Use of 1-deoxymannojirimycin to show that complex oligosaccharides regulate cellular distribution of the alpha 1-adrenergic receptor glycoprotein in BC3H1 muscle cells.
We have previously shown that alpha 1-adrenergic receptors in BC3H1 muscle cells are glycoproteins containing complex but not high mannose oligosaccharides. In the present study we investigated the role of the complex sugars in functional aspects of the receptor by treating BC3H1 cells with 1-deoxymannojirimycin (dMM), which blocks conversion of high mannose oligosaccharides to complex chains. Receptors were photoaffinity labeled in intact cells with 125I-azido prazosin; drug treatment with dMM resulted in conversion of the 87-kDa receptor to 62 kDa. The 62-kDa protein was sensitive to mannosidase, indicating loss of complex sugars. Radioligand ([3H]prazosin) binding analysis carried out at 37 degrees to intact cells indicated that dMM treatment increased the affinity of the alpha 1-receptors for [3H]prazosin 2-fold and decreased the number of total cellular receptors by 15%. In order to distinguish between surface and sequestered receptors, we assessed [3H]prazosin binding to intact cells at 4 degrees using competition by the hydrophilic agonist epinephrine to define surface receptors and by nonradioactive antagonists (prazosin and phentolamine) to define total receptors. In control cells, epinephrine competed for 90% of the total receptors, whereas for dMM-treated cells this value was only 60%. In addition, dMM treatment caused a 40% reduction in epinephrine-stimulated phosphatidylinositol turnover when compared with untreated cells. The results indicate that dMM treatment reduces the number of functional alpha 1-adrenergic receptors on the cell surface while increasing the number of sequestered receptors. We conclude that complex oligosaccharides are important for cellular localization and function of alpha 1-adrenergic receptors in BC3H1 cells.
Terman
B I
BI
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Insel
P A
PA
D017485:1-Deoxynojirimycin; D000345:Affinity Labels; D000818:Animals; D002478:Cells, Cultured; D004837:Epinephrine / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis*; D051379:Mice; D009132:Muscles / Q000032:analysis; D009844:Oligosaccharides / Q000502:physiology*; D010646:Phentolamine / Q000494:pharmacology; D011224:Prazosin / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000032:analysis*
D000345:Affinity Labels; D006023:Glycoproteins; D009844:Oligosaccharides; D011942:Receptors, Adrenergic, alpha; D017485:1-Deoxynojirimycin; D005944:Glucosamine; D011224:Prazosin; D004837:Epinephrine; D010646:Phentolamine