The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

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Abstract
Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr ∼64,000. Treatment of purified [125I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endo-glycosidase F results initially in the formation of a Mr ∼57,000 peptide which is further converted to Mr ∼49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (∼10% of which are fucosylated) on ∼45% of the receptors. The remaining ∼55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.