Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC1) receptor

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To study desensitization and glycosylation of the type I pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC1R), a hemagglutinin (HA) epitope was inserted within the N-terminal extracellular domain, allowing immunological detection of PAC1R both in intact and permeabilized cells. PAC1R was tagged without loss of functions in ligand binding and ligand-stimulated cAMP production. In transiently transfected COS-7 cells, PAC1R was localized both in the plasma membrane and the cytoplasm around the nucleus. By immunoblot analysis, the immunoreactive bands with relative molecular masses ranging from 45 to 70 kDa were detected in the membrane fractions of PAC1R-expressing COS-7 cells. Digestion of the membranes with endoglycosidase F or treatment of the cells with tunicamycin decreased the size of the receptor to major bands of smaller size (approximately 45 and 48 kDa), suggesting that these two forms of PAC1R represent core proteins. Flow cytometric analysis indicated that the agonist promoted a disappearance of cell surface receptor. In accordance with this observation, preexposure of cells to PACAP38 induced a desensitization of PAC1R to the agonist response, although it did not cause a reduction in PAC1R mRNA or protein level and even slightly elevated them. These results suggest that agonist-induced desensitization of PAC1R involves the receptor sequestration. (C) 2000 Elsevier Science B.V.




Shintani, N., Hashimoto, H., Kunugi, A., Koyama, Y., Yamamoto, K., Tomimoto, S., … Baba, A. (2000). Desensitization, surface expression, and glycosylation of a functional, epitope-tagged type I PACAP (PAC1) receptor. Biochimica et Biophysica Acta - Biomembranes, 1509(1–2), 195–202.

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