Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type IPACAP (PAC(1)) receptor

To study desensitization and glycosylation of the type I pituitary adenylat e cyclase-activating polypeptide (PACAP) receptor (PAC(1)R), a hemagglutini n (HA) epitope was inserted within the N-terminal extracellular domain, all owing immunological detection of PAC(1)R both in intact and permeabilized c ells. PAC(1)R was tagged without loss of functions in ligand binding and li gand-stimulated cAMP production. In transiently transfected COS-7 cells, PA C(1)R was localized both in the plasma membrane and the cytoplasm around th e nucleus. By immunoblot analysis, the immunoreactive bands with relative m olecular masses ranging from 45 to 70 kDa were detected in the membrane fra ctions of PAC(1)R-expressing COS-7 cells. Digestion of the membranes with e ndoglycosidase F or treatment of the cells with tunicamycin decreased the s ize of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC(1)R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearanc e of cell surface receptor. In accordance with this observation, preexposur e of cells to PACAP38 induced a desensitization of PAC(1)R to the agonist r esponse, although it did not cause a reduction in PAC(1)R mRNA or protein l evel and even slightly elevated them. These results suggest that agonist-in duced desensitization of PAC(1)R involves the receptor sequestration. (C) 2 000 Elsevier Science B.V. All rights reserved.