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Spatial Regulation of Gi Protein Signaling in Clathrin-Coated Membrane Microdomains Containing GAIP

Departments of Cellular and Molecular Medicine (E.E., T.F., I.N., T.H., T.M., M.G.F.) and Pathology (T.H., M.G.F.), University of California San Diego, La Jolla, California; and Department of Psychiatry, University of California San Francisco, San Francisco, California

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to G subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous G-interacting protein (GAIP) (RGS19) binds to Gi3-YFP and its fate after activation of -opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Gi3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Gi3-YPF and DOR are located in non–clathrin-coated microdomains of the PM. Upon addition of agonist, Gi3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and Gi3-YFP with GAIP in CCPs. Moreover, Gi3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Gi3 move together into CCPs where Gi3 and GAIP meet and turn off G protein signaling. Subsequently, Gi3 returns to non–clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of G signaling through spatial segregation of a GAP in clathrin-coated pits.

Address correspondence to: Dr. Marilyn G. Farquhar, Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0651. E-mail: mfarquhar{at}ucsd.edu

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