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.
Elenko
Eric
E
Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0651, USA.
Fischer
Thierry
T
Niesman
Ingrid
I
Harding
Tim
T
McQuistan
Tammie
T
Von Zastrow
Mark
M
Farquhar
Marilyn G
MG
D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D018699:Coated Vesicles / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D021962:Membrane Microdomains / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D020710:RGS Proteins; D017465:Receptors, Opioid, delta / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
D033962:Adaptor Protein Complex 2; D001426:Bacterial Proteins; D002966:Clathrin; D020690:GTPase-Activating Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D020710:RGS Proteins; D017465:Receptors, Opioid, delta; C097187:regulator of G-protein signalling 19; C054562:yellow fluorescent protein, Bacteria; D019204:GTP-Binding Proteins; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins; D034281:Dynamins