The β2-adrenergic receptors of human epidermoid carcinoma cells bear two different types of oligosaccharides which influence expression on the cell surface

Abstract

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.

Structure, function, and regulation of adrenergic receptors

“…Lack of polysaccharide addition (as is the case for PAR receptors functionally expressed in Escherichia coli Strosberg & Marullo, 1992]), partial or complete inhibition of glycosylation by monensin or tunicamycin, or removal by specific enzymes (reviewed in Ostrowski et al [1992]) does not seem to alter ligand binding or signal transmission in any G protein-coupled receptor yet examined. Absence of carbohydrates does appear, however, to reduce considerably the density of P2AR expressed at the surface of A431 cells (Cervantes et al, 1985(Cervantes et al, , 1988. Carbohydrates may thus play a role in receptor trafficking.…”

Mapping of a functional autoimmune epitope on the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.

“…The clinical characterization of the patients is summarized in Table I. Peptides. Peptides were synthesized by the solid phase method of (9), and the fl2-peptide to the sequence 172-197 of the human fl2-adrenergic receptor ( (19). The membrane proteins were subjected to electrophoresis on a 10% polyacrylamide gel in SDS according to Laemmli (20), subjected to electrotransfer to nitrocellulose (21), and the latter was saturated for 2 h with PMT.…”

Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties.

“…Five milliliters of a solution of 10 mM Tris-HCl (pH 7.5) and 5 mM EDTA was then added and the cells or membranes were centrifuged, respectively, at 5000 x g for 10 min or 300,000 x g for 20 min. After suspension in 5 ml of the same buffer, photolysis was performed and cells or membranes were washed as described (15). Labeled membranes and cells were solubilized in Laemmli's sample buffer (16) containing 10% (wt/vol) NaDodSO4, incubated for 2 hr at room temperature, subjected to electrophoresis in 10% polyacrylamide gels in the presence of NaDodSO4 (16), and autoradiographed.…”