The VPAC1 receptor: structure and function of a class B GPCR prototype.

The class B G protein-coupled receptors (GPCRs) represents a small sub-family encompassing 15 members, and are very promising targets for the development of drugs to treat many diseases such as chronic inflammation, neurodegeneration, diabetes, stress, and osteoporosis. The VPAC1 receptor which is an archetype of the class B GPCRs binds Vasoactive Intestinal Peptide (VIP), a neuropeptide widely distributed in central and peripheral nervous system modulating many physiological processes including regulation of exocrine secretions, hormone release, foetal development, immune response … VIP appears to exert beneficial effect in neurodegenerative and inflammatory diseases. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC1 receptors. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of VPAC1 plays a pivotal role in VIP recognition. The use of different approaches such as directed mutagenesis, photoaffinity labeling, Nuclear Magnetic Resonance (NMR), molecular modeling, and molecular dynamic simulation has led to demonstrate that: (1) the central and C-terminal part of the VIP molecule interacts with the N-ted of VPAC1 receptor which is itself structured as a « Sushi ¼ domain; (2) the N-terminal end of the VIP molecule interacts with the first transmembrane domain of the receptor where three residues (K(143), T(144), and T(147)) play an important role in VPAC1 interaction with the first histidine residue of VIP.

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.

Orexins/hypocretins and orexin receptors in apoptosis: a mini-review.

An unexpected and fascinating aspect of the neuropeptides orexins has recently emerged when it was shown that orexins acting at orexin receptors OX1R or OX2R induce dramatic apoptosis resulting in massive reduction in cell growth in various cancer cell lines. This mini-review will provide the reader with recent findings related to the proapoptotic actions of orexins and the entirely novel mechanism whereby the seven membrane-spanning G-protein-coupled receptor (GPCR) OX1R triggers apoptosis. Recent data show that orexins induce tyrosine phosphorylation of the tyrosine-based motifs - immunoreceptor tyrosine-based inhibitory motif and immunoreceptor tyrosine-based switch motif - in OX1R. These phosphorylations result in the recruitment and activation of the phosphotyrosine phosphatase SHP-2 and subsequent cytochrome c-mediated mitochondrial apoptosis. Finally, this mini-review will also speculate on: (1) the potential importance of tyrosine-based motifs in the large family of GPCRs; (2) the interest of orexin receptors as therapeutic targets in cancer therapy; (3) the possible role of orexin receptor-mediated apoptosis in physiology and pathophysiology in the brain (neurodevelopment, neurodegenerative diseases) and in the periphery.

Orexins and their receptors: structural aspects and role in peripheral tissues.

Orexins, also named hypocretins, were discovered in 1998 by subtractive cDNA cloning or orphan receptor technologies. Prepro-orexin is enzymatically matured into two peptides, orexin-A and orexin-B which are 33- and 28-amino-acid peptides, respectively. Two cloned orexin receptors OX1R and OX2R are serpentine G-protein-coupled receptors, both of which bind orexins and are coupled to Ca2+ mobilization. Orexins are neuropeptides present in hypothalamic neurons that project throughout the central nervous system to nuclei involved in the control of feeding, sleep-wakefulness, neuroendocrine homeostasis and autonomic regulation. The interest of investigators in orexins has focused on narcolepsy, since genetic or experimental alterations of the orexin system are associated with this sleep disorder. However, orexins are not restricted to the hypothalamus and together with their receptors they are expressed in peripheral tissues. This new multifaceted aspect of orexin biology is reviewed here in descriptions of (i) the proform, maturation and structure of orexins, (ii) the structure, signal transduction and pharmacology of orexin receptors and (iii) the expression of orexins and orexin receptors as well as their biological role in the hypothalamus-pituitary-adrenal axis, gastrointestinal tract, endocrine pancreas and other peripheral tissues.

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.

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.

PepT1-mediated epithelial transport of dipeptides and cephalexin is enhanced by luminal leptin in the small intestine.

Dietary proteins are mostly absorbed as di- and tripeptides by the intestinal proton-dependent transporter PepT1. We have examined the effects of leptin on PepT1 function in rat jejunum and in monolayers of the human enterocyte-like 2 cell Caco-2. Leptin is produced by the stomach and secreted in the gut lumen. We show here that PepT1 and leptin receptors are expressed in Caco-2 and rat intestinal mucosal cells. Apical (but not basolateral) leptin increased Caco-2 cell transport of cephalexin (CFX) and glycylsarcosine (Gly-Sar), an effect that was associated with increased Gly-Sar uptake, increased membrane PepT1 protein, decreased intracellular PepT1 content, and no change in PepT1 mRNA levels. The maximal velocity (Vmax) for Gly-Sar transport was significantly increased by leptin, whereas the apparent Michaelis-Menten constant (Km) did not change. Furthermore, leptin-stimulated Gly-Sar transport was completely suppressed by colchicine, which disrupts cellular translocation of proteins to plasma membranes. Intrajejunal leptin also induced a rapid twofold increase in plasma CFX after jejunal perfusion with CFX in the rat, indicating enhanced intestinal absorption of CFX. These data revealed an unexpected action of gastric leptin in controlling ingestion of dietary proteins.

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2.

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 microM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 microM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.

The peptide YY-preferring receptor mediating inhibition of small intestinal secretion is a peripheral Y(2) receptor: pharmacological evidence and molecular cloning.

A peptide YY (PYY)-preferring receptor [PYY > neuropeptide Y (NPY)] was previously characterized in rat small intestinal crypt cells, where it mediates inhibition of fluid secretion. Here, we investigated the possible status of this receptor as a peripheral Y(2) receptor in rats. Typical Y(2) agonists (PYY(3-36), NPY(3-36), NPY(13-36), C2-NPY) and very short PYY analogs (N-alpha-Ac-PYY(22-36) and N-alpha-Ac-PYY(25-36)) acting at the intestinal PYY receptor were tested for their ability to inhibit the binding of (125)I-PYY to membranes of rat intestinal crypt cells and of CHO cells stably transfected with the rat hippocampal Y(2) receptor cDNA. Similar PYY preference was observed and all analogs exhibited comparable high affinity in both binding assays. The same held true for the specific Y(2) antagonist BIIE0246 with a K(i) value of 6.5 and 9.0 nM, respectively. BIIE0246 completely abolished the inhibition of cAMP production by PYY in crypt cells and transfected CHO cells. Moreover, the antagonist 1) considerably reversed the PYY-induced reduction of short-circuit current in rat jejunum mucosa in Ussing chamber and 2) completely abolished the antisecretory action of PYY on vasoactive intestinal peptide (VIP)-induced fluid secretion in rat jejunum in vivo. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that Y(2) receptor transcripts were present in intestinal crypt cells (3 x 10(2) molecules/100 ng RNA(T)) with no expression in villus cells, in complete agreement with the exclusive binding of PYY in crypt cells. Finally, a full-length Y(2) receptor was cloned by RT-PCR from rat intestinal crypt cells and also from human small intestine. We conclude that the so-called PYY-preferring receptor mediating inhibition of intestinal secretion is a peripheral Y(2) receptor.

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.

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.

Structure-activity studies including a Psi(CH(2)-NH) scan of peptide YY (PYY) active site, PYY(22-36), for interaction with rat intestinal PYY receptors: development of analogues with potent in vivo activity in the intestine.

Peptide YY (PYY) is a gut hormone that inhibits secretion and promotes absorption and growth in the intestinal epithelium. We have performed structure-activity studies with the active site, N-alpha-Ac-PYY(22-36)-NH(2), for interaction with intestinal PYY receptors. Investigation of aromatic substitutions at position 27 resulted in analogues that exhibited potent in vitro antisecretory potencies with N-alpha-Ac-[Trp(27)]PYY(22-36)-NH(2) exhibiting even greater potency than intact PYY. In vivo studies in dogs revealed that this analogue also promoted intestinal absorption of water and electrolytes during continuous intravenous and intraluminal infusion. Investigations carried out to identify features that would enhance stability revealed that incorporation of Trp(30) increased affinity for PYY receptors. A "CH(2)-NH" scan revealed that incorporation of reduced bonds at position 28-29 or 35-36 imparted greater receptor affinity. In general, disubstituted analogues designed based on the results of single substitutions exhibited good receptor affinity with N-alpha-Ac-[Trp(27),CH(2)-NH(35-36)]PYY(22-36)-NH(2) having the greatest affinity (IC(50) = 0.28 nM). Conservative multiple substitutions with Nle-->Leu and Nva-->Val also imparted good affinity. An analogue designed to encompass most of the favored substitutions, N-alpha-Ac-[Nle(24,28),Trp(30),Nva(31), CH(2)-NH(35-36)]PYY(22-36)-NH(2), exhibited a proabsorptive effect in dogs comparable to, but longer lasting than, that of intact hormone. Selected analogues also exhibited good antisecretory potencies in rats with N-alpha-Ac-[Trp(30)]PYY(22-36)-NH(2) being even more potent than PYY. However, the potencies did not correlate well with the PYY receptor affinity or the proabsorptive potencies in dogs. These differences could be due to species effects and/or the involvement of multiple receptors and neuronal elements in controlling the in vivo activity of PYY compounds. PYY(22-36) analogues exhibited good affinity for neuronal Y2 receptors but poor affinity for Y1 receptors. Also, crucial analogues in this series hardly bound to Y4 and Y5 receptors. In summary, we have developed PYY(22-36) analogues which, via interacting with intestinal PYY receptors, promoted potent and long-lasting proabsorptive and antisecretory effects in in vivo models. These compounds or analogues based on them may have useful clinical application in treating malabsorptive disorders observed under a variety of conditions.

alpha(2)-adrenergic receptors stimulate oligopeptide transport in a human intestinal cell line.

Di- and tripeptides, as well as peptidomimetic drugs such as cephalexin (CFX), are absorbed by enterocytes via the oligopeptide transporter PepT1. We recently showed that the alpha(2)-adrenergic agonist clonidine increases CFX absorption in anaesthetized rats. Herein, we investigated whether alpha(2)-adrenergic receptors can directly affect PepT1 activity in a clone of the differentiated human intestinal cell line Caco-2 (Caco-2 3B) engineered to stably express alpha(2A)-adrenergic receptors at a density similar to that found in normal mucosa. Measurement of CFX fluxes across cell monolayers cultured on transwell filters demonstrated that the alpha(2)-agonists clonidine and UK14304 caused a 2-fold increase of CFX transport in Caco-2 3B cells, but not in Caco-2 (expressing PepT1 but not alpha(2)-adrenergic receptors) or in the HT29 19A clone (expressing alpha(2)-adrenergic receptors but not PepT1). The stimulatory effect of clonidine was abolished by glycyl-sarcosine (a competitor for the transporter) and blocked by yohimbine or RX821002 (alpha(2)-antagonists). Analysis of the kinetics of CFX transport in control and clonidine-treated Caco-2 3B cells showed that clonidine increased V(max) of CFX transport without changing K(m). Clonidine action was abolished by colchicine but not altered by amiloride, demonstrating that microtubule integrity but not Na(+)/H(+) exchanger activity is necessary for the effect of alpha(2)-agonists to occur. In conclusion, clonidine can directly activate alpha(2)-adrenergic receptors located on epithelial cells. The precise molecular mechanisms whereby these receptors modulate PepT1 activity remain to be elucidated but an increased translocation to the apical membrane of preformed cytoplasmic transporter molecules is likely to be involved.

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.

Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization.

After stable transfection of Chinese hamster ovary cells with the human Y4 receptor, clone 29 was isolated and studied for receptor properties. The following data were obtained: 1) one class of binding site was identified by analysis of (125)I-human pancreatic polypeptide (hPP) binding to cell membranes with a K(d) value of 0. 26 nM and a B(max) value of 1.44 pmol/mg protein; 2) the K(i) values for inhibition of (125)I-hPP binding by hPP, human peptide YY (hPYY), human neuropeptide Y (hNPY), and analogs were hPP (0.7 nM) < rat PP (47 nM) < hPYY (94 nM) < h[Leu(31)-Pro(34)]NPY (124 nM) << hNPY = porcine NPY(13-36) = rat D-[Trp(32)]NPY (>1 microM); 3) cross-linking experiments using (125)I-hPP identified a single M(r) 60,000 glycosylated Y4 receptor; and 4) the natural peptides hPP, hPYY, and hNPY inhibited forskolin-stimulated cAMP production in clone 29 cells with EC(50) values of 0.56 nM, 218 nM, and >1 microM, respectively. The inhibitory effect of hPP was abolished when cells were incubated with pertussis toxin, indicating a pertussis toxin-sensitive G(i) protein-mediated event. 5) Exposure of cells to 10 nM hPP for 24 h resulted in the absence of modification of binding capacity (1.38 versus 1.44 pmol/mg protein in control cells) or affinity (0.31 versus 0.26 nM in control cells); there also was no modification in the potency and efficacy of hPP in inhibiting forskolin-stimulated cAMP. Immunofluorescence indicated that the Y4 receptor was not internalized within the cells after 24-h treatment with 10 nM hPP. These data support that Y4 receptors are resistant to agonist-promoted desensitization and internalization. Clone 29 cells provide a valuable tool to further characterize the pharmacological aspects of human Y4 receptor.

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.