INTERACTION OF AMINO-ACID-RESIDUES AT POSITIONS 8-15 OF SECRETIN WITHTHE N-TERMINAL DOMAIN OF THE SECRETIN RECEPTOR

The ability of secretin, PACAP-(1-27)-peptide, and ten hybrid peptides to recognize and activate the rat secretin and vasoactive intestinal polypeptide (PACAP type II VIP1) receptors was tested on recombinant C hinese hamster ovary (CHO) cell lines. PACAP had a 2500-fold lower aff inity than secretin for the secretin receptor, and secretin had a 300- fold lower affinity than PACAP for the VIP1 receptor. Amino acids 8, 1 3, and 15 of the PACAP molecule contributed significantly to the low a ffinity of PACAP for the secretin receptor. The amino acids at positio ns 5, 9, 10, 15, 16, and unidentified amino acid(s) between positions 17-20 made limited contributions to the low affinity of secretin for t he VIP1 receptor. To identify the receptor region that interacts with these amino acids, we constructed chimeric receptors, which consist ei ther of the N-terminal extracellular part of the secretin receptor and the core of the VIP1 receptor (N-Sn/VIP(1)r) or the N-terminal extrac ellular part of the VIP1 receptor and the core of the secretin recepto r (N-VIP1/Snr), and tested the ability of the hybrid ligands to activa te the adenylate cyclase of CHO cells expressing these chimeric recept ors. The N-Sn/VIP1 receptors had a higher affinity for secretin than f or PACAP. The hybrid peptide 6 that consists of the PACAP-(1-8)-Sn-(9- 15)-PACAP-(16-27)-peptide sequence had a 30-fold to 200-fold higher po tency than either parent peptide for the chimeric receptor, which sugg ests that while the N- and/ or C-terminal part of the peptide interact with the transmembrane domain of the receptor, the discriminator regi on 9-15 recognizes the extracellular N-terminal domain of the receptor . This was confirmed by the observation that, out of all the peptides tested, hybrid 6 had the weakest potency for activation of the N-VIP1/ Sn chimeric receptors.