Tonic plasma membrane association increases the function of RGS proteins as inhibitors of M1 muscarinic receptor signaling

Abstract

RGS2 is a more potent attenuator of M1 muscarinic receptor signaling than other small RGS proteins. To explain differences, the function of RGS2 and RGS5 were compared in HEK293 cells stably expressing the M1 muscarinic receptor. Results show that the carboxyl terminal GAP domains of RGS2 and RGS5 show comparable inhibitory function. Plasma membrane-targeted RGS GAP domains show enhanced function relative to non-targeted proteins suggesting that membrane association is a determinant of M1 muscarinic receptor inhibition. Amino terminal domain swaps of RGS2 and RGS5 reveal that the RGS2 amino terminus is sufficient to confer increased plasma membrane association and function. Mutation of a single hydrophobic residue in the amino terminal amphipathic helix of RGS2 and the RGS2/5 chimera reduce their plasma membrane association and inhibitory function without disrupting interaction with the third intracellular loop of the M1 muscarinic receptor. Replacement of two unique leucine residues adjacent to the amphipathic helix of RGS2 with the corresponding residues from RGS5 disrupts membrane localization and function. Together, these data suggest that the amphipathic alpha helix in RGS2 displays an elongated hydrophobic surface that facilitates its increased tonic plasma membrane targeting and greater inhibition of M1 muscarinic signaling relative to RGS5.