Analysis of the role of N-glycosylation in cell-surface expression and binding properties of angiotensin II type-2 receptor of rat pheochromocytoma cells

Abstract

We previously demonstrated that the AT₂ receptor is a glycoprotein containing N-linked oligosaccharide side chains and that the marked disparity between the sizes of AT₂ receptors from different tissues was related to different degrees of N-glycosylation. In the present study, we used an inhibitor of N-glycosylation, tunicamycin, as well as an endoglycosidase, glycopeptidase-F, to examine the contribution of carbohydrate moieties to the ligand-binding properties, cell-surface expression and apparent molecular mass of AT₂ receptors of rat pheochromocytoma cells (PC-12 cells). Photoaffinity labelling of cell-surface AT₂ receptors revealed that PC-12 cells grown in the presence of tunicamycin expressed, in addition to the previously described 140 kDa receptor, lower-molecular-mass receptors of 63 kDa, 47 kDa and 32 kDa. Lectin affinity chromatography revealed that the 63 kDa and the 47 kDa receptors are partially glycosylated and that the 32 kDa receptor is completely deglycosylated. Competitive binding experiments were carried out on tunicamycin-treated cells that expressed predominantly the 63 kDa or the 47 kDa receptors. Both receptor forms exhibited a high affinity for angiotensin II, although a slight decrease (of about 2-fold) was consistently observed on tunicamycin-treated cells as compared with control cells. Endoglycosidase digestion of AT₂ receptors of PC-12 cells also yielded smaller receptor forms of 47 kDa and 32 kDa. Similarly, angiotensin II showed a high but slightly decreased binding affinity (of about 2-fold) for deglycosylated membranes as compared with control membranes. In conclusion, the stepwise action of tunicamycin suggests the presence of at least three N-linked oligosaccharide side chains on the AT₂ receptor of PC-12 cells. These oligosaccharide side chains have a minor contribution to the affinity of the receptor. Interestingly, glycosylation is not an essential requirement for the expression of AT₂ receptor at the surface of PC-12 cells.