Abstract
Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Gα 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 Gα-interacting protein (GAIP) (RGS19) binds to Gαi3-YFP and its fate after activation of δ-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Gαi3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Gαi3-YPF and DOR are located in non–clathrin-coated microdomains of the PM. Upon addition of agonist, Gαi3 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 Gαi3-YFP with GAIP in CCPs. Moreover, Gαi3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Gαi3 move together into CCPs where Gαi3 and GAIP meet and turn off G protein signaling. Subsequently, Gαi3 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 Gα signaling through spatial segregation of a GAP in clathrin-coated pits.
- Received August 2, 2002.
- Accepted March 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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