Amino-terminal cysteine residues of RGS16 are required for palmitoylation and modulation of Gi- and Gq-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 alpha subunits 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 [3H]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 for Gialpha. 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 Galpha subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Galpha subunits on cellular membranes.
Druey
K M
KM
Molecular Signal Transduction Section, Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20852, USA.
Ugur
O
O
Caron
J M
JM
Chen
C K
CK
Backlund
P S
PS
Jones
T L
TL
D000215:Acylation; D000818:Animals; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D016384:Consensus Sequence; D003545:Cysteine / Q000502:physiology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D010168:Palmitates / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D020710:RGS Proteins; D015398:Signal Transduction; D014162:Transfection
D010168:Palmitates; D010750:Phosphoproteins; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; C097187:regulator of G-protein signalling 19; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D003545:Cysteine