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 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.