RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles.
RGS-GAIP (Galpha-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Galphai/Galphaq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Galphai3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP 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 carrier fractions; it was not found in either Golgi- or PM-enriched fractions. 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 represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.
De Vries
L
L
Division of Cellular and Molecular Medicine and Department of Pathology, University of California, San Diego, La Jolla, California 92093-0651, USA.
Elenko
E
E
McCaffery
J M
JM
Fischer
T
T
Hubler
L
L
McQuistan
T
T
Watson
N
N
Farquhar
M G
MG
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D018699:Coated Vesicles / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D020710:RGS Proteins; D051381:Rats
D002966:Clathrin; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D020710:RGS Proteins; C097187:regulator of G-protein signalling 19; D019206:GTP-Binding Protein alpha Subunits, Gi-Go