Glycosylation of asparagine 24 of the natriuretic peptide receptor-B is crucial for the formation of a competent ligand binding domain.
UV cross-linking studies of the natriuretic peptide receptor-B (NPR-B) using radiolabeled C-type natriuretic peptide (CNP) indicate that only fully glycosylated receptors are capable of binding ligand. We therefore used site-directed mutagenesis to determine which potential glycosylation sites are occupied by carbohydrate, and the relevant mutants were characterized in order to understand the function of carbohydrate addition at those sites. Our results suggest that five of seven potential N-linked glycosylation sites are modified. In addition, mutation of asparagine 24 results in a loss of approximately 90% of receptor activity. This mutant is expressed at levels comparable to the wild-type receptor, and its activity is not significantly different from that of wild-type NPR-B in terms of EC50 for CNP. Ligand binding studies on this mutant further show that although there is no change in affinity for ligand, approximately 90% of receptor binding is lost. These data suggest that many of the mutant receptors are simply not properly folded. Our results indicate that glycosylation of asparagine 24 of NPR-B receptors may be critical for the formation of a competent ligand binding domain.
Fenrick
R
R
Department of Biochemistry, University of Montreal, Quebec, Canada.
Bouchard
N
N
McNicoll
N
N
De Léan
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D001667:Binding, Competitive; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002417:Cattle; D003432:Cross-Linking Reagents; D006152:Cyclic GMP / Q000096:biosynthesis; D006031:Glycosylation; D006162:Guanylate Cyclase; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D020098:Natriuretic Peptide, C-Type; D011485:Protein Binding / Q000502:physiology* / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012016:Reference Values; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays; D000099242:Atrial Natriuretic Factor Receptor B
D001216:Asparagine; D003432:Cross-Linking Reagents; D006152:Cyclic GMP; D006162:Guanylate Cyclase; D007457:Iodine Radioisotopes; D008024:Ligands; D020098:Natriuretic Peptide, C-Type; D011506:Proteins; D017461:Receptors, Atrial Natriuretic Factor; D011994:Recombinant Proteins; D000099242:Atrial Natriuretic Factor Receptor B