In Vitro Properties of a High Affinity Selective Antagonist of the VIP1 Receptor

Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. In vitro properties of a high affinity selective antagonist of the VIP₁ receptor. Peptides 18(10) 1555–1560, 1997.—A selective high affinity VIP₁ receptor antagonist [Acetyl-His¹, D-Phe², Lys¹⁵, Arg¹⁶, Leu¹⁷] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP₁ receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP₁-, rat VIP₂- or the human VIP₂- recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP₁ receptor. The IC₅₀ values of ¹²⁵I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP₁-, rat VIP₂-, human VIP₁- and human VIP₂ receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP₁ receptor mediated stimulation of adenylate cyclase activity. The K_i values were respectively of 15 ± 5 nM and 2 ± 1 nM for the rat and human VIP₁ receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP₁ receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.     

Inhibition of expression of the type I G protein-coupled receptor for vasoactive intestinal peptide (VPAC1) by hammerhead ribozymes

Vasoactive intestinal peptide (VIP) is a neuromediator expressed widely in the nervous, gastrointestinal, respiratory, and immune systems. Two G protein-coupled receptors (GPCRs), designated VPAC1 and VPAC2, bind VIP with high affinity and transduce increases in [cyclic AMP](i) and [Ca(2+)](i). As there are no potent VPAC1- or VPAC2-selective antagonists, a hammerhead ribozyme (Rz) strategy capable of in vivo application was adopted to inactivate individual domains of VPAC1. Three Rzs were designed to cleave mRNA encoding the amino terminus, the third intracellular loop, and the cytoplasmic tail of human VPAC1 and were introduced by transfection into HEK-293 cells expressing recombinant human VPAC1. Each Rz specifically degraded VPAC1 mRNA and down-regulated VPAC1 protein and VIP-binding activity, as assessed by ribonuclease protection assays, Western blots, and binding of (125)I-VIP. Rz-mediated down-regulation of VPAC1 was associated with up to 75% suppression of VIP signaling of increases in [cyclic AMP](i) and [IP3](i), and of cyclic AMP response element-luciferase reports. The Rz specific for the amino terminus inhibited VPAC1 expression and signaling to the greatest extent. VIP-evoked cellular responses thus appear to be proportional to the level of VPAC1 expression. Specific Rzs may be powerful tools for manipulating tissue-specific contributions of GPCRs in vitro and in vivo.     

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.     

(N-stearyl, norleucine17) VIP hybrid inhibits the growth of pancreatic cancer cell lines

The effects vasoactive intestinal peptide (VIP) antagonists were investigated on pancreatic cancer cell lines. (N-Stearyl, Norleucine17) VIP hybrid ((SN)VIPhyb) inhibited 125I-VIP binding to human Capan-2 cells with an IC50 value of 0.01 microM whereas VIP hybrid had an IC50 value of 0.2 microM. By RT-PCR and Northern blot, VPAC1 receptor mRNA was detected in CAPAN-2 cells. One microM (SN)VIPhyb and 10 microM VIPhyb inhibited the ability of 30 nM VIP to elevate cyclic AMP and increase c-fos mRNA. (SN)VIPhyb, 1 microM inhibited the clonal growth of CAPAN-2 cells in vitro. In vivo, (SN)VIPhyb (10 microg/day s.c.) inhibited CAPAN-2 xenograft growth in nude mice. These results indicate that (SN)VIPhyb is a pancreatic cancer VPAC receptor antagonist.     

Atypical nuclear localization of VIP receptors in glioma cell lines and patients

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.     

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

VIP activates G(s) and G(i3) in rat alveolar macrophages and G(s) in HEK293 cells transfected with the human VPAC(1) receptor

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.     

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten microg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys15, Arg16, Leu27)VIP1-7GRF8-27 (VPAC1 selective) but not Ro25-1553 (VPAC2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC1 receptors predominate. By RT-PCR, VPAC1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC1 receptor antisera. By immunocytochemistry, VPAC1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.     

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 VPAC(1) and VPAC(2). 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 (125)I-VIP binding (K(i)) and to stimulate adenylyl cyclase activity (EC(50)) in membranes from cell clones stably expressing human recombinant VPAC(1) or VPAC(2) receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of K(i) or EC(50) at the VPAC(1) receptor. Modeling of the three-dimensional structure of native VIP (central alpha-helice from Val(5) to Asn(24) with random coiled N and C terminus) and analogs shows that substitutions of His(1), Val(5), Arg(14), Lys(15), Lys(21), Leu(23), and Ile(26) decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC(1) receptor. The interaction of the analogs with human VPAC(2) receptor is similar to that observed with VPAC(1) receptor, with three remarkable exceptions: substitution of Thr(11) and Asn(28) by alanine increased K(i) for binding to VPAC(2) receptor; substitution of Tyr(22) by alanine increased EC(50) for stimulating adenylyl cyclase activity through interaction with the VPAC(2) receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala(11,22,28)]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC(1) receptor agonist derived from VIP ever described.     

VIP as a trophic factor in the CNS and cancer cells

The effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and differentiation of brain neurons. Addition of VIP to embryonic mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic factor (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain growth and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell growth factor. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC(1) receptor antagonist. VIPhyb inhibits the basal growth of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC(1) receptors in cancer cells, VIP has been radiolabeled with 123I, 18F and 99mTc to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.     

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.     

Tryptophan 67 in the human VPAC(1) receptor: crucial role for VIP binding

The human receptor subtype for VIP and PACAP, referred to as VPAC(1) receptor, has a large N-terminal extracellular domain which is critical for VIP binding. We further investigated this domain by mutating 12 amino acid residues which could participate in the formation of a tight bend (W67) or a coiled coil motif. They were changed to alanine (A) and the cDNAs were transiently transfected into Cos cells. All mutants but W67A exhibited K(d) values similar to that of the wild-type receptor. For the W67A mutant, no specific (125)I-VIP binding could be observed. Mutants at the W67 site were further characterized after stable transfection of epitope-tagged VPAC(1) receptor-GFP fusion proteins into CHO cells. W67A, W67E, W67H, and W67K mutants neither bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F mutant mediated stimulation of adenylyl cyclase only at high VIP concentrations. Microscopic analysis and antibody binding experiments showed that all mutants were similarly expressed at the cell surface of CHO cells. Therefore tryptophan 67 in the human VPAC(1) receptor plays a crucial role in VIP binding due, in part, to its aromatic moiety.     

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Novel extended and branched N-terminal analogs of VIP

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an alpha helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.