Alain Couvineau

VPAC Receptors for VIP and PACAP

VIP and PACAP are two prominent neuropeptides that share two common G protein-coupled receptors, VPAC1 and VPAC2, while PACAP has an additional specific receptor, PAC1. This article reviews the present knowledge regarding various aspects of VPAC receptors including: 1) receptor specificity toward natural VIP-related peptides and pharmacology of synthetic agonists or antagonists; 2) genomic organization and chromosomal localization; 3) signaling and established or putative interactions with G proteins or accessory proteins such as RAMPs or PDZ-containing proteins; 4) molecular basis of ligand-receptor interaction as determined by site-directed mutagenesis, construction of receptor chimeras, and structural modeling; 5) constitutively active receptor mutants; 6) short-term (desensitization, internalization, phosphorylation) and long-term (transcription) regulations and transgenic models; 7) receptor polymorphisms.

Molecular pharmacology and structure of VPAC receptors for VIP and PACAP

VIP and PACAP are two prominent neuropeptides which share two common G protein-coupled receptors VPAC1 and VPAC2 while PACAP has an additional specific receptor PAC1. This paper reviews the present knowledge regarding three aspects of VPAC receptors including: (i). receptor specificity towards natural VIP-related peptides and pharmacology of synthetic agonists or antagonists; (ii). receptor signaling; (iii). molecular basis of ligand-receptor interaction as determined by site-directed mutagenesis, construction of receptor chimeras and structural modeling.

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 of key residues for interaction of vasoactive intestinal peptide with human VPAC1 and VPAC2 receptors and development of a highly selective VPAC1 receptor agonist. Alanine scanning and molecular modeling of the peptide.

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC1 and VPAC2. Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initio molecular modeling; (ii) ability to inhibit 125I-VIP binding (K i ) and to stimulate adenylyl cyclase activity (EC50) in membranes from cell clones stably expressing human recombinant VPAC1or VPAC2 receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase ofK i or EC50 at the VPAC1receptor. Modeling of the three-dimensional structure of native VIP (central α-helice from Val5 to Asn24 with random coiled N and C terminus) and analogs shows that substitutions of His1, Val5, Arg14, Lys15, Lys21, Leu23, and Ile26 decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC1 receptor. The interaction of the analogs with human VPAC2 receptor is similar to that observed with VPAC1 receptor, with three remarkable exceptions: substitution of Thr11 and Asn28 by alanine increased K i for binding to VPAC2receptor; substitution of Tyr22 by alanine increased EC50 for stimulating adenylyl cyclase activity through interaction with the VPAC2 receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala11,22,28]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC1 receptor agonist derived from VIP ever described.

Class-B GPCR activation: is ligand helix-capping the key?

The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.

Novel stable PACAP analogs with potent activity towards the PAC1 receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.

Structural requirements for VIP interaction with specific receptors in human and rat intestinal membranes: Effect of nine partial sequences

Nine VIP sequences have been tested for their ability to inhibit the specific binding of 125I-VIP and to stimulate adenylate cyclase activity in intestinal epithelial membranes from rat and man. They are VIP 2-28; VIP 1-14; VIP 2-14; VIP 14-28; VIP 15-28; VIP 20-28; VIP 21-28 and two sequences where the N-terminal VIP 1-6 or VIP 1-9 have been joined covalently with the C-terminal VIP 20-28 or VIP 21-28. It appears that only VIP 2-28, VIP 14-28 and VIP 15-18 are able to inhibit competitively the binding of 125I-VIP to human and rat membranes. These analogues are respectively 88, 8,300 and 25,000 times less potent than VIP 1-28 in rat; they are respectively 70, 7,900 and 13,000 times less potent than VIP 1-28 in man. With respect to adenylate cyclase activation, VIP 14-28 and VIP 15-28 are very weak stimulators in the membranes from both species. VIP 2-28 behaves as a full VIP agonist in man whereas it is a partial VIP agonist in rat. These results indicate the structural importance of the whole VIP sequence for interacting with human and rat VIP receptors and further argue for a different structural requirement of rat and human receptors.

PTHR1 mutations associated with Ollier disease result in receptor loss of function

PTHR1-signaling pathway is critical for the regulation of endochondral ossification. Thus, abnormalities in genes belonging to this pathway could potentially participate in the pathogenesis of Ollier disease/Maffucci syndrome, two developmental disorders defined by the presence of multiple enchondromas. In agreement, a functionally deleterious mutation in PTHR1 (p.R150C) was identified in enchondromas from two of six unrelated patients with enchondromatosis. However, neither the p.R150C mutation (26 tumors) nor any other mutation in the PTHR1 gene (11 patients) could be identified in another study. To further define the role of PTHR1-signaling pathway in Ollier disease and Maffucci syndrome, we analyzed the coding sequences of four genes (PTHR1, IHH, PTHrP and GNAS1) in leucocyte and/or tumor DNA from 61 and 23 patients affected with Ollier disease or Maffucci syndrome, respectively. We identified three previously undescribed missense mutations in PTHR1 in patients with Ollier disease at the heterozygous state. Two mutations (p.G121E, p.A122T) were present only in enchondromas, and one (p.R255H) in both enchondroma and leukocyte DNA. Assessment of receptor function demonstrated that these three mutations impair PTHR1 function by reducing either the affinity of the receptor for PTH or the receptor expression at the cell surface. These mutations were not found in DNA from 222 controls. Including our data, PTHR1 functionally deleterious mutations have now been identified in five out 31 enchondromas from Ollier patients. These findings provide further support for the idea that heterozygous mutations in PTHR1 that impair receptor function participate in the pathogenesis of Ollier disease in some patients.

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 α helix, a disordered N-terminal His1-Phe6 segment and a 310 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 2β as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel β 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.

VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins

The vasoactive intestinal peptide (VIP) is a neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP displays a large biological functions including regulation of exocrine secretions, hormone release, fetal development, immune responses, etc. VIP appears to exert beneficial effect in neuro‐degenerative and inflammatory diseases. The mechanism of action of VIP implicates two subtypes of receptors (VPAC1 and VPAC2), which are members of class B receptors belonging to the super‐family of GPCR. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC receptors. The structure–function relationship of VPAC1 receptor has been extensively studied, allowing to understand the molecular basis for receptor affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies have clearly demonstrated the crucial role of the N‐terminal ectodomain (N‐ted) of VPAC1 receptor in VIP recognition. By using different approaches including directed mutagenesis, photoaffinity labelling, NMR, molecular modelling and molecular dynamic simulation, it has been shown that the VIP molecule interacts with the N‐ted of VPAC1 receptor, which is itself structured as a ‘Sushi’ domain. VPAC1 receptor also interacts with a few accessory proteins that play a role in cell signalling of receptors. Recent advances in the structural characterization of VPAC receptor and more generally of class B GPCRs will lead to the design of new molecules, which could have considerable interest for the treatment of inflammatory and neuro‐degenerative diseases.