Vasoactive Intestinal Peptide Binding Research Articles

Heparin Oligosaccharides as Vasoactive Intestinal Peptide Inhibitors via their Binding Process Characterization.

It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al. found that by an alanine scan, the heparin-binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP. The binding process between heparin oligosaccharides and VIPA variety of experiments was designed to study the structure-activity relationship between heparin oligosaccharides and VIP. Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro. The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP. The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity.

Heme and Cu2+-induced vasoactive intestinal peptide (VIP) tyrosine nitration: A possible molecular mechanism for the attenuated anti-inflammatory effect of VIP in inflammatory diseases

Vasoactive intestinal peptide (VIP) is a neuropeptide that play an important role in immunoregulation and anti-inflammation. Numerous inflammatory/autoimmune disorders are associated with decreased VIP binding ability to receptors and diminished VIP activation of cAMP generation in immune cells. However, the mechanisms linking oxidative/nitrative stress to VIP immune dysfunction remain unknown. It has been reported that the elevated heme or Cu2+ in inflammatory diseases can cause oxidative and nitrative damage to nearby biological targets under high oxidative stress conditions, which affects the structure and activity of linked peptides or proteins. Thus, the VIP down-regulated immune response may be interfered by redox metal catalyzed VIP tyrosine nitration. To explore this, we systematically investigated the possibility of heme or Cu2+ to catalyze VIP tyrosine nitration. The results showed that Tyr10 and Tyr22 of VIP can both be nitrated in heme/H2O2/NO2− system as well as in Cu2+/H2O2/NO2− system. Then, we used synthetic mutant VIPs with tyrosine residues substituted by 3-nitrotyrosine to study the impact of tyrosine nitration on VIP activity in SHSY-5Y cells. Our findings demonstrated that VIP nitration dramatically decreased the content of its α-helix and random coil, suggesting that VIP nitration might reduce its affinity to the receptor. This was further confirmed in the cAMP assay. The results showed that 10 nM of these tyrosine nitrated VIPs could significantly (p < 0.01) decrease cAMP secretion compared to the wild type VIP. Our data reveal that the attenuation of the neuroprotective effect of VIP in inflammation-related diseases might be attributed to metal-catalyzed VIP tyrosine nitration.

Administration of a VIP-antagonist in vivo modifies ovarian hormone secretion in a rat model with polycystic ovary syndrome

AimsIn the cyclic rat in estrus, the vasoactive intestinal peptide (VIP) has an impact on ovarian function, which depends on the endocrine status of the animal. In this work, we aimed to clarify the participation of VIP in the pathophysiological condition of polycystic ovary syndrome (PCOS) using a model of PCOS induced by estradiol valerate (EV-PCOS) in rats. Main methodsIn the cyclic rat in estrus and in the EV-PCOS model, we analyzed the acute effects of blocking VIP receptors with the use of an antagonist (Ant-VIP) injected into the left or right ovarian bursa on the steroidogenic response and ovarian catecholamine levels. Key findingsIn the cyclic animal in estrus, the treatment with Ant-VIP in the left ovarian bursa resulted in a reduction in testosterone serum levels and in ovarian levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), without changes in 4-hydroxy-3-methoxyphenyl (MHPG) and norepinephrine (NE). When the treatment was applied on the right side, only MHPG levels increased.In the EV-PCOS model, the treatment with Ant-VIP in the left ovarian bursa increased testosterone, estradiol, MHPG, and NE levels. When the treatment was performed on the right side, progesterone levels decreased and estradiol increased, without changes in ovarian catecholamines. SignificanceThe binding of VIP to its receptors differentially regulates steroidogenesis in the cyclic animal in estrus and in the EV-PCOS model. The blocking of VIP signaling produces changes in ovarian catecholamines.

Heterogeneity of Vasoactive Intestinal Peptide Binding Sites in Hut‐78 T‐Lymphocytes

Vasoactive Intestinal Peptide (VIP) is a neuropeptide that possesses potent cell cycle arrest activity in primary healthy T‐Lymphocytes. Transformed T‐cells from human cancer patients and over 25 cell lines (human, mouse, and rat) have consistently shown reduced VIP receptor expression, suggesting that cancer cells obtain a growth advance with reduced VIP receptor signaling. Utilizing the human Hut‐78 T‐lymphoblastic T‐cell line that has reduced levels but functional VIP binding sites, we carried out growth curves +/‐ VIP utilizing alamar blue. Surprisingly, these results were inconclusive as they showed opposite effects of growth from exogenous addition of VIP ligand. To better understand the discrepancy in growth rates by VIP we measured the homogeneity of VIP binding sites in Hut‐78s by VIP‐FAM labeling and flow cytometry analysis. Curiously, three definable subpopulations possessing varying levels of VIP binding sites were observed. These three populations were sorted, cultured, and showed a stable homogenous population with intermediate levels of VIP binding sites. Collectively, we conclude that the heterogeneity of VIP binding sites in HUT‐78s may explain the disparate growth results collected above. Future studies will utilize the stable sorted homogenous VIP binding site sorted populations to investigate the cell cycle arresting abilities of VIP.

Protected graft copolymer excipient leads to a higher acute maximum tolerated dose and extends residence time of vasoactive intestinal Peptide significantly better than sterically stabilized micelles.

To determine and compare pharmacokinetics and toxicity of two nanoformulations of Vasoactive Intestinal Peptide (VIP). VIP was formulated using a micellar (Sterically Stabilized Micelles, SSM) and a polymer-based (Protected Graft Copolymer, PGC) nanocarrier at various loading percentages. VIP binding to the nanocarriers, pharmacokinetics, blood pressure, blood chemistry, and acute maximum tolerated dose (MTD) of the formulations after injection into BALB/c mice were determined. Both formulations significantly extend in vivo residence time compared to unformulated VIP. Formulation toxicity is dependent on loading percentage, showing major differences between the two carrier types. Both formulations increase in vivo potency of unformulated VIP and show acute MTDs at least 140 times lower than unformulated VIP, but still at least 100 times higher than the anticipated highest human dose, 1-5μg/kg. These nanocarriers prevented a significant drop in arterial blood pressure compared to unformulated VIP. While both carriers enhance in vivo residence time compared to unformulated VIP and reduce the drop in blood pressure immediately after injection, PGC is the excipient of choice to extend residence time and improve the safety of potent therapeutic peptides such as VIP.

Conformational switches in the VPAC1 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.

Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe6, Tyr10, Leu13, and Met17 found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water–micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.

Evidence that Interaction between Conserved Residues in Transmembrane Helices 2, 3, and 7 Are Crucial for Human VPAC1 Receptor Activation

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

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 <<Sushi>> 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.

Development of peptide receptor binding assays: Methods to avoid false negatives

Selection of appropriate ligand receptor binding assay conditions is critical for peptides, where the possibility of obtaining false negative results is pertinent due to their inherent adsorption and instability characteristics, as well as high response-sensitivity to operational conditions. The aim of this study was thus to develop a cost-effective multivariate screening method for determination of the influence of different factors on the outcome of such studies, using 125I-labelled vasoactive intestinal peptide binding on lung homogenate as a model. The study was divided into two parts: investigation of filtration for bound–unbound ligand separation, and screening of sample incubation variables. Experimental designs were used (including Plackett–Burman) to evaluate adsorption, total binding, non-specific binding, specific binding and (non-)specific/total binding ratio. Several significant factors were identified. For filtration, a combination of polyethylenimine and BSA filter pretreatment was best, whereas albumin-containing washing solvent negatively influenced the amount of specific bound radioligand. For sample incubation, significant effects on one or more of the studied responses were observed for several factors. Bacitracin protease inhibitor also decreased adsorption. We report here multivariate experimental designs for screening of peptide (radio)ligand receptor binding assay conditions. This approach efficiently minimizes the risk on false negative results due to inappropriate operational conditions.

The 5TM VPAC1 Isoform Blocks cAMP Signaling Evoked from the Full‐Length 7TM receptor

Vasoactive intestinal peptide receptor 1 (VPAC1) couples heterotrimeric G proteins, Gαs and Gαi, that activate and inhibit adenylate cyclase cAMP production. The 7 transmembrane (TM) VPAC1 isoform, increases intracellular cAMP ([cAMP]i) upon binding its ligand, vasoactive intestinal peptide (VIP). In contrast, the 5TM VPAC1 isoform fails to increase [cAMP]i over a range of 10‐6‐10‐12 M VIP. Our hypothesis is that the dual expression of both the 5 and 7TM isoforms cause a lack of [cAMP]i elevation due to an antagonist effect by the 5TM on 7TM signaling. To investigate this, human HuT 78 cells, a malignant T cell, which express both 5 and 7TM VPAC1 isoforms. Levels of [cAMP]i were measured by a competitive ELISA after treating with increasing concentrations of VIP. Interesting, we show a VIP dependent decrease in [cAMP]i, suggesting that the 5TM VPAC1 isoform coexpressed in HuT 78 cells suppress 7 TM induced [cAMP]i levels. The ratio of the 5 and 7TM receptors on the cell membrane may explain the unique level of [cAMP]i upon VIP binding. Future studies will inhibit Gαi coupling with pertussis toxin and RNAi to knockdown the 5TM VPAC1 to measure [cAMP]i response evoked by the 7TM VPAC‐1 receptor in coexpressing cells. Research supported by NIH‐KO1 1KO1DK664828 and COBRE 2P20RR015566.

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.

Detection of the Five Transmembrane Vasoactive Intestinal Peptide Receptor‐1 Isoform in Mouse Tissue

Vasoactive intestinal peptide (VIP) is a pleiotropic secretagogue involved in crucial biological processes necessary for life. Its biological effects are mediated by two structurally similar integral membrane receptors called vasoactive intestinal peptide receptor‐1 and 2 (VPACR‐1/2). VPACR‐1 is a member of the group IIA G protein coupled receptors (GPCR) which are characterized by 7 transmembrane domains (7TM). VIP binding to VPACR‐1 elicits adenylate cyclase/cAMP/PKA and phospholipase C/D signaling cascades. VPACR‐1 has a wide expression profile in the mouse, including the nervous and immune systems. Recently, a novel VPACR‐1 isoform was identified that lacks exons 10 and 11 to form a 5 transmembrane receptor (5TM), and in contrast, elicits significant tyrosine phosphorylation through a G protein independent mechanism. The biological effects mediated by this recently discovered non‐GPCR linked isoform, 5TM, are not known. To determine the expression profile of this isoform, mRNA will be collected from various mouse tissues, cDNA generated with anchored primers and the expression profile of the 7TM and 5TM quantitated using qPCR. Identifying which mouse tissues express the 5 TM VPACR‐1 isoform is a major first step in understanding its contribution to cellular function and physiology. Supported by NIH‐KO1 1K01DK064828 and COBRE 2P20RR05566.

Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2receptors in smooth muscle

The smooth muscle of the gut expresses mainly G(s) protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC(2) receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC(2) receptors via the phosphorylation of GRK2 at Ser(685). VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to G betagamma. VIP-induced phosphorylation of VPAC(2) receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC(2) receptor internalization (determined from residual (125)I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC(2) receptor degradation (determined from residual (125)I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC(2) receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC(2) receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC(2) receptor phosphorylation, internalization, desensitization, and degradation.

Decrease in Binding for the Neuropeptide VIP in Response to Marked Inflammation of the Mucosa in Ulcerative Colitis

The neuropeptide vasoactive intestinal peptide (VIP) is involved in the neuroimmunomodulation of the intestine. In the present study, specimens from the sigmoid colon of ulcerative colitis (UC) and non-UC patients were examined for immunohistochemistry and in vitro receptor autoradiography. Marked occurrence of VIP binding was observed in the mucosa. However, there were very low levels of binding in areas showing pronounced inflammation/derangement. The study shows that marked derangement of the mucosa leads to a distinct decrease in VIP binding. Thus, it is possible that a decrease in trophic and anti-inflammatory VIP effects occurs in areas exhibiting a very marked inflammation.

Blockage of VIP during mouse embryogenesis modifies adult behavior and results in permanent changes in brain chemistry

Vasoactive intestinal peptide (VIP) regulates growth and development during the early postimplantation period of mouse embryogenesis. Blockage of VIP with a VIP antagonist during this period results in growth restriction, microcephaly, and developmental delays. Similar treatment of neonatal rodents also causes developmental delays and impaired diurnal rhythms, and the adult brains of these animals exhibit neuronal dystrophy and increased VIP binding. These data suggest that blockage of VIP during the development of the nervous system can result in permanent changes to the brain. In the current study, pregnant mice were treated with a VIP antagonist during embryonic days 8 through 10. The adult male offspring were examined in tests of novelty, paired activity, and social recognition. Brain tissue was examined for several measures of chemistry and gene expression of VIP and related compounds. Glial cells from the cortex of treated newborn mice were plated with neurons and examined for VIP binding and their ability to enhance neuronal survival. Treated adult male mice exhibited increased anxiety-like behavior and deficits in social behavior. Brain tissue exhibited regionally specific changes in VIP chemistry and a trend toward increased gene expression of VIP and related compounds that reached statistical significance in the VIP receptor, VPAC-1, in the female cortex. When compared to control astrocytes, astrocytes from treated cerebral cortex produced further increases in neuronal survival with excess synaptic connections and reduced VIP binding. In conclusion, impaired VIP activity during mouse embryogenesis resulted in permanent changes to both adult brain chemistry/cell biology and behavior with aspects of autism-like social deficits.

VIP regulation of embryonic growth.

Vasoactive intestinal peptide (VIP) plays a regulatory role in the growth of early postimplantation rodent embryos through its action on receptors localized to the central nervous system (CNS). However, the origin of the VIP influencing embryonic growth is unknown. VIP binding sites have been found prenatally; however, VIP mRNA was not detected in the rat CNS before birth and has been detected in peripheral organs only during the final third of gestation. Recent studies have revealed that VIP receptors were limited to the CNS in the embryonic day 11 (E11) rat embryo/trophoblast, which, in addition, had almost four times the VIP concentration of the E17 fetus. However, neither in situ hybridization or reverse transcriptase-polymerase chain reaction methods detected VIP mRNA in the E11 rat embryo or embryonic membranes. Rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP levels 6- to 10-fold higher than later during pregnancy. Radiolabeled VIP, administered intravenously to pregnant female mice, was found in the E10 embryo. These results suggest that VIP produced by extraembryonic tissues may regulate embryonic growth during the early postimplantation stage of development in the rodent.

New Nonradioactive Technique for Vasoactive Intestinal Peptide‐Receptor‐Ligand‐Binding Studies

We describe fluorescent-labeled peptide (FLP) studies on living cells. The new technique is nonradioactive and it allows monitoring of the binding and internalization of Vasoactive Intestinal Peptide (VIP) in VIP receptor-expressing cells. The technique is easy to perform and the observed reaction is peptide sequence specific.

Characterization of the New Photoaffinity Probe (Bz2‐K24)‐VIP

Site-directed mutagenesis and molecular modeling demonstrated that the N-terminal ectodomain of the VPAC1 receptor is a major site of vasoactive intestinal peptide (VIP) binding. Previous studies with the [Bpa6]-VIP and [Bpa22]-VIP probes (substitution with the photoactivable Bpa for the residues 6 and 22 in VIP) showed spatial approximation between the amino acids 6 and 22 of VIP and the 104-108 and 109-119 sequences within the N-terminal ectodomain of the receptor, respectively. Here, we characterize the new probe (Bz2-K24)-VIP (substitution with the photoreactive Bz2-K for the residue 24 in VIP). After photolabeling and sequential digestions of the receptor, the 121-133 sequence of the N-terminal ectodomain was identified as the site of interaction. The N-terminal ectodomain of the VPAC1 receptor is therefore an affinity trap for the central part of VIP, at least between residues 6 and 24.

Shark rectal gland vasoactive intestinal peptide receptor: cloning, functional expression, and regulation of CFTR chloride channels

Vasoactive intestinal peptide (VIP) is a secretagogue that mediates chloride secretion in intestinal epithelia. We determined the relative potency of VIP and related peptides in the rectal gland of the elasmobranch dogfish shark and cloned and expressed the VIP receptor (sVIP-R) from this species. In the perfused rectal gland, VIP (5 nM) stimulated chloride secretion from 250 +/- 66 to 2,604 +/- 286 microeq x h(-1) x g(-1); the relative potency of peptide agonists was VIP > PHI = GHRH > PACAP > secretin, where PHI is peptide histidine isoleucine amide, GHRH is growth hormone-releasing hormone, and PACAP is pituitary adenylate cylase activating peptide. The cloned sVIP-R from shark rectal gland (SRG) is only 61% identical to the human VIP-R1. It maintains a long, extracellular NH2 terminus with seven cysteine residues, and has three N-glycosylation sites and eight other residues implicated in VIP binding. Two amino acids considered important for peptide binding in mammals are not present in the shark orthologue. When sVIP-R and the CFTR chloride channel were coexpressed in Xenopus oocytes, VIP increased chloride conductance from 11.3 +/- 2 to 127 +/- 34 microS. The agonist affinity for activating chloride conductance by the cloned receptor was VIP > GHRH = PHI > PACAP > secretin, a profile mirroring that in the perfused gland. The receptor differs from previously cloned VIP-Rs in having a low affinity for PACAP. Expression of both sVIP-R and CFTR mRNA was detected by quantitative PCR in shark rectal gland, intestine, and brain. These studies characterize a unique G protein-coupled receptor from the shark rectal gland that is the oldest cloned VIP-R.