N-glycosylation in expression and function of beta-adrenergic receptors.

ABSTRACT

Through the use of specific staining and the analysis of the interaction of pure beta-adrenergic receptor of S49 mouse lymphoma cells with lectins immobilized to insoluble matrices, we establish that this beta-adrenergic receptor is a glycoprotein. The effects of swainsonine (0.2 microgram/ml), an inhibitor of Golgi mannosidase II, as well as those of tunicamycin (0.2 microgram/ml), an inhibitor of N-glycosylation, on the expression and function of this integral membrane glycoprotein were investigated in S49 mouse lymphoma cells grown in culture. Preexisting receptors on the cells were inactivated by alkylation with the beta-adrenergic antagonist ligand N-(2-hydroxy-3-naphthoxylpropyl)-N'-bromoacetyl-ethylenediamine. Swainsonine did not alter the number of beta-receptors measured in intact cells, the Bmax, or Kd of receptors measured in membranes prepared from these cells as assayed by [125I]iodocyanopindolol binding or their functional coupling to adenylate cyclase. Autoradiograms of membranes photoaffinity-labeled with [125I]iodoazidobenzylpindolol and subjected to electrophoresis on polyacrylamide gels reveal a reduction of 6,000 in the Mr of beta-receptors in membranes prepared from swainsonine-treated cells. This form of receptor was sensitive to endoglycosaminidase H, indicating its high mannose hybrid oligosaccharide nature. The number and affinity of beta-receptors in tunicamycin-treated S49 cells were normal. Whereas stimulation of cyclic AMP accumulation in cells or adenylate cyclase in membranes by prostaglandin E1 was essentially abolished by tunicamycin treatment, stimulation by isoproterenol was largely unaffected. The nonglycosylated receptor displays an Mr that is approximately 8,000-11,000 smaller than the native receptor. Thus, N-glycosylation does not affect the expression (steady-state) or function of the beta-adrenergic receptor, whereas prostaglandin E1 receptor function is lost. The role of N-glycosylation in receptor function is not universal among receptors coupled to adenylate cyclase.