title: Decreased transcript expression coincident with impaired glycosylation in the beta2-adrenergic receptor gene does not result from differences in the primary sequence.
abstract: Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to beta2-adrenergic stimulation. Two such variants, beta(p) and beta(d), are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of beta2-adrenergic receptor synthesis was measured and found to be decreased in the beta d cells vis-a-vis the rate in wild type cells. The molecular mass of the beta2-adrenergic receptor in the S49 wild-type, beta(p) and beta(d) variant cells was estimated by labeling the receptor with the photoaffinity probe [125I]iodocyanopindololdiazirine. Receptor size was found to be 67,000 and 47,000 Da in the wild-type and 60,000 and 42,000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N-glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the beta2-adrenergic receptor gene and two kilobases of the 5'-non-coding region. No differences were found in the coding region of the gene from wild-type, beta(p) and beta(d) S49 cells suggesting that both the diminished expression and the decreased size of beta2-adrenergic receptor in the beta(p) and beta(d) S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' beta2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the alpha-mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame.
authors.affiliation: Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Hughes
authors.forename: M
authors.initials: M
authors.lastname: Pasillas
authors.forename: J
authors.initials: J
authors.lastname: Saiz
authors.forename: J
authors.initials: J
authors.lastname: Jasper
authors.forename: P A
authors.initials: PA
authors.lastname: Insel
mesh_terms: D000581:Amidohydrolases; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D006031:Glycosylation; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics*; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase