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

Recent evidence suggests that many signaling molecules localize in microdom ains of the plasma membrane, particularly caveolae. In this study, overexpr ession of adenylyl cyclase was used as a functional probe of G protein-coup led receptor (GPCR) compartmentation. We found that three endogenous recept ors in neonatal rat cardiomyocytes couple with different levels of efficien cy to the activation of adenylyl cyclase type 6 (AC6), which localizes to c aveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta (1)-adrenergic receptor (beta (1)AR)-selective activ ation 3.7-fold, to beta (2)AR-selective activation only 1.6-fold and to pro staglandin E-2 (PGE(2)) not at all. Therefore, the rank order of efficacy i n coupling to AC6 is beta (1)AR > beta (2)AR > prostaglandin E-2 receptor ( EP2R). beta (2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing beta ARKct, an inhib itor of G protein-coupled receptor kinase activation, but was not significa ntly greater when cells were treated with pertussis toxin. Assessment of re ceptor and AC expression indicated co-localization of AC5/6,beta (1)AR, and beta (2)AR, but not EP2R, in caveolin-rich membranes and caveolin-3 immuno precipitates, likely explaining the observed activation of AC6 by beta AR s ubtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated wit h a beta AR agonist, beta (2)AR were no longer found in caveolin-3 immunopr ecipitates; an effect that was blocked by expression of beta ARKet. Thus, a gonist-induced translocation of beta (2)AR out of caveolae causes a sequest ration of receptor from effector and likely contributes to the lower effica cy of beta (2)AR coupling to AC6 as compared with beta (1)AR, which do not similarly translocate. Therefore, spatial co-localization is a ey determina nt of efficiency of coupling by particular extracellular signals to activat ion of GPCR-linked effectors.