N-linked glycosylation is required for optimal AT(1a) angotensin receptor expression in COS-7 cells

The nature and role of glycosylation in AT, angiotensin receptor (AT(1)-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT(1a)-Rs (HA-AT(1a)-Rs) in COS-7 cel ls. All three asparagine residues (Asn(4), Asn(176) Asn(188)) contained wit hin consensus sites for N-linked glycosylation could be glycosylated in Cos -7 cells and appeared to be glycosylated on the endogenous AT(1)-R in bovin e adrenal glomerulosa cells. Heterogeneity of glycosylation at each site ac counted for the broad migration pattern of the AT(1)-R in SDS-PAGE. Mutatio n at each glycosylation site, either alone or in combination, had little ef fect on Ligand binding parameters (although the N4K mutant had higher affin ity) or signaling activity. However, an increasing number of mutated glycos ylation sites was associated with decreasing cell surface receptor expressi on, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decr eased surface expression of mutant HA-AT(1a)-Rs was correlated with decreas ed total cell receptor content as revealed by immunoblotting with an anti-H A antibody. These findings suggest that glycosylation enhances receptor sta bility, possibly by protecting nascent receptors from proteolytic degradati on.