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 ␤ subunit, G␤ 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 RGS11⅐G␤ 5 complex at G␣ o . Single turnover GTPase assays reveal that R9AP co-localizes RGS11⅐G␤ 5 and G␣ o on the membrane and allosterically potentiates the GTPase-accelerating function of RGS11⅐G␤ 5 . Reconstitution of mGluR6-G␣ o signaling in Xenopus oocytes indicates that RGS11⅐G␤ 5 -mediated GTPase acceleration in this system requires co-expression of R9AP. The results provide new insight into the regulation of mGluR6-G␣ o signaling by the RGS11⅐G␤ 5 ⅐R9AP complex and establish R9AP as a general GTPase-accelerating protein activity regulator of R7 RGS complexes. 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). Most RGS proteins act by stimulating the rate of the GTP hydrolysis of the ␣ subunits of G proteins (G␣), thus speeding up their inactivation and limiting the duration of GPCR signaling (2). R7 subfamily of RGS proteins plays a central role in regulating fundamental neuronal functions including vision, nociception, reward processing, and motor control (3-6). The subfamily consists of four homologous proteins, RGS6, RGS7, RGS9, and RGS11, that are enriched in the nervous system and share a common multidomain architecture that, in addition to the catalytic RGS domain, includes an N-terminal DEP/DHEX (Dishevelled, EGL-10, Pleckstrin/DEP helical extension) module and a GGL (G protein ␥ subunit-like) domain (7).In vivo, R7 RGS proteins are found in complexes with two types of proteins that are becoming increasingly accepted as their subunits. The GGL forms a complex with the atypical G protein ␤ subunit, G␤ 5 (8). The DEP/DHEX module binds to either R7BP (R7 family binding protein) or R9AP (RGS9 anchor protein) (9, 10), a two-member family of small SNARE (soluble N-ethylmaleimide factor attachment protein receptor)-like proteins. Association with both G␤ 5 (11, 12) and R7BP/R9AP (13-15) protects complexes from proteolytic degradation and, thus, plays an important role in regulating the expression levels of the R7 RGS proteins. In addition, membrane proteins R9AP and R7BP can localize R7 RGS proteins to cell membranes in transfected cells and native neurons (16 -18). Finally, R9AP has been shown to enhance the GTPase-accelerating protein (GAP) activity of RGS9-1 toward transducin, suggesting that membrane anchors also play a role in regulation of R7 RGS activity (10, ...