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 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.
Robberecht
P
P
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
Gourlet
P
P
De Neef
P
P
Woussen-Colle
M C
MC
Vandermeers-Piret
M C
MC
Vandermeers
A
A
Christophe
J
J
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009479:Neuropeptides / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D009479:Neuropeptides; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011956:Receptors, Cell Surface; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; C075647:pituitary adenylate-cyclase-activating-peptide (6-38); D000262:Adenylyl Cyclases