DECREASED TRANSCRIPT EXPRESSION COINCIDENT WITH IMPAIRED GLYCOSYLATION IN THE BETA(2)-ADRENERGIC RECEPTOR GENE DOES NOT RESULT FROM DIFFERENCES IN THE PRIMARY SEQUENCE

Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to beta(2)-adre nergic stimulation. Two such variants, beta(p) and beta(d), are charac terized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of beta(2)-a drenergic 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 beta(2)-adrenergic receptor in the S49 wild-type, beta(p) and beta(d) variant cells was estimated by labeling the receptor with the photoaffinity probe [I-125]iodocyanopindololdiazirine. Receptor s ize was found to be 67 000 and 47 000 Da in the wild-type and 60 000 a nd 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 alter ed glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the beta(2)-adrenergic recep tor 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) a nd beta(d) S49 cells suggesting that both the diminished expression an d the decreased size of beta(2)-adrenergic receptor in the beta(p) and beta(d) S49 variants are related to impaired glycosylation of the rec eptor. This hypothesis was substantiated by the reduced retention of t he variant cells' beta(2)-adrenergic receptor on immobilized WGA. Furt hermore, growth of the S49 cells in the presence of the alpha-mannosid ase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These res ults imply that altered expression and glycosylation of G-protein-link ed receptors occur as a consequence of one or more mutations outside t he receptor's open reading frame. (C) 1997 Elsevier Science B.V.