STRUCTURAL REQUIREMENTS FOR THE OCCUPANCY OF PITUITARY ADENYLATE-CYCLASE-ACTIVATING-PEPTIDE (PACAP) RECEPTORS AND ADENYLATE-CYCLASE ACTIVATION IN HUMAN NEUROBLASTOMA NB-OK-1 CELL-MEMBRANES - DISCOVERY OF PACAP(6-38) AS A POTENT ANTAGONIST

In these structure activity studies, the 46 analogs of the 27-amino-acid form of the pituitary-adenylate-cyclase-activating peptide, PACAP(1 - 27), and the 38-amino-acid form, PACAP(1 - 38), were either monosubstituted or bisubstituted at positions 1 - 3, 20 and 21 or N-terminally shortened. All analogs were compared on human neuroblastoma NB-OK-1 cell membranes for their ability to occupy I-125-[AcHis1]PACAP(1 -27)-labelled receptors (AcHis, N(alpha)-acetylhistidine) and to activate adenylate cyclase (in terms of potency and intrinsic activity). The monophasic slope of dose/effect curves on both parameters suggested interaction with one class of PACAP receptor. Residues 28-38 in the C-terminally extended peptide, PACAP(1 - 38), played a favorable role in recognition, in that receptors coupled to adenylate cyclase were, in general, more sensitive to PACAP(1 - 38) analogs than to the corresponding PACAP(1 - 27) analogs. At variance with PACAP(6 - 27), PACAP(6 - 38) was well recognized and acted as a potent competitive antagonist (K(i) 1.5 nM). Residues 1 - 3 were all important in enzyme activation: modification of the beta-turn potential gave full agonists (the LAla2 and DAla2 derivatives) or partial agonists (LPhe2 and DPhe2; LArg2 and DArg2; Glu3 and Asn3). Finally, a proper alpha-helix was also important: the combined substitution of Lys21/Lys22 by Gly21/Gly22 decreased the binding affinity sharply.