Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of G(i)- and G(q)-mediated signaling

RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein a subunits of the i, q, and 12 classes. The proteins share a homolo gous core domain but have divergent amino-terminal sequences that are the s ite of palmitoylation for RGS-GAIP and RGS4. We investigated the function o f palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blo cked the incorporation of [H-3]palmitate into RGS16 in metabolic labeling s tudies of transfected cells or into purified RGS proteins in a cell-free pa lmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for G(i)alpha. Inhibit ion or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both G(i)-and G(q)- linked signaling pathways when expressed in HEK293T cells. These findings s uggest that the aminoterminal region of RGS16 may affect the affinity of th ese proteins for Ga subunits in vivo or that palmitoylation localizes the R GS protein in close proximity to Ga subunits on cellular membranes.