Antagonistic properties are shifted back to agonistic properties by further N‐terminal shortening of pituitary adenylate‐cyclase‐activating peptides in human neuroblastoma NB‐OK‐1 cell membranes

Abstract

) -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

Functional Characterization of Structural Alterations in the Sequence of the Vasodilatory Peptide Maxadilan Yields a Pituitary Adenylate Cyclase-activating Peptide Type 1 Receptor-specific Antagonist

“…To identify which part of the PACAP molecule is responsible for the recognition of PACAP type 1-binding sites, PACAP and VIP sequences have been compared. It has been reported that replacement of Asp 9 and Leu 13 in VIP by serine and tyrosine, respectively, increased, by two to five times, the affinity of VIP for I-PACAP-binding sites (13,15). Maxadilan does not possess histidine in the corresponding site.…”

“…Our results, as well as those of others, reveal that receptor binding affinity was higher with PACAP-38 than with PACAP-27. It has also been reported that the helix-breaking Gly 20 -Gly 21 substitution in PACAP-27 reduced the affinity of the resulting peptide 500-fold, indicating that the two highly hydrophilic residues (Lys 20 and Lys 21 ), located in the central portion of a hydrophobic domain in PACAP-27, were important for PACAP receptor recognition (13,15).…”

Photoaffinity Labeling Analysis of the Interaction of Pituitary Adenylate‐Cyclase‐Activating Polypeptide (PACAP) with the PACAP Type I Receptor

“…Accordingly, PACAP-(1 -27)-peptide has a disordered N-terminal domain consisting of eight amino acids, followed by an a-helical structure stretching from Ser9 to Va126, which contains a discontinuity between Lys20 and Lys21. Previous data concerning the structural requirements of PACAP to recognize its receptor are primarily based on modifications of PACAP at its N-terminal part Vandermeers et al, 1992;Bitar et al, 1994). In the PACAP derivatives that we synthesized in this study, the amino acids Phe6 and Tyr22 were displaced by the photoreactive BzPhe thus leading to benzophenone-containing PACAP analogues with modifications in both domains of the peptide.…”

“…A characteristic feature of these receptors is a relatively large N-terminal extracellular region consisting of about 1.50 amino acids with several highly conserved cysteine residues. Some structural requirements for the occupancy of the PACAP type I receptors have been reported and led to the development of receptor antagonists Vandermeers et al, 1992;Bitar et al, 1994). By using chemical modification and deletion mutagenesis, we have previously demonstrated that the N-terminal fragment of the PACAP type I receptor contains a major binding site for its ligand (Cao et al, 1994(Cao et al, , 1995.…”

The Different Effects of I.c.v. Injection of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) on Prolactin Secretion in Adult Male and Lactating Rats

“…PACAP38 and PACAP6-38 (Peptide Institute Inc., Osaka, Japan), a specific and selective receptor antagonist [13,17] were dissolved (0.25, 1, or 4 nmol/10 µl) in artificial cerebral spinal fluid [15,16]. Control animals received the same volume (10 µl) of vehicle.…”