N-linked glycosylation is required for optimal AT1a angiotensin receptor expression in COS-7 cells.
The nature and role of glycosylation in AT1 angiotensin receptor (AT1-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT1a-Rs (HA-AT1a-Rs) in COS-7 cells. All three asparagine residues (Asn4, Asn176, Asn188) contained within consensus sites for N-linked glycosylation could be glycosylated in Cos-7 cells and appeared to be glycosylated on the endogenous AT1-R in bovine adrenal glomerulosa cells. Heterogeneity of glycosylation at each site accounted for the broad migration pattern of the AT1-R in SDS-PAGE. Mutation at each glycosylation site, either alone or in combination, had little effect on ligand binding parameters (although the N4K mutant had higher affinity) or signaling activity. However, an increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decreased surface expression of mutant HA-AT1a-Rs was correlated with decreased total cell receptor content as revealed by immunoblotting with an anti-HA antibody. These findings suggest that glycosylation enhances receptor stability, possibly by protecting nascent receptors from proteolytic degradation.
Jayadev
S
S
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510, USA.
Smith
R D
RD
Jagadeesh
G
G
Baukal
A J
AJ
Hunyady
L
L
Catt
K J
KJ
D000345:Affinity Labels; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002236:Carbohydrate Conformation; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D016296:Mutagenesis; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II