Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of 'tyrosine toggle switch' required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist - cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

Are heterobivalent GRPR- and VPAC1R-bispecific radiopeptides suitable for efficient in vivo tumor imaging of prostate carcinomas?

Receptor-specific peptides labeled with positron emitters play an important role in the clinical imaging of several malignancies by positron emission tomography (PET). Radiolabeled heterobivalent bispecific peptidic ligands (HBPLs) can target more than one receptor type and by this - besides exhibiting other advantages - increase tumor imaging sensitivity. In the present study, we show the initial in vivo evaluation of the most potent heterobivalent gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC1R)-bispecific radiotracer and determined its tumor visualization potential via PET/CT imaging. For this purpose, the most potent described HBPL was synthesized together with its partly scrambled heterobivalent monospecific homologs and its monovalent counterparts. The agents were efficiently labeled with 68Ga3+ and evaluated in an initial PET/CT tumor imaging study in a human prostate carcinoma (PCa) xenograft rat tumor model established for this purpose. None of the three 68Ga-HBPLs enabled a clear tumor visualization and a considerably higher involvement in receptor-mediated uptake was found for the GRPR-binding part of the molecule than for the VPAC1R-binding one. Of the monovalent radiotracers, only [68Ga]Ga-NODA-GA-PESIN could efficiently delineate the tumor, confirming the results. Thus, this work sets the direction for future developments in the field of GRPR- and VPAC1R-bispecific radioligands, which should be based on other VPAC1R-specific peptides than PACAP-27.

A Molecular Dynamics Study of Vasoactive Intestinal Peptide Receptor 1 and the Basis of Its Therapeutic Antagonism.

Vasoactive intestinal peptide receptor 1 (VPAC1) is a member of a secretin-like subfamily of G protein-coupled receptors. Its endogenous neuropeptide (VIP), secreted by neurons and immune cells, modulates various physiological functions such as exocrine and endocrine secretions, immune response, smooth muscles relaxation, vasodilation, and fetal development. As a drug target, VPAC1 has been selected for therapy of inflammatory diseases but drug discovery is still hampered by lack of its crystal structure. In this study we presented the homology model of this receptor constructed with the well-known web service GPCRM. The VPAC1 model is composed of extracellular and transmembrane domains that form a complex with an endogenous hormone VIP. Using the homology model of VPAC1 the mechanism of action of potential drug candidates for VPAC1 was described. Only two series of small-molecule antagonists of confirmed biological activity for VPAC1 have been described thus far. Molecular docking and a series of molecular dynamics simulations were performed to elucidate their binding to VPAC1 and resulting antagonist effect. The presented work provides the basis for the possible binding mode of VPAC1 antagonists and determinants of their molecular recognition in the context of other class B GPCRs. Until the crystal structure of VPAC1 will be released, the presented homology model of VPAC1 can serve as a scaffold for drug discovery studies and is available from the author upon request.

3D structure prediction of VAPC1 and identification of dual natural inhibitors for VPAC1 and EGFR.

Vasoactive intestinal polypeptide receptor 1 (VPAC1) and epidermal growth factor receptor (EGFR) are associated with signal transduction pathways relevant to neuroblastoma, cancer of breast, prostate and lungs. In order to identify appropriate ligand analogues for simultaneous inhibition of EGFR and VPAC1, in-silico homology modelling of VPAC1 and its characterization by molecular interaction studies have been undertaken. Homology modelling was performed with the Swiss Model and validation of the predicted 3D structure was carried out using PROCHECK and RAMPAGE. Ramachandran's plot of the predicted structure from this two software revealed that 92% and 94% of the residues were in the most favoured region, respectively. Compounds screened from Naturally Occurring Plant-based Anti-Cancerous Compound-Activity-Target (NPACT) database having strong interactions with EGFR were further checked for ADMET properties. Molecular interaction studies revealed four compounds namely Fisetin, Genistein, Tectorigenin, and Tephrosin docked with VPAC1 having respective binding energies of -7.1, -6.98, -6.9 and - 6.61 kcal/mol. Fisetin and Genistein with a rotatable bond and lower molecular weight increased their drug-likeness than the others. Therefore, simultaneous inhibition of VPAC1 and EGFR, in turn, might inhibit the progression of breast carcinoma. The results obtained were further substantiated by comparing them with positive and negative controls. Quercetin was used as positive control, and strong binding energy of -7.54 kcal/mol with EGFR is in accordance with experimental evidence. 3-O-cis-p coumaroyl alphitolic acid was used as negative control, where docking was not possible in absence of binding with either EGFR or VIPR1.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Increased Conformational Flexibility of HLA-B*27 Subtypes Associated With Ankylosing Spondylitis.

OBJECTIVE: Dissimilarities in antigen processing and presentation are known to contribute to the differential association of HLA-B*27 subtypes with the inflammatory rheumatic disease ankylosing spondylitis (AS). In support of this notion, previous x-ray crystallographic data showed that peptides can be displayed by almost identical HLA-B*27 molecules in a subtype-dependent manner, allowing cytotoxic T lymphocytes to distinguish between these subtypes. For example, a human self-peptide derived from vasoactive intestinal peptide receptor type 1 (pVIPR; sequence RRKWRRWHL) is displayed in a single conformation by B*27:09 (which is not associated with AS), while B*27:05 (which is associated with AS) presents the peptide in a dual binding mode. In addition, differences in conformational flexibility between these subtypes might affect their stability or antigen presentation capability. This study was undertaken to investigate B*27:04 and B*27:06, another pair of minimally distinct HLA-B*27 subtypes, to assess whether dual peptide conformations or structural dynamics play a role in the initiation of AS. METHODS: Using x-ray crystallography, we determined the structures of the pVIPR-B*27:04 and pVIPR-B*27:06 complexes and used isotope-edited infrared (IR) spectroscopy to probe the dynamics of these HLA-B*27 subtypes. RESULTS: As opposed to B*27:05 and B*27:09, B*27:04 (which is associated with AS) displays pVIPR conventionally and B*27:06 (which is not associated with AS) presents the peptide in a dual conformation. Comparison of the 4 HLA-B*27 subtypes using IR spectroscopy revealed that B*27:04 and B*27:05 possess elevated molecular dynamics compared to the nonassociated subtypes B*27:06 and B*27:09. CONCLUSION: Our results demonstrate that an increase in conformational flexibility characterizes the disease-associated subtypes B*27:04 and B*27:05.

Strategies for studying the ligand binding site of GPCRs: photoaffinity labeling of the VPAC1 receptor, a prototype of class B GPCRs.

G protein-coupled receptors (GPCRs) are crucial receptors acting as molecular sensors for many physiological and pathological processes. Class B GPCRs represent a small GPCR subfamily encompassing 15 members, and are very promising targets for the development of drugs to improve many diseases such as chronic inflammation, neurodegeneration, diabetes, stress, and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of class B GPCRs plays a pivotal role in natural ligand recognition. The N-ted structure of some class B GPCRs folds into a Sushi domain consisting of two antiparallel ß sheets stabilized by three disulfide bonds and a salt bridge. The VPAC1 receptor is an archetype of class B GPCRs that binds vasoactive intestinal peptide (VIP), a neuropeptide modulating many physiological processes. The structure-function relationship of VPAC1 has been extensively studied. The use of a photoaffinity labeling strategy has been a powerful approach to determine the physical contacts between the functional receptor and its ligand. Those studies, coupled with 3D molecular modeling techniques, have clearly demonstrated the crucial role of the VPAC1 receptor N-ted in VIP recognition.

Conformational switches in the VPAC(1) receptor.

The vasoactive intestinal peptide receptor 1 (VPAC(1) ) belongs to family B of GPCRs and is activated upon binding of vasoactive intestinal peptide (VIP) and pituitary AC-activating polypeptide neuropeptides. Widely distributed throughout body, VPAC(1) plays important regulatory roles in human physiology and physiopathology. Like most members of the GPCR-B family, VPAC(1) receptor is predicted to follow the actual paradigm of a common 'two-domain' model of natural ligand action. However the precise structural basis for ligand binding, receptor activation and signal transduction are still incompletely understood due in part to the absence of X-ray crystal structure of the whole receptor and to significant structural differences with the most extensively studied family of receptor, the GPCR-A/rhodopsin family. Here, we try to summarize the current knowledge of the molecular mechanisms involved in VPAC(1) receptor activation and signal transduction. This includes search for amino acids involved in the two-step process of VIP binding, in the stabilization of VPAC(1) inactive and active conformations, and in binding and activation of G proteins.

VPAC1 receptor binding site: contribution of photoaffinity labeling approach.

The vasoactive intestinal peptide (VIP) is a prominent 28 aminoacid neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP has a large spectrum of biological functions including exocrine secretions, hormone release, foetal development, immune response and also exerts beneficial effect in neuro-degenerative and inflammatory diseases. Few years ago, it has been shown that VIP can be a promising anti-inflammatory agent. VIP mechanisms of action implicate two sub-types of receptors (VPAC1 and VPAC2) which are members of class B receptors belonging to the super-family of G protein-coupled receptor (GPCR). Because, VPAC1 receptor plays an important role in the modulation of the ant-inflammatory response and represent an archetype of class B GPCR, we have extensively studied the structure-function relationship of this receptor, which allowed us to define the molecular basis of that receptor in term of affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies showed the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP binding. Using different techniques including photoaffinity labeling, NMR, molecular modeling and molecular dynamic simulation, it has been possible to define how VIP interacts with its receptor. We have shown that most of the VIP molecule, 1-28 (alpha-helix) sequence, tightly binds the N-ted part of the receptor which is himself structured as a <> domain. In contrast, the N-terminal part of the specific antagonist PG97-269 is in physical contact with the N-ted but in different region. These studies define the molecular mechanism implicated in the activation of class B VPAC1 receptor and should allow the development of new VIP pharmacology using rational synthesis of agonist molecules.

Grasping molecular structures through publication-integrated 3D models.

Although the need for communicating 3D data using simple and intuitive means extends to disciplines as diverse as biology, engineering sciences and the visual arts, none of the currently available molecular-visualization programs depicting potentially highly complex structures are compatible with the portable document format (PDF), the current gold standard of electronic publishing. Therefore, it is highly desirable for authors to be able to provide their readers with a basic 3D display of structures that can be accessed without the need for specialized visualization software. Here, we describe how an interactive 3D model of a molecular complex can be embedded directly into a PDF, thus providing readers with important and educational visual information that would otherwise be more difficult to disseminate.

Production and purification of large quantities of the functional N-terminal ectodomain of human VPAC1 receptor.

Vasoactive intestinal peptide (VIP) is implicated in many physiological and pathophysiological processes, and its receptors are promising targets for the development of new drugs. The human VPAC1 receptor for VIP and pituitary adenylate cyclase-activating polypeptide is a class II G protein coupled receptor. The N-terminal ectodomain (N-ted) of the VPAC1 receptor is a major VIP binding site. To determinate the high resolution structure of the VPAC1 receptor N-ted, large quantities of purified recombinant N-ted produced are required. The N-ted sequence (31-144), which is fused to thioredoxin protein and 6xHis tag, was expressed into Origami Escherichia coli strain. Purification of recombinant N-ted using Ni-NTA affinity column associated to Nu-polyacrylamide gel electrophoresis analysis reveals the presence of one single band of Mw 19,000 corresponding to the purified recombinant N-ted. The purified N-ted was able to recognize VIP and the selective antagonist PG96-269. About 5-10 mg of functional purified protein/liter of bacterial culture is currently produced. This is a crucial step to determine the structure of functional human VPAC1 receptor N-ted by nuclear magnetic resonance.

Cell surface targeting of VPAC1 receptors: evidence for implication of a quality control system and the proteasome.

Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.

HLA-B27 subtypes differentially associated with disease exhibit conformational differences in solution.

Human leukocyte antigen (HLA) class I molecules consist of a heavy chain, beta(2)-microglobulin, and a peptide that are noncovalently bound. Certain HLA-B27 subtypes are associated with ankylosing spondylitis (such as HLA-B*2705), whereas others (such as HLA-B*2709) are not. Both differ in only one residue (Asp116 and His116, respectively) in the F pocket that accommodates the peptide C-terminus. An isotope-edited IR spectroscopy study of these HLA-B27 subtypes complexed with the self-peptide RRKWRRWHL was carried out, revealing that the heavy chain is more flexible in the HLA-B*2705 than in the HLA-B*2709 subtype. In agreement with these experimental data, molecular dynamics simulations showed an increased flexibility of the HLA-B*2705 binding groove in comparison with that of the HLA-B*2709 subtype. This difference correlates with an opening of the HLA-B*2705 binding groove, accompanied by a partial detachment of the C-terminal peptide anchor. These combined results demonstrate how the deeply embedded polymorphic heavy-chain residue 116 influences the flexibility of the peptide binding groove in a subtype-dependent manner, a feature that could also influence the recognition of the HLA-B27 complexes by effector cells.

Effect of positive charge in VIP 16gamma-glutamyl diamino derivatives on hVPAC1 and hVPAC2 receptor function.

Increase of VPAC receptor s binding to the (16)gamma-glutamyl diaminopropane vasoactive intestinal peptide (VIP-DAP) agonist, a vasoactive intestinal polypeptide (VIP) structural analogue containing a positive charge at position 16, has confirmed the importance of a positive charge at this site. By investigating the effect of distance from the peptide backbone Calpha of a positive charge in position 16, data are reported here concerning: (i) a novel chemical method used for the synthesis of a new family of (16)gamma-glutamyl diamine VIP derivatives differing among them for single carbon atoms and including diaminoethane (VIP-DAE2), diaminopropane (VIP-DAP3), diaminobutane (VIP-DAB4), diaminopentane (VIP-DAP5), and diaminohexane (VIP-DAH6); (ii) functional characterization of these compounds on human VPAC1 and VPAC2 receptors. In more detail, the EC50 and IC50 values, when measured as a function of the alkylic chain length, show in more detail, that the use of VIP-DAB4 derivative changes the IC50 but not the EC50, thus indicating on hVPAC2 receptor an unexpected relationship between binding and activity that differs from that obtained on hVPAC1.

The vasoactive intestinal peptide (VIP) alpha-Helix up to C terminus interacts with the N-terminal ectodomain of the human VIP/Pituitary adenylate cyclase-activating peptide 1 receptor: photoaffinity, molecular modeling, and dynamics.

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors. We characterized the C terminus-binding site of VIP in the N-terminal ectodomain (N-ted) of the human VPAC1 receptor: 1) The probe [(125)I-Bpa(28)]VIP in which the C-terminal residue (Asn(28)) is substituted by a photoreactive p-benzoyl-l-Phe (Bpa) was used to photolabel the receptor. After receptor cleavage and Edman sequencing, it was shown that Asn(28) of VIP is in contact with Lys(127) in the receptor N-ted. Taking into account previous data, it follows that the C-terminal and central parts of VIP from Asn(28) to Phe(6) lie in the N-ted. 2) A three-dimensional model of the N-ted was constructed, the fold being identified as a Sushi domain with two antiparallel beta-sheets and three disulfide bonds. The nuclear magnetic resonance structure of VIP was then docked into this model by taking into account the constraint provided by photoaffinity experiments with [(125)I-Bpa(28)]VIP. It appeared that VIP runs parallel to the beta3-beta4 antiparallel sheets. 3) We performed molecular dynamic simulations over 14 nsec of the complex between VIP and receptor N-ted and the free N-ted. The structural model of the free N-ted is stable, and VIP tends to further stabilize the N-ted structure more especially in the loops connecting the beta-sheets. These structural studies provide a detailed molecular understanding of the VIP-receptor interaction.

Class II G protein-coupled receptors for VIP and PACAP: structure, models of activation and pharmacology.

VIP and PACAP impact strongly on human pathophysiology. Their receptors are very promising targets for developing new drugs in the treatment of inflammatory and neurodegenerative diseases. This article reviews the present knowledge regarding VIP and PACAP receptors, i.e. VPAC1, VPAC2 and PAC1. This includes: (I) a critical review of instrumental peptide agonists and antagonists; (II) a survey of recent data regarding the structure of VPAC1 receptor and the docking of VIP in the receptor binding domain. Structural models for the VPAC2 and PAC1 receptor N-terminal ectodomains are also described; (III) A critical description of the two models of VPAC1 receptor activation in the general context of class II/family B G protein-coupled receptors.

Identification and characterization of five-transmembrane isoforms of human vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors.

The seven-transmembrane (7TM) G-protein-coupled neuroendocrine receptors VPAC1 (HGNC approved gene symbol VIPR1) and VPAC2 (HGNC approved gene symbol VIPR2) are expressed in different tissues and involved in the regulation of important biological functions. We now report the identification and characterization of novel five-transmembrane(5TM) forms of both human VPAC1 and human VPAC2. These alternatively spliced variant mRNAs result from the skipping of exons 10/11, spanning the third intracellular loop, the fourth extracellular loop, and the transmembrane regions 6 and 7, producing in-frame 5TM receptors predicted to lack a G-protein-binding motif. RT-PCR showed that these 5TM receptors are differentially expressed in transformed and normal cells. Translation of the 5TM protein was demonstrated by transfection and expression in CHO cells. Following agonist stimulation, differential signaling of the 7TM versus 5TM forms was shown both for the activation of adenylate cyclase and for tyrosine phosphorylation. The identification of these splice variants in various cells and their expression and differential signal transduction compared to the 7TM form suggest that these novel receptors have biological relevance.

The human VPAC1 receptor: identification of the N-terminal ectodomain as a major VIP-binding site by photoaffinity labeling and 3D modeling.

The human VPAC1 receptor for VIP and PACAP is a class II Gprotein-coupled receptor (GPCR). The N-terminal ectodomain of the VPAC1 receptor plays a crucial role in VIP binding. Photoaffinity experiments clearly indicated that the 6-28 part of VIP physically interacts with the N-terminal ectodomain. Construction of a 3D model of the N-terminal ectodomain of VPAC1 receptor based on the NMR structure of the mouse CRF receptor 2 indicated the presence of short consensus repeat/Sushi domain. Docking of VIP in the N-terminal ectodomain structural model was performed taking into account the severe constraints provided by photoaffinity. A VIP-binding site was identified on the side of the structured core of the N-terminal ectodomain of the receptor.

Mutation of the phosphorylatable residue Thr429 in Glu of the human VPAC1 led to a constitutively desensitized receptor.

The hVPAC1 receptor is rapidly phosphorylated and internalized by agonists but not re-expressed at the membrane after washing. Mutation of Ser/Thr residues in the C-terminus reduced phosphorylation but not internalization that was abolished only when all the phosphorylatable residues were mutated. Substitution of Thr429 by Glu mimicking a phosphothreonin led to a mutant with unchanged binding properties, decreased coupling to adenylate cyclase consisting in a reduced VIP potency, increased basal and VIP stimulated phosphorylation, preserved internalization followed by a rapid receptor re-expression. These are the expected characteristics of a constitutively desensitized receptor, putting forward the role of Thr429 phosphorylation in that process.

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling.

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.