GAIP IS MEMBRANE-ANCHORED BY PALMITOYLATION AND INTERACTS WITH THE ACTIVATED (GTP-BOUND) FORM OF G-ALPHA(I) SUBUNITS

GAIP (G Alpha Interacting Protein) is a member of the recently describ ed RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein G alpha(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 carbon ate treatment but was digested by proteinase K, When Cos cells were tr ansiently transfected with GAIP and metabolically labeled with [S-35]m ethionine, two pools of GAIP-a soluble and a membrane-anchored pool-we re found, Since the N terminus of GAIP contains a cysteine string moti f and cysteine string proteins are heavily palmitoylated, we investiga ted the possibility that membrane-anchored GAIP might be palmitoylated , We found that after labeling with [H-3] 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 membe rs of the G alpha(i) subfamily, G alpha(i1), G alpha(i2), G alpha(i3), G alpha(z), and G alpha(o), but not with members of other G alpha sub families, G alpha(s), G alpha(q), and G alpha(12/13). The C terminus o f G alpha(i3) is important for binding because a 10-aa C-terminal trun cation and a point mutant of G alpha(i3) showed significantly diminish ed interaction, GAIP interacted preferentially with the activated (GTP ) form of G alpha(i3), which is in keeping with its GAP activity, We c onclude that GAIP is a membrane-anchored GAP with a cysteine string mo tif, This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, sugge sts GAIP's possible involvement in membrane trafficking.