Anti-RGS8 (full length) polyclonal antibody

Melanin-concentrating hormone receptor I (MCHR1) is a G protein coupled receptor (GPCR) highly expressed in the central nervous system. MCHR1 mediates many physiological functions including energy homeostasis and emotional processing. By acting as GTPase-activating proteins, regulators of G protein signaling (RGS) proteins are negative modulators of GPCRs. We previously elucidated that RGS8 of the B/R4 RGS subfamily potently inhibits the action of both G alpha q- and G alpha i/o-dependent MCHR1 signaling. In the present study of living cells, we provide evidence that another B/R4 protein, RGS2, is an efficient regulator of MCHR1-mediated calcium signaling exclusively via the G alpha q-dependent pathway. This effect was not observed for RGS4 and RGS5 proteins. Cotransfection of RGS2 with RGS8 additively increased the potency for inhibition of MCHR1 signaling. Truncation experiments revealed that an internal sequence within the N-terminal region of RGS2 (amino acids 28-80) was involved in the RGS2 modulation of MCHR1 activity. Our data suggest that RGS2 and RGS8 differentially associate with MCHR1 and may represent two distinct modes of signaling mechanisms in vivo.

Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G protein-coupled receptor activation. RGS proteins exert their effect by directly binding to the activated G alpha subunit of the G protein, catalyzing the accelerated hydrolysis of GTP and returning the G protein to its inactive, heterotrimeric form. In previous studies, we have sought to inhibit this GTPase-accelerating protein activity of the RGS protein by using small molecules. In this study, we investigated the mechanism of CCG-4986 [methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitro-benzenesulfinimidoate], a previously reported small-molecule RGS inhibitor. Here, we find that CCG-4986 inhibits RGS4 function through the covalent modification of two spatially distinct cysteine residues on RGS4. We confirm that modification of Cys132, located near the RGS/G alpha interaction surface, modestly inhibits G alpha binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/G alpha interaction through an allosteric mechanism that almost completely inhibits the G alpha-RGS protein-protein interaction. These findings demonstrate three important points: 1) the modification of the Cys148 allosteric site results in significant changes to the RGS interaction surface with G alpha; 2) this identifies a "hot spot" on RGS4 for binding of small molecules and triggering an allosteric change that may be significantly more effective than targeting the actual protein-protein interaction surface; and 3) because of the modification of a positional equivalent of Cys148 in RGS8 by CCG-4986, lack of inhibition indicates that RGS proteins exhibit fundamental differences in their responses to small-molecule ligands.