O-glycosylation of the V2 vasopressin receptor

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino termina l segment of the protein. This segment also contains clusters of serines an d threonines that are potential sites for O-glycosylation. Mutagenesis of a sparagine 22 to glutamine abolished N-linked glycosylation of the V2 recept or (N22Q-V2R), without altering its function or level of expression. The N2 2Q-V2R expressed in transfected cells migrated in denaturing acrylamide gel s as two protein bands with a difference of 7000 Da. Protein labeling exper iments demonstrated that the faster band could be chase to the slower one s uggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels dem onstrating the existence of O-glycosylation, the first time this type of gl ycosylation has been found in a G protein coupled receptor, Synthesis of me tabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha- D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose an d N-acetylneuraminic acid transferases, also produced a receptor that migra ted faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acce ptor sites. O-glycosylation was found at most serines and threonines presen t in the amino terminus. Because the disappearance of a site opened the ava ilability of others to the transferases, the exact identification of the ac ceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bear ing all serine/threonine substitutions by alanine at the amino terminus yie lded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase t reatment.