Decreased transcript expression coincident with impaired glycosylation in the β2-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 β 2-adrenergic stimulation. Two such variants, β p and β d, are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of β 2-adrenergic receptor synthesis was measured and found to be decreased in the βd cells vis-à-vis the rate in wild type cells. The molecular mass of the β 2-adrenergic receptor in the S49 wild-type, β p and β 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 β 2-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, β p and β d S49 cells suggesting that both the diminished expression and the decreased size of β 2-adrenergic receptor in the β p and β d S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' β 2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the α-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. ©1997 Elsevier Science B.V. All rights reserved.