GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of G i subunits

L. De Vries, E. Elenko, L. Hubler, T. L. Z. Jones, M. G. Farquhar
1996 Proceedings of the National Academy of Sciences of the United States of America  
GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of Gprotein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G␣ i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently
more » ... cted with GAIP and metabolically labeled with [ 35 S]methionine, two pools of GAIP-a soluble and a membrane-anchored pool-were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membraneanchored GAIP might be palmitoylated. We found that after labeling with [ 3 H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the G␣ i subfamily, G␣ i1 , G␣ i2 , G␣ i3 , G␣ z , and G␣ o , but not with members of other G␣ subfamilies, G␣ s , G␣ q , and G␣ 12/13 . The C terminus of G␣ i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G␣ i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G␣ i3 , which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP's possible involvement in membrane trafficking.
doi:10.1073/pnas.93.26.15203 pmid:8986788 pmcid:PMC26381 fatcat:s74zgzer3rg5xdlktzzr4ixxbm