Fluorescence Studies of the Bradykinin 2 and µ-Opioid Receptors Suggest that Caveolae Localization of GPCRs is Mediated by their Attached G Proteins

Abstract

Caveolae are 50-100 nm plasma membrane invaginations that have several proposed functions. Some studies suggest that caveolae may influence cell signaling by sequestering certain signaling proteins such as G-protein coupled receptors. We have previously found that Caveolin-1, the main structural protein of caveolae specifically binds to Gαq and that their binding is strengthened upon Gαq activation. Here, we have used fluorescence methods to determine the effect of caveolae on the functional properties and localization of two G-protein coupled receptors: the bradykinin receptor type 2 (B2R), which is coupled to Gαq, and the µ opioid receptor (µOR), which is coupled to Gαi. While caveolae do not affect cAMP signals mediated by µOR, they prolong Ca2+ signals mediated by B2R. In A10 cells, down-regulation of Caveolin-1 ablates the prolonged calcium signal in accord with idea that caveolae binds to Gαq. Immunofluorescence and FRET studies show that a significant fraction of B2R resides at or close to caveolae domains while none or very little µOR resides in caveolae domains. FRET between B2R and caveolae is reduced by down-regulation of Gαq or by addition of a peptide that interferes with Gαq/Caveolin-1 interactions suggesting that Gαq promotes localization of B2R to caveolae domains. Fluorescence Correlation Spectroscopy studies on live cells show that the presence of caveolae changes the distribution of the apparent diffusion coefficients of B2R and Gαq but not µOR and Gαi. Our results suggest that Gαq can localize its associated receptors to caveolae domains to enhance their signals.