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 described 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 carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]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 membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]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 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 subfamilies, G alpha s, G alpha q, and G alpha 12/13. The C terminus of G alpha i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of G alpha i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of G alpha 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.
De Vries
L
L
Division of Cellular and Molecular Medicine, University of California at San Diego, La Jolla 92093-0651, USA.
Elenko
E
E
Hubler
L
L
Jones
T L
TL
Farquhar
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000378:metabolism*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D020710:RGS Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D014101:Torpedo; D014162:Transfection
D046911:Macromolecular Substances; D010750:Phosphoproteins; D020710:RGS Proteins; D011994:Recombinant Proteins; C097187:regulator of G-protein signalling 19; D019308:Palmitic Acid; D019204:GTP-Binding Proteins