Role of N-glycosylation in the expression and functional properties of human AT1 receptor.
The role of N-glycosylation in the pharmacological properties and cell surface expression of AT1 receptor was evaluated. Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences (N-X-S/T) of AT1 receptor (positions 4, 176, and 188) with aspartic acid. Expression of these mutant receptors in COS-7 cells followed by photolabeling with [125I]-[p-benzoyl-Phe8]AngII and SDS-PAGE revealed ligand-receptor complexes of four different molecular sizes, indicating that the three N-glycosylation sites are actually occupied by oligosaccharides. Binding studies showed that the affinity of each mutant receptor for [Sar1,Ile8]Ang II was not significantly different from that of wild-type AT1 receptor. Moreover, the functional properties of all mutant receptors were unaffected as evaluated by inositol phosphate production. However, the expression levels of the aglycosylated mutant were 5-fold lower than that of the wild-type AT1 receptor. Use of green fluorescent protein-AT1 receptor fusion proteins in studying the cellular location of the aglycosylated mutant demonstrated that it was distributed at a much higher density to the ER-Golgi complex than to the plasma membrane in HEK 293 cells. Together, these results suggest an important role of N-glycosylation in the proper trafficking of AT1 receptor to the plasma membrane.
Lanctôt
P M
PM
Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Canada.
Leclerc
P C
PC
Escher
E
E
Leduc
R
R
Guillemette
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D014162:Transfection
D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins