title: RGS14 regulates the lifetime of Gα-GTP signaling but does not prolong Gβγsignaling following receptor activation in live cells.
abstract: RGS14 is a multifunctional scaffolding protein possessing two distinct G protein interaction sites including a regulator of G protein signaling (RGS) domain that acts as a GTPase activating protein (GAP) to deactivate Gαi/o-GTP proteins, and a G protein regulatory (GPR) motif that binds inactive Gαi1/3-GDP proteins independent of Gβγ. GPR interactions with Gαi recruit RGS14 to the plasma membrane to interact with Gαi-linked GPCRs and regulate Gαi signaling. While RGS14 actions on Gαproteins are well characterized, consequent effects on Gβγsignaling remain unknown. Conventional RGS proteins act as dedicated GAPs to deactivate Gαand Gβγsignaling following receptor activation. RGS14 may do the same or, alternatively, may coordinate its actions to deactivate Gα-GTP with the RGS domain and then capture the same Gα-GDP via its GPR motif to prevent heterotrimer reassociation and prolong Gβγsignaling. To test this idea, we compared the regulation of G protein activation and deactivation kinetics by a conventional RGS protein, RGS4, and RGS14 in response to GPCR agonist/antagonist treatment utilizing bioluminescence resonance energy transfer (BRET). Co-expression of either RGS4 or RGS14 inhibited the release of free Gβγafter agonist stimulation and increased the deactivation rate of Gα, consistent with their roles as GTPase activating proteins (GAPs). Overexpression of inactive Gαi1 to recruit RGS14 to the plasma membrane did not alter RGS14's capacity to act as a GAP for a second Gαo protein. These results demonstrate the role of RGS14 as a dedicated GAP and suggest that the G protein regulatory (GPR) motif functions independently of the RGS domain and is silent in regulating GAP activity in a cellular context.
authors.affiliation: Department of Pharmacology Emory University School of Medicine Atlanta Georgia 30322.
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Brown
authors.affiliation: Department of Pharmacology and Toxicology Medical College of Georgia at Augusta University Augusta Georgia 30912.
authors.forename: Nevin A
authors.initials: NA
authors.lastname: Lambert
authors.affiliation: Department of Pharmacology Emory University School of Medicine Atlanta Georgia 30322.
authors.forename: John R
authors.initials: JR
authors.lastname: Hepler
keywords: G protein; GPCR; Gαi1; Gβγ; RGS protein; RGS14.