Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor.
GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT(1) receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.
Lanctot
Pascal M
PM
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
Leclerc
Patrice C
PC
Clément
Martin
M
Auger-Messier
Mannix
M
Escher
Emanuel
E
Leduc
Richard
R
Guillemette
Gaétan
G
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium; D002522:Chlorocebus aethiops; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry / Q000378:metabolism*
D044140:Receptor, Angiotensin, Type 1; D002118:Calcium