Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to β1-adrenergic receptor (β 1AR)-selective activation 3.7-fold, to β 2AR-selective activation only 1.6-fold and to prostaglandin E 2 (PGE2) not at all. Therefore, the rank order of efficacy in coupling to AC6 is β1AR > β2AR > prostaglandin E2 receptor (EP2R). β2AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing βARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, β1AR, and β 2AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by β2AR subtypes but lack thereof by PGE2. When cardiomyocytes were stimulated with a βAR agonist, β2AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of βARKct. Thus, agonist-induced translocation of β2AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of β2AR coupling to AC6 as compared with β1AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.
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