Abstract
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 beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, 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, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, 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.
Highlights
Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, caveolae
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
We previously reported that overexpression of AC6 in neonatal rat cardiac myocytes selectively enhances responses to AR activation but not to activation of prostanoid, adenosine, glucagon, or histamine receptors and found that AC6 and 1AR are co-localized in caveolar membranes of cardiomyocytes (13)
Summary
Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, caveolae. The distinct physiological actions of 1AR and 2AR may represent coupling to different signaling pathways (5– 8) and/or different spatial localization within the heart or within single cells (9, 10) Expression of both AR and AC in distinct caveolar microdomains of the plasma membrane has been recently demonstrated in both non-cardiac and cardiac cells (10 –14). We previously reported that overexpression of AC6 in neonatal rat cardiac myocytes selectively enhances responses to AR activation but not to activation of prostanoid, adenosine, glucagon, or histamine receptors and found that AC6 and 1AR are co-localized in caveolar membranes of cardiomyocytes (13). Taken together with the fact that prostanoid receptors, which do not couple to AC6, were not found in caveolae, we conclude that GPCR ability to couple to the stimulation of AC6 depends upon the co-localization of sufficient active receptors in caveolin-rich membrane microdomains, the predominant subcellular site of AC6 expression
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