(8 -27). This is the first demonstration that, to activate the VPAC 1 receptor, the Asp 3 side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2. The neuropeptides vasoactive intestinal polypeptide (VIP)1 and pituitary adenylate cyclase-activating polypeptide (PACAP) contribute to the regulation of intestinal secretion and motility, of the vascular tone, of the exocrine and endocrine secretions, of immunological responses, and to the development of the central nervous system (1-3). The effects of VIP are mediated through interaction with two receptor subclasses named the VPAC 1 and VPAC 2 receptors; the effects of PACAP are also mediated through interactions with the same receptors, as well as through a selective receptor named PAC 1 (3, 4). VPAC 1 , VPAC 2 , and PAC 1 receptors are encoded by different genes and expressed in different cell populations in both the central nervous system and peripheral tissues (3, 5, 6). They are preferentially coupled to G␣ s proteins that stimulate adenylate cyclase activity. The PAC 1 and VPAC 1 receptors may stimulate, in addition, inositol trisphosphate synthesis and calcium mobilization (7,8). This effect is however detected only at high VPAC 1 receptor expression levels (8). VIP and PACAP receptors are members of a large family of G protein-coupled receptors, often referred as the GPCR-B family (4, 9), that includes the secretin, glucagon, glucagon-like peptide-1, calcitonin, parathyroid hormone, and growth hormone releasing factor (GRF) receptors. The VIP, PACAP, secretin, and GRF receptors constitute a subfamily based on the homology of the ligands and of the receptors. Each receptor recognizes its own cognate ligand with a high affinity but recognizes at least one other parent peptide with a comparable or a lower affinity (4). Because of the sequence homology of the ligands and the receptors, the information obtained on one receptor-ligand pair can be anticipated to be relevant also in the other systems.The positioning of the ligand on the receptors is still poorly understood. Investigations of chimeric receptors and mutants have indicated that the large amino-terminal domain (10 -13) structured by disulfide bridges (14 -16) makes a key contribution to ligand recognition that several other highly conserved residues play a role in the general structure (17,18) and that creating constitutively active receptors through mutations in the intracellular part of the receptor is possible (19).The amino-terminal part of the ligand is necessary for high affinity binding and for second messenger activation; its deletion in VIP, PACAP, and secretin reduced both the affinity and the intrinsic activity of the peptide (20). The identification of the receptor residues interacting with the amino terminus of the ligand is a prerequisite to model the active form of the receptor and conceive new ligands, preferably non-peptidic, that could be of therapeutic interest. We focused in this work on the ...