N-Glycosylation in expression and function of β-adrenergic receptors

Through the use of specific staining and the analysis of the interaction of pure β-adrenergic receptor of S49 mouse lymphoma cells with lectins immobilized to insoluble matrices, we establish that this β-adrenergic receptor is a glycoprotein. The effects of swainsonine (0.2 μg/ml), an inhibitor of Golgi mannosidase II, as well as those of tunicamycin (0.2 μg/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. Pre-existing receptors on the cells were inactivated by alkylation with the β-adrenergic antagonist ligand N-(2-hydroxy-3-naphthoxylpropyl)-N'-bromoacetylethylenediami ne. Swainsonine did not alter the number of β-receptors measured in intact cells, the B(max), or K(d) 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 M(r) of β-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 β-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 M(r) 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 β-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.