RGS14 regulates the lifetime of G α ‐GTP signaling but does not prolong G βγ signaling following receptor activation in live cells

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 G α i/o‐GTP proteins, and a G protein regulatory (GPR) motif that binds inactive G α i1/3‐GDP proteins independent of G βγ . GPR interactions with G α i recruit RGS14 to the plasma membrane to interact with G α i‐linked GPCRs and regulate G α i signaling. While RGS14 actions on G α proteins are well characterized, consequent effects on G βγ signaling remain unknown. Conventional RGS proteins act as dedicated GAPs to deactivate G α and G βγ signaling following receptor activation. RGS14 may do the same or, alternatively, may coordinate its actions to deactivate G α ‐GTP with the RGS domain and then capture the same G α ‐GDP via its GPR motif to prevent heterotrimer reassociation and prolong G βγ signaling. 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 G βγ after agonist stimulation and increased the deactivation rate of G α , consistent with their roles as GTPase activating proteins (GAPs). Overexpression of inactive G α i1 to recruit RGS14 to the plasma membrane did not alter RGS14′s capacity to act as a GAP for a second G α o 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.