期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：12
页码：6722-6727
DOI：10.1073/pnas.96.12.6722
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：G-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for G subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIP-enriched vesicles could accelerate the GTPase activity of Gi proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5x) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant Gi3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant Gi3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for Gi3 and that membrane-associated GAIP is capable of interacting with Gi3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.
关键词：heterotrimeric G proteins ; RGS proteins ; GAP
