G-protein-coupled receptor signaling components localize in both sarcolemmal and intracellular caveolin-3-associated microdomains in adult cardiac myocytes

This study tests the hypothesis that G- protein- coupled receptor ( GPCR) signaling components involved in the regulation of adenylyl cyclase ( AC) localize with caveolin ( Cav), a protein marker for caveolae, in both cellsurface and intracellular membrane regions. Using sucrose density fractionation of adult cardiac myocytes, we detected Cav- 3 in both buoyant membrane fractions ( BF) and heavy/ non- buoyant fractions ( HF); beta (2)- adrenergic receptors ( AR) in BF; and AC5/ 6, beta(1)- AR, M-4- muscarinic acetylcholine receptors ( mAChR), mu- opioid receptors, and G alpha(s) in both BF and HF. In contrast, M-2- mAChR, G alpha(i3), and G alpha(i2) were found only in HF. Immunofluorescence microscopy showed co- localization of Cav- 3 with AC5/ 6, G alpha s, beta(2)- AR, and mu- opioid receptors in both sarcolemmal and intracellular membranes, whereas M-2-mAChR were detected only intracellularly. Immunofluorescence of adult heart revealed a distribution of Cav- 3 identical to that in isolated adult cardiac myocytes. Upon immunoelectron microscopy, Cav- 3 co- localized with AC5/ 6 and G alpha(s) in sarcolemmal and intracellular vesicles, the latter closely allied with T- tubules. Cav- 3 immunoprecipitates possessed components that were necessary and sufficient for GPCR agonist- promoted stimulation and inhibition of cAMP formation. The distribution of GPCR, G- proteins, and AC with Cav- 3 in both sarcolemmal and intracellular T- tubule- associated regions indicates the existence of multiple Cav- 3- localized cellular microdomains for signaling by hormones and drugs in the heart.