Localization and Coupling of Adenylyl Cyclase Isoforms 2, 3 and 6 With G Protein-Coupled Receptors in Mouse Bronchial Smooth Muscle Cells
Several GPCR stimulate adenylyl cyclase (AC) to increase cAMP and promote relaxation in smooth muscle. β-adrenergic receptors only couple to certain AC isoforms due to compartmentation in lipid rafts. Previous data show AC2, AC3, AC4 and AC6 are expressed in primary mouse bronchial smooth muscle cells (mBSMC). We overexpressed AC2, AC3 or AC6 in mBSMC using recombinant adenoviruses and assessed their localization by cell fractionation and confocal microscopy then determined the coupling of various GPCR to each AC isoform by examining the arborization response to receptor-specific agonists. Lipid rafts were isolated using a detergent-based fractionation method and fractions were analyzed by immunoblotting. AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas the AC2 expression was excluded from these domains. Forskolin-induced arborization of mBSMC, measured by time-lapse microscopy, was enhanced by overexpression of all three AC isoforms. Isoproterenol- or beraprost-mediated arborization was only accelerated by AC6 overexpression. In contrast, butaprost-mediated arborization was only accelerated by AC2 overexpression. AC3 overexpression did not significantly alter the response to any of these agonists. Our results imply that particular GPCR couple to discreet AC isoforms, likely based upon their colocalization.
T Muthusamy, P Adris and RS Ostrom. Localization and coupling of adenylyl cyclase isoforms 2, 3 and 6 with G protein-coupled receptors in mouse bronchial smooth muscle cells. FASEB J, 23(1):582.3, 2009.
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