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 125I‐[AcHis1]PACAP(1–27)‐labelled receptors (AcHis, Nα‐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 (Ki 1.5 nM). Residues 1–3 were all important in enzyme activation: modification of the β‐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 α‐helix was also important: the combined substitution of Lys21/Lys22 by Gly21/Gly22 decreased the binding affinity sharply.

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) -EJB 92 0760 N-terminally shortened analogs of the 27-amino-acid and 38-amino-acid forms of the pituitaryadenylate-cyclase-activating neuropeptide, PACAP( 1 -27) and PACAP( 1 -38), were synthesized by a solid-phase method. Systematic deletion of the first 13 amino acids of both PACAP was tested by evaluating their ability to occupy the specific and selective PACAP receptor of human neuroblastoma NB-OK-1 cell membranes and to stimulate adenylate cyclase or, when inactive per se, to inhibit PACAP-stimulated adenylate cyclase activity. For each peptide, the K,,, (concentration required for half-maximal adenylate cyclase activation) or Ki [concentration required to shift the dose/response curve of PACAP(1-27) twofold to the right] was in good agreement with the corresponding IC50 [concentration inhibiting 50% of 1251-[AcHisl]PACAP(1 -27) binding to membranes], suggesting interaction with the same homogeneous class of adenylate cyclase-coupled receptors.The deletion of the two first amino acids (Hisl and Ser2) sufficed to decrease the affinity for receptors and to suppress the capacity to activate adenylate cyclase. The shorter fragments 3 -27 and 3-38,4-27and4-38,5-27and5-38,6-27and6-38,7-27and7-38,8-27and8-38,and 9 -27 and 9 -38 were all competitive antagonists of PACAP(1-27)-stimulated activity with the Nterminally shortened PACAP(1-38) derivatives being 4-30-fold more potent than the equivalent PACAP(1-27) derivatives. In this group PACAP(6-38) was the most potent antagonist (Ki 1.5 nM).Surprisingly, the N-terminally shorter fragments 10 -27 and 10 -38, 11 -27 and 11 -38, 12 -27 and 12-38, 13-27 and 13-38, and 14-27 and 14-38 were again able to stimulate adenylate cyclase, the smallest fragments, PACAP(14 -27) and PACAP(14 -38), being the most potent and efficient (K,,, 2 pM and 0.1 pM, respectively). In this group of agonists, PACAP(1-38) derivatives deleted at the N-terminus were also more potent than the equivalent PACAP(1-27) derivatives.PACAP (pituitary adenylate-cyclase-activating peptides) [l, 21 are neuropeptides that belong to a family including secretin, vasoactive intestinal peptide (VIP), growth-hormone-releasing factor and glucagon. They interact with highaffinity selective receptors coupled to adenylate cyclase in rat brain [3-51, rat pituitary [6], cultured rat astrocytes [7], rat adrenal pheochromocytoma cells [8], human neuroblastoma NB-OK-1 cells [9], and rat cancerous pancreatic acinar AR 4-2J cells [lo]. PACAP(1-27) and PACAP(1-38) (the 27-amino-acid and 38-amino-acid forms corresponding, respectively, to HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH2 and to HSDGIFTDSYSRYRKQMAVKKYLAAVLG-KRYKQRVKNK-NH2) are equally potent, while VIP, despite 68% similarity to PACAP(1-27), is 3000-fold less potent than PACAP with the human neuroblastoma NB-OK-1 cell PACAP receptor, considered as a prototype [ll]. Using

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