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

• Published in: European journal of biochemistry Vol. 207; no. 1; pp. 239 - 246
• Main Authors: ROBBERECHT, P, GOURLET, P, DE NEEF, P, WOUSSEN-COLLE, M.-C, VANDERMEERS-PIRET, M.-C, VANDERMEERS, A, CHRISTOPHE, J
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01-07-1992
• Subjects: Adenylyl Cyclases - metabolism; Amino Acid Sequence; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Cell Membrane - metabolism; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; Molecular Sequence Data; Neuroblastoma; Neuropeptides - pharmacology; Peptide Fragments - pharmacology; Pituitary Adenylate Cyclase-Activating Polypeptide; Proteins; Receptors, Cell Surface - antagonists & inhibitors; Receptors, Cell Surface - drug effects; Receptors, Cell Surface - metabolism; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Sequence Homology, Nucleic Acid; Signal Transduction - drug effects; Structure-Activity Relationship; Tumor Cells, Cultured; Human; Adenylate cyclase; Cell culture; Signal transduction; Membrane receptor; Cell line; Structure activity relation; Enzyme; Analog; Molecular interaction; Neuroblastoma
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1992.tb17043.x

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, 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 (Ki 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.