title: Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.
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.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Ostrom
authors.forename: C
authors.initials: C
authors.lastname: Gregorian
authors.forename: R M
authors.initials: RM
authors.lastname: Drenan
authors.forename: Y
authors.initials: Y
authors.lastname: Xiang
authors.forename: J W
authors.initials: JW
authors.lastname: Regan
authors.forename: P A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000737:chemistry*; D000242:Cyclic AMP / Q000096:biosynthesis; D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000032:analysis* / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015232:Dinoprostone; D002118:Calcium