There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that β1- and β2-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from β1AR and β2AR knockout mice, we found that stimulation of the β1AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the β2AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the β2AR but not the β1AR in neonatal cardiac myocytes. Evidence for β2AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the β2AR and caveolin 3, and co-migration of the β2AR with a caveolin-3-enriched membrane fraction. The β2AR-stimulated increase in the myocyte contraction rate is increased by ∼2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of β2AR and caveolin 3 and comigration of β2AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on β1AR signaling. These observations suggest that β2ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.
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