PHARMACOLOGICAL PROPERTIES OF 2 RECOMBINANT SPLICE VARIANTS OF THE PACAP TYPE-I RECEPTOR, TRANSFECTED AND STABLY EXPRESSED IN CHO CELLS

Two splice variants of the pituitary adenylate cyclase activating poly peptide (PACAP) type I receptor (PACAP receptor and PACAP/HOP receptor isoform) were stably expressed in Chinese hamster ovary (CHO) cells t hat did not express constitutively receptors for this family of peptid es. The PACAP/HOP receptor protein had a 28 amino acid extension in th e C-terminal part of the third intracellular loop. The two cell lines studied, CHO 2-10 (PACAP receptor) and CHO 4-12 (PACAP/HOP receptor) e xpressed a receptor density of 4.6 +/- 0.3 and 2.6 +/- 0.2 pmol/mg pro tein, respectively, with corresponding K-d values of 14.2 +/- 2.0 and 8.2 +/- 1.0 nM for [Ac-His(1)]PACAP-27 used as a tracer. Tracer bindin g was slightly decreased by GTP in both clones. The K-d values of PACA P-27, PACAP-38, vasoactive intestinal peptide (VIP), PACAP-27 fragment s and analogues evaluated by binding competition curves, were higher i n CHO 2-10 than in CHO 4-12, whereas the K-d for PACAP-38 fragments di d not differ. The receptors were coupled to adenylate cyclase and the EC(50) values were lower than the K-d values in both cell lines, sugge sting an amplification process due to the existence of spare receptors . Pretreatment of the CHO 4-12 cells with increasing concentrations of PACAP-27 for 24 h induced an increase in the K-act values and a decre ase in the maximal stimulation; the same pretreatment of CHO 2-10 cell s also induced an increase in the K-act values, but a marked increase in the adenylate cyclase activity in the absence of added peptide, sug gesting that PACAP pretreatment had induced a permanent coupling of th e receptor to the G(s) site. Thus, the two splice variants differed in their capacity to recognize the ligand, and in their coupling to the G(s) sites.