Spatial regulation of Galphai protein signaling in clathrin-coated membrane microdomains containing GAIP.

Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Galpha 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 Galpha-interacting protein (GAIP) (RGS19) binds to Galphai3-YFP and its fate after activation of delta-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Galphai3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Galphai3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, Galphai3 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 Galphai3-YFP with GAIP in CCPs. Moreover, Galphai3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Galphai3 move together into CCPs where Galphai3 and GAIP meet and turn off G protein signaling. Subsequently, Galphai3 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 Galpha signaling through spatial segregation of a GAP in clathrin-coated pits.