RGS2 modulates the selection of Gαi/o subtype involved in dopamine D2 receptor signaling

Abstract

IntroductionRegulator of G‐protein signaling (RGS) proteins modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. It is believed that RGS proteins are recruited by and bind to activated Gα subunits to exert GTPase‐activating (GAP) activity. However, recent literature has indicated that certain RGS proteins bind to GPCRs and this binding is critical for receptor‐mediated G protein signaling. Previously, we reported that RGS2 is a negative modulator of dopamine D2 receptor‐mediated Gαi/o signaling. The present study aimed to investigate the mechanism underlying the interaction of RGS2 with D2Rs and Gαi/o proteins.MethodsIn the present study, we utilized a neuroblastoma 2a cell line stably expressing the short form of dopamine D2 receptors (N2A‐D2S). RGS2 N‐terminus truncation (partial and full truncation) mutants, RGS2 siRNA and corresponding controls were transiently transfected into N2A‐D2S cells. Immunoprecipitation was performed to evaluate the coupling between D2Rs and RGS2. Immunocytochemistry and confocal microscopy were utilized to study agonist (quinpirole) stimulated recruitment of Gαi/o subtypes to surface D2Rs. [35S] GTPγS binding was performed to measure D2R‐stimulated Gαi/o activation.ResultsOur data indicate that RGS2 interacts with D2Rs via its N‐terminus and modulates D2R‐stimulated recruitment of Gαi/o subtypes. Specifically, we found that: 1) RGS2 couples to D2Rs via its N‐terminus. RGS2 N‐terminus truncation abolishes the co‐localization of RGS2 with surface D2Rs 2) RGS2 N‐terminus truncation attenuated D2R‐stimulated Gαi/o activation; and 3) RGS2 knockdown alters the selection of Gα subtype recruited to activated D2Rs. D2R stimulation by quinpirole (5 min, 1 μM) induced translocation of Gαi2, not Gαo, towards the plasma membrane in the control cells. However, RGS2 knockdown by siRNA resulted in the translocation of Gαo, not Gαi2, towards the plasma membrane after D2R stimulation.These results suggest that the interaction between RGS2 and D2Rs mediates the selection of the Gαi/o subtype engaged in D2R‐mediated signaling. The finding of this study highlights a novel mechanistic action of RGS2 in regulation of D2R‐mediated Gαi/o signaling.Support or Funding InformationThis work is supported by NIH NIAAA training grant T32 AA007565, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.