RGS-GAIP, A GTPASE-ACTIVATING PROTEIN FOR G-ALPHA(I) HETEROTRIMERIC G-PROTEINS, IS LOCATED ON CLATHRIN-COATED VESICLES

RGS-GAIP (G alpha-interacting protein) is a member of the RGS (regulat or of G protein signaling) family of proteins that functions to down-r egulate G alpha(i)/G alpha(q)-linked signaling. GAIP is a GAP or guano sine triphosphatase-activating protein that was initially discovered b y virtue of its ability to bind to the heterotrimeric G protein G alph a(i3) which is found on both the plasma membrane (PM) and Golgi membra nes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cel l fractionation and immunocytochemistry to determine with what particu lar membranes GAIP is associated. In pituitary cells we found that GAI P fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular car rier fractions; it was not found in either Golgi-or PM-enriched fracti ons. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP repr esents the first GAP found on CCVs or any other intracellular membrane s. The presence of GAIP on CCVs suggests a model whereby a GAP is sepa rated in space from its target G protein with the two coming into cont act at the time of vesicle fusion.