Interaction of amino acid residues at positions 8-15 of secretin with the 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 Chinese hamster ovary (CHO) cell lines. PACAP had a 2500-fold lower affinity than secretin for the secretin receptor, and secretin had a 300-fold lower affinity than PACAP for the VIP1 receptor. Amino acids 8, 13, and 15 of the PACAP molecule contributed significantly to the low affinity of PACAP for the secretin receptor. The amino acids at positions 5, 9, 10, 15, 16, and unidentified amino acid(s) between positions 17-20 made limited contributions to the low affinity of secretin for the VIP1 receptor. To identify the receptor region that interacts with these amino acids, we constructed chimeric receptors, which consist either of the N-terminal extracellular part of the secretin receptor and the core of the VIP1 receptor (N-Sn/VIP1r) or the N-terminal extracellular part of the VIP1 receptor and the core of the secretin receptor (N-VIP1/Snr), and tested the ability of the hybrid ligands to activate the adenylate cyclase of CHO cells expressing these chimeric receptors. The N-Sn/VIP1 receptors had a higher affinity for secretin than for 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 potency than either parent peptide for the chimeric receptor, which suggests that while the N- and/or C-terminal part of the peptide interact with the transmembrane domain of the receptor, the discriminator region 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.