The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high-mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site.

A serum-albumin-alprenolol conjugate was used to isolate beta-adrenergic receptors from the human A431 cell lysates. Three monoclonal antibodies were obtained from BALB/c mice immunized with these receptors. These antibodies: BRK-1, BRK-2, BRK-3, were respectively of the IgM, IgG2a and IgG3 classes. All three antibodies recognized photoaffinity-labelled receptors, immunoprecipitated ligand-binding activity and identified the 65-kDa and 55-kDa polypeptides corresponding to the beta 2-adrenergic receptors of A431 cells. BRK-2 and BRK-3 recognized both beta 1 and beta 2-adrenergic receptors of several mammalian cells. All three antibodies visualized, by immunofluorescence, the beta 2-adrenergic receptors at the surface of A431 cells. The monoclonal antibodies are directed against the protein portion of the beta-adrenergic receptors since partial or complete removal of the carbohydrate moieties by treatment with endoglycosidase such as endo-F (endo-beta-N-acetylglucosaminidase F) and periodate oxidation did not affect the immunoreactivity. These antibodies will be of value to immunopurify the beta-adrenergic receptors.

The beta 2-adrenergic receptors of the human epidermoid carcinoma A431 cells reside on two polypeptide chains revealed by photoaffinity labelling with [125I]iodocyanopindolol-diazirine. These proteins correspond to two distinct populations of N-asparagine-linked glycoproteins: the 55-52 kDa molecules are associated with complex carbohydrate chain(s), the 65-63 kDa component with polymannosidic carbohydrate chain(s). Both types of receptors are present in preconfluent cells, but only the polymannosidic type is found in the postconfluent cells. Moreover, complex chains appear to be associated with the receptors with the highest affinity for (-)-isoproterenol and polymannosidic chains with the receptors with the lowest affinity for this agonist. the carbohydrate moiety of the beta-adrenergic receptor is involved in the expression and function of the beta 2-adrenergic receptors at the surface of the A431 cells, since tunicamycin and monensin, complete and partial inhibitors of glycosylation respectively, diminish the number of binding sites at the cell surface and increase the total number of sites in the cell. In these conditions a diminution of cyclic AMP accumulation is also observed.