The human VPAC1 receptor: three-dimensional model and mutagenesis of the N-terminal domain.

The human VPAC(1) receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC(1) receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1-144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC(1) receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC(1) receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu(36), Trp(67), Asp(68), Trp(73), and Gly(109)) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro(74), Pro(87), Phe(90), and Trp(110) are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC(1) receptor.

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.

The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20.

The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2 cells.

Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice.

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.

The human vasoactive intestinal Peptide/Pituitary adenylate cyclase activating peptide receptor 1 (VPAC1): constitutive activation by mutations at threonine 343.

The human vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide receptor1 (VPAC1) belongs to the class II subfamily of G protein-coupled receptors. Specific changes by mutagenesis of a strictly conserved threonine (H) into lysine (K), proline (P) or alanine (A) at position 343 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells evoked a 3.5 fold-increase in the cAMP level as compared to cells transfected with the wild-type receptor. In contrast other mutants such as T343C, T343E or T343F were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the T343K mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants T343K-E36A and T343K-D68A were no longer constitutively activated. A control double mutant (T343K-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VPAC1 receptor can be evoked by specific mutations of T343 at the junction of the second intracellular loop and fourth transmembrane segment. This constitutive activation appears to require the functional integrity of the N-terminal extracellular VIP binding domain.

Constitutive activation of the human vasoactive intestinal peptide 1 receptor, a member of the new class II family of G protein-coupled receptors.

The human vasoactive intestinal peptide (VIP) 1 receptor belongs to the new class II subfamily of G protein-coupled receptors. Specific change by mutagenesis of a strictly conserved histidine into arginine at position 178 of the human VIP1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of the H178R mutant into COS cells resulted in a 3.5-fold increase in the cAMP level as compared with cells transfected with the wild type receptor or the vector alone. This increase was proportional to the amount of transfected cDNA. The H178R mutant exhibited an otherwise normal cAMP response to VIP as well as a dissociation constant similar to that of the wild type receptor. Other mutants at position 178 such as H178K, H178A, and H178D were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the H178R mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production, i.e. E36A or D68A. The corresponding double mutants H178R/E36A and H178R/D68A were no longer constitutively activated. A control double mutant (H178R/D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP was still constitutively activated. Our findings demonstrate that constitutive activation of the VIP1 receptor by mutation of His178 into R requires the functional integrity of the N-terminal extracellular VIP binding domain. They might provide interesting generalities about the activation process of G protein-coupled receptors.

Cloning and functional characterization of the human VIP1/PACAP receptor promoter.

The 5'-flanking region (1.5 kb) of the gene coding for the human VIP1/PACAP receptor was isolated, sequenced, and characterized. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the VIP1/PACAP receptor fused to a luciferase reporter gene showed that this sequence was active as a promoter in the intestinal cancer cell line, HT-29, expressing endogenous VIP1/PACAP receptor. The shortest DNA fragment with significant promoter activity encompassed the region from -205 to +76 bp. Deletion of a CCAAT-box sequence in the construction corresponding to -173 to +76 bp dramatically reduced the promoter activity. The promoter -205 to +76 bp has a housekeeping gene structure without TATA-box. It contains GC-rich regions characterized by potential Sp1 and AP2 sites and some potential regulatory elements, such as CRE and ATF, and a CCAAT-box sequence (-182 to -178) crucial for gene transcription.

Vasoactive intestinal peptide (VIP)1 receptor. Three nonadjacent amino acids are responsible for species selectivity with respect to recognition of peptide histidine isoleucineamide.

Vasoactive intestinal peptide (VIP)1 receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting 125I-VIP binding was shown to be >1,000 and <40, respectively. Construction of eight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to human and rat sequences, respectively, e.g. Gln207 (human) versus His208 (rat), Gly211 versus Ala212 and Met219 versus Val220. Site-directed mutagenesis introducing individual, double, or triple mutations in a chimeric construct revealed that all three amino acids were involved in the recognition of PHI. Triple mutations were then introduced in the wild-type receptors i.e. Q207H, G211A, M219V human VIP1 receptor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, respectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.

Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: pharmacological, functional and molecular properties.

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP)1 receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [125I]VIP binding to cell membranes with a Kd of 0.41 nM and a Bmax of 1.62 pmol/mg protein; (2) the constant Ki for the inhibition of [125I]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 microM) < peptide histidine methionineamide (2.0 microM) < secretin (> 10 microM); (3) cross-linking experiments using [125I]VIP identified a single M(r) 67000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells with an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe6, Leu17]VIP, [Ac-Tyr1,D-Phe2]GRF-(1-29) amide and VIP-(10-28) inhibited [125I]VIP binding with a Ki of 0.7, 1.6 and 2.5 microM, respectively. They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in Bmax in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structures within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.

Mutational analysis of cysteine residues within the extracellular domains of the human vasoactive intestinal peptide (VIP) 1 receptor identifies seven mutants that are defective in VIP binding.

We have used site-directed mutagenesis to investigate the requirement of cysteine residues in the extracellular domains of the human VIP 1 receptor for binding VIP. Cys37, Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 (N-terminal extracellular domain), Cys208 and Cys215 (second extracellular loop) and Cys288 (third extracellular loop) were mutated into glycine, and mutated cDNAs transfected into Cos-7 cells. It appeared that mutants C50G, C63G, C72G, C86G, C105G, C122G, and C288G did not bind VIP whereas mutants C37G, C208G and C215G bound VIP with the same dissociation constant (#0.5 nM) as the wild-type receptor. All mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and delivered at the plasma membrane level (confocal microscopy). The fact that C208G and C215G mutants retained a complete binding activity while the C288G mutant was inactive does not suggest the presence of a functionally relevant disulfide bond between the second and third extracellular loop of the human VIP receptor contrary to what has been shown in several other heptahelical receptors. It is also concluded that the six crucial cysteine residues, e.g., Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 in the N-terminal extracellular domain, may be functionally important by forming intramolecular disulfide bonds which help to maintain the topology for ligand binding in human VIP 1 receptors.

Highly conserved aspartate 68, tryptophane 73 and glycine 109 in the N-terminal extracellular domain of the human VIP receptor are essential for its ability to bind VIP.

The human VIP receptor belongs to a subfamily of G protein-coupled receptors that includes secretin, glucagon and several other receptors. We have used site-directed mutagenesis to investigate the requirement of highly conserved aspartate 68 (mutant D68G), tryptophane 73 (mutant W73G), proline 87 (mutant P87G), glycine 109 (mutant delta 109) and tryptophane 110 (mutant W110G) for the ability of the human VIP receptor to bind VIP. After transfection of mutated cDNAs in Cos-7 cells, it appeared that mutants G87P and W110G bound VIP with the same dissociation constant as the wild type receptor whereas mutants W73G, P87G and delta 109 did not bind VIP. Since all mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and expressed at the plasma membrane level (immunofluorescence studies), it is concluded that the N-terminal extracellular domain of the human VIP receptor contains highly conserved amino acid residues which are essential for its intrinsic binding activity.

Regional expression of epithelial dipeptidyl peptidase IV in the human intestines.

The expression of DPP IV/CD26 was studied in epithelial cells from different regions of the human small intestine and colon. The following data were obtained: 1) DPP IV/CD26 activity was highest in ileum and jejunum, low in duodenum and not detectable in colon; 2) The amount of DPP IV, as analyzed by Western blotting, was very low in duodenum and increased up to the ileum. No DPP IV protein was detected in colon; 3) RT-PCR analysis showed the expected 315-bp product in the small intestine with the highest signal in ileum and jejunum and a low signal in duodenum; a faint PCR signal could be detected in colon. Similar data were obtained by Northern blot analysis which revealed a 3.6-kb transcript only in small intestine. It is concluded that DPP IV/CD26 gene expression in human intestine is highest in the distal small intestine, and that is regulated at the mRNA level.

Neurotensin receptor and its mRNA are expressed in many human colon cancer cell lines but not in normal colonic epithelium: binding studies and RT-PCR experiments.

Neurotensin receptor expression was studied in 19 human colon cancer cell lines and normal human colon by i) binding experiments using [125I-Tyr3]-neurotensin; ii) RT-PCR analysis. The following data were obtained: 1) A single class of receptor (Kd ranging from 0.23 to 1.21 nM) was found in 9 out of 19 cell lines but not in normal colonic epithelium; 2) The Bmax was in the range between 1000 and 85 fmoles/mg protein with SW48 > WiDR > Cl 19A > HCT116 > SW480 > SW620 > Cl 16E > Cl 27H > HT-29. No specific binding was measurable in Caco-2, FRI, CBS, EB, HCT-8, 320HRS, 320DM and LS174T cell lines; 3) A single RT-PCR product was observed in HT-29, SW48, WIDR, Cl 19A, SW480, Cl 16E, Cl 27H, SW620 and HCT116, but not in other cell lines or in normal human colon. It is concluded that the expression of neurotensin receptors in human colon cancer cells is regulated at the mRNA level and occurs upon malignancy in > 40% of colon cancer cell lines.

Human intestinal VIP receptor: cloning and functional expression of two cDNA encoding proteins with different N-terminal domains.

We describe here two human VIP receptor cDNA clones isolated from a jejunal epithelial cell cDNA library. The hIVR8 cDNA encodes a human VIP receptor consisting of 460 amino acids and has seven putative transmembrane domains like other G protein-coupled receptors. When expressed in COS-7 cells, hIVR8 conferred specific [125I]VIP binding sites (dissociation constant = 0.6 nM) with displacement patterns characteristic of the human common VIP/PACAP receptor, i.e., VIP = PACAP-27 > PACAP-38 > helodermin > growth hormone-releasing factor = peptide methionineamide > secretin. It also conferred stimulation of cAMP production by VIP (half-maximal stimulation for 0.5 nM peptide). Another clone, hIVR5, encodes a 495 amino acid VIP receptor-related protein exhibiting 100% homology with the functional VIP receptor (hIVR8) over the 428 amino acids at the C-terminus but a completely divergent 67 amino acid N-terminal domain. When expressed in COS-7 cells, this VIP receptor-related protein does not bind 125I-VIP, although it is normally addressed at the plasma membrane as assessed by immunofluorescence studies.

Characterization of a common VIP-PACAP receptor in human small intestinal epithelium.

Vasoactive intestinal peptide (VIP) receptors were characterized in epithelial plasma membranes from human small intestine. Native VIP inhibited the binding of 125I-labeled VIP to jejunal membranes, and Scatchard analysis of these data was consistent with the existence of one class of receptor with a dissociation constant of 42 pM and a maximal binding of 256 fmol/mg membrane protein. VIP stimulated adenylyl cyclase activity in human jejunal membranes in the 0.01 nM-1 microM range [half-maximal effective dose = 0.7 nM]. Coupling of VIP receptors with a Gs protein was further assessed by the ability of GTP (10(-8) to 10(-3) M) to inhibit 125I-VIP binding to membranes. 125I-VIP binding was seven to eight times higher in villus cells than in crypt cells. Finally, 125I-VIP binding was detectable throughout the small and large intestines with the highest binding in jejunum. Among the natural peptides structurally related to VIP, some inhibited 125I-VIP binding with the following order of potency: VIP = pituitary adenylate cyclase-activating peptide (PACAP)-27 = PACAP-38 > helodermin >> peptide histidine methionineamide = human growth hormone-releasing factor > secretin. The same order of potency of peptides for inhibiting 125I-VIP or 125I-labeled PACAP was observed, supporting that the two tracers bound to a common VIP-PACAP receptor site. This order of potency was also observed for the stimulation of adenylyl cyclase activity by these peptides. 125I-VIP was cross-linked to membranes using disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, one single band of 70,000 mol wt was observed.(ABSTRACT TRUNCATED AT 250 WORDS)