Abstract
RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys95 in RGS4, Cys66 in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys2 or Cys12. The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [3H]palmitate labeling of Cys95. Membrane-bound RGS4 is palmitoylated both at Cys95 and Cys2/12, but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys95 on RGS4 or Cys66 on RGS10 inhibits GAP activity 80-100% toward either Gα(i) or Gα(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity ≥20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Gα(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.