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

Abstract

Regulators of G-protein signaling (RGS) proteins are GTPaseactivating 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 G␣i3-YFP and its fate after activation of ␦-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from G␣i3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas G␣i3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, G␣i3 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 G␣i3-YFP with GAIP in CCPs. Moreover, G␣i3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and G␣i3 move together into CCPs where G␣i3 and GAIP meet and turn off G protein signaling. Subsequently, G␣i3 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.

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2Receptor Signaling

“…In addition, GFPGAIP colocalized with clathrin mostly at the PM ( Figure 4K, see arrows) and faintly with EEA1 upon D 2 R activation ( Figure 4L), despite Information 2). This suggests that GFPGAIP moved to clathrin-coated pits to meet with GIPC and active D 2 R in agreement with previous data reporting the codistribution of GIPC and GAIP within clathrin-coated pits, where GAIP interacted with G␣i3 (Lou et al, 2002;Elenko et al, 2003). Collectively, these results indicate that D 2 R activation regulates the spatial distribution of GFPGAIP.…”

“…The formation of a GPCR-GAIP complex through GIPC could favor targeting to the correct G␣ substrate generated by receptor activation. For instance, GAIP interacts with its cognate G i ␣3 subunit upon ␦-opioid receptor (DOR) activation (Elenko et al, 2003). Whether GIPC links GAIP to DOR as it clustered GAIP with D 2 R, despite the absence of apparent PDZ-binding motif in DOR C-terminus, is currently unknown.…”

Molecular Pharmacology ofδ-Opioid Receptors

“…In this case, ligand-activated DOPrs and their associated Gai3 proteins translocated to clathrin pits where Gai3 met RGS19, which turned off DOPr signaling before internalization took place (Elenko et al, 2003).…”

GIPC Is Recruited by APPL to Peripheral TrkA Endosomes and Regulates TrkA Trafficking and Signaling

“…PC12(615) cells were fixed with 2% paraformaldehyde in PBS on ice for 30 min, permeabilized with 0.1% Triton X-100 containing 1% BSA, and incubated with primary antibodies for 1 h and goat anti-rabbit Alexa-594 or anti-mouse Alexa-488 F(abЈ) 2 for 1 h. Fluorescence images were taken with a Zeiss Axiophot or AxioImager M1 (Carl Zeiss, Thornwood, NY) equipped with a digital ORCA-ER camera (Hamamatsu, Bridgewater, NJ) and processed with Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA). Fluorescence images of double-labeled samples were quantified as described previously (17). Cell membrane areas were traced for early time points (0 and 5 min), or entire cells were traced at 30-min time points and selected using Adobe Photoshop 5.0.…”