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Photoaffinity labeling of beta-adrenergic receptors in mammalian tissues
Abstract:
Photoaffinity labeling of beta 1- and beta 2-adrenergic receptors in plasma membranes from various mammalian tissues has been been performed utilizing the recently developed beta-adrenergic antagonist probe [125I]para-azidobenzylcarazolol. Tissues studied and their proportions of beta 1 and beta 2 receptors were: rat lung (18% beta 1, 82% beta 2), rabbit lung (72% beta 1, 28% beta 2), guinea pig lung (15% beta 1, 85% beta 2), dog lung (20% beta 1, 80% beta 2) and rabbit skeletal muscle (10% beta 1, 90% beta 2). As assessed by autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two to three specifically protected bands of Mr 62,000-65,000, 50,000-55,000 and 38,000-42,000 were observed in each tissue system. In each case, beta-adrenergic agonists and antagonists protected against photolabeling with appropriate beta 1 and beta 2 selectivity. Thus, in rat lung the beta 2 selective antagonist ICI-118,551 was more potent in blocking incorporation than the beta 1 selective antagonist betaxolol, whereas in rat, dog and guinea pig lung and rabbit skeletal muscle epinephrine was more potent than norepinephrine in blocking labeling, indicating a beta 2 specificity in these tissues. Conversely, in rabbit lung membranes, norepinephrine was approximately equipotent with epinephrine in blocking photoincorporation, indicating a beta 1 selectivity. In some systems protease inhibitors, especially those specific for metalloproteases (EDTA, EGTA), markedly diminished the amount of the smaller Mr peptides. For example, in rat lung the ratio of Mr 62,000:47,000:36,000 peptides changed from 30:40:30 to 60:35:5 in the presence of inhibitors. These results demonstrate the applicability of using [125I]para-azidobenzylcarazolol to covalently label mammalian beta-adrenergic receptors and suggest that mammalian beta 1 and beta 2 receptor binding sites primarily reside on peptides of Mr 62,000-65,000 and that smaller ligand binding fragments may arise by proteolysis.