino-acids 144 –223) [28], sheep anti-RGS11 CT [29], and rabbit anti-RGS11 CT [30] antibodies has been described previously. For the purification of the RGS111⁄7G5S complex, Sf9 cells from 1 liter of Sf9 culture were harvested 48 h after co-infection with amplified recombinant baculoviruses encoding His-tagged RGS11 and G5S, resuspended in 40 ml of lysis buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 10 mM imidazole, 5% glycerol, 10 mM -mercaptoethanol), and lysed by sonication. The final concentrations in the binding reactions were 50 nM RGS9-11⁄7G5L complexes, 27 nM RGS111⁄7G5S, 1.0 M GST, 1.0 M GSTR9AP⌬TM, 0.5 mg/ml Sf9 membranes, 0.5 mg/ml R9AP membranes, and uROS and V8-uROS containing 20 M rhodopsin. To reveal inward currents through the inwardly rectifying GIRK channels, recordings were performed in oocyte saline buffer with elevated (16 mM) KCl concentrations (other components were 82 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM HEPES, pH 7.5). Two-tailed p value Ͻ0.01 is defined as significantly different
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