RGS proteins (Regulators of G protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G-protein of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of tritiated palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase activating protein (GAP) for G alpha i1. Inhibition 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 Gi and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino terminal region of RGS16 may affect the affinity of these proteins for G alpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to G alpha subunits on cellular membranes. We are currently testing the palmitoylation and palmitate turnover of other RGS proteins and the role of palmitoylation in the subcellular localization of these proteins. - RGS proteins, heterotrimeric G proteins, signal transduction, palmitoylation, GTPase activity
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