Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase

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 ß₁-adrenergic receptor (ß₁AR)-selective activation 3.7-fold, to ß₂AR-selective activation only 1.6-fold and to prostaglandin E₂ (PGE₂) not at all. Therefore, the rank order of efficacy in coupling to AC6 is ß₁AR > ß₂AR > prostaglandin E₂ receptor (EP₂R). ß₂AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing ßARKct, 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, ß₁AR, and ß₂AR, but not EP₂R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by ßAR subtypes but lack thereof by PGE₂. When cardiomyocytes were stimulated with a ßAR agonist, ß₂AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of ßARKct. Thus, agonist-induced translocation of ß₂AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of ß₂AR coupling to AC6 as compared with ß₁AR, 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.