L. Devries et al., GAIP IS MEMBRANE-ANCHORED BY PALMITOYLATION AND INTERACTS WITH THE ACTIVATED (GTP-BOUND) FORM OF G-ALPHA(I) SUBUNITS, Proceedings of the National Academy of Sciences of the United Statesof America, 93(26), 1996, pp. 15203-15208
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.