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Distinct themes exist for ligand-binding domains of G protein-coupled receptors. The secretin receptor is prototypic of a recently described family in this superfamily which binds moderate-sized peptides possessing a diffuse pharmacophore. We recently demonstrated the importance of the N terminus and first loop of this receptor for secretin binding (Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) J. Biol. Chem. 270:14394-14398). Here, we extend those findings to define another receptor domain important for agonist recognition and to focus on critical determinants within each of these domains. Extending the secretin-vasoactive intestinal polypeptide (VIP) chimeric receptor approach, we confirmed and refined the critical importance of the N terminus and the need to complement this with other domains of the secretin receptor. There was redundancy in the complementary determinants required, with the second extracellular loop able to compensate for the absence of the first loop. The first 10 residues of the N terminus of the secretin receptor were critical. Sequential segmental and site replacements permitted focusing on the His189-Lys190 sequence at the C terminus of the first extracellular loop, and on four residues (Phe257, Leu258, Asn260, and Thr261) in the N-terminal half of the second loop as providing critical determinants. All receptor constructs which expressed sensitive cAMP responses to secretin (EC50

Secretin and vasoactive intestinal polypeptide (VIP) receptors are closely related G protein-coupled receptors in a recently described family possessing a large amino-terminal ectodomain. We postulated that this domain might be critical for agonist recognition and therefore constructed a series of six chimeric receptors, exchanging the amino terminus, the first extracellular loop, or both in secretin and VIP receptors. Constructs were expressed in COS cells and characterized by cAMP generation and binding of both secretin and VIP radioligands. Wild type receptors demonstrated high affinity binding of respective ligands (IC50 values (in nM): at the secretin receptor: 2.2 for secretin, >1000 for VIP; at the VIP receptor: 2.2 for VIP, >1000 for secretin) and appropriately sensitive and selective biological responses (EC50 values (in nM): at the secretin receptor: 1.5 for secretin, 127 for VIP; at the VIP receptor: 1.0 for VIP, 273 for secretin). Replacement of the secretin receptor amino terminus with that of the VIP receptor resulted in biological responsiveness typical of the VIP receptor (EC50 = 120 nM for secretin, 1.7 nM for VIP). The converse was not true, with this domain of the secretin receptor not able to provide the same response when incorporated into the VIP receptor (EC50 = 50 nM for VIP, 30 nM for secretin). The addition of both the first loop and the amino terminus of the secretin receptor was effective in yielding a secretin receptor-like response (EC50 = 2.0 nM for secretin, 47 nM for VIP). All chimeric constructs expressing selectivity for secretin-stimulated activity bound this hormone with high affinity (IC50 = 0.2-2.2 nM); however, there was divergence between VIP binding and biological activity. Thus, the amino terminus of secretin and VIP receptors plays a key role in agonist recognition and responsiveness, with the first loop playing a critical complementary role for the secretin receptor.