Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

Comparative mapping of human thyrotropin, gonadotropins, and free subunits with antipeptide antibodies.

To compare the structural topology of the human TSH to that of the structurally related gonadotropins, 10 peptides covering the entire primary sequence of the alpha- and beta-subunits of TSH were synthesized and used as antigens for the preparation of polyclonal antibodies. The alpha-subunit was synthesized as 4 nonoverlapping peptides (1-25, 26-51, 49-73, 72-92) while the beta-subunit was segmented in 6 overlapping sequences (2-18, 10-38, 31-51, 53-76, 77-96, 92-112). Most of the peptide sequences were predicted to contain a putative antigenic determinant. All antipeptide antisera were found to bind to the corresponding synthetic sequence in an enzyme-linked immunosorbent assay as well as to denatured TSH subunits after Western blotting. The N-terminal half of the alpha-subunit was found differentially accessible in TSH and gonadotropins compared to the free subunit: antipeptide-alpha 1-25 antibodies exhibited variable affinity for the four glycoprotein hormones whereas anti-alpha 26-51 displayed a remarkable recognition of free alpha-subunit. Four peptides proved to be accessible in the TSH beta-subunit: the N-terminal peptide (beta 2-18) elicited antibodies that bound to free TSH-beta and poorly to the dimer while antibodies against the C-terminal sequence (beta 92-112) recognized equally well free beta-subunit and TSH. Antipeptide-beta 31-51 antibodies proved to be specific for TSH while the beta 53-76 contiguous peptide appeared accessible in both TSH and gonadotropins. The current findings therefore demonstrate that most of the sequences predicted to contain antigenic sites in the alpha- or the beta-subunits are indeed accessible at the surface of these proteins. Additionally, both subunits appear to contain amino acid sequences that are differentially expressed in TSH and gonadotropins as well as in free and combined subunits.

[Radioactive analogs of neurotensin. I. Solid phase synthesis and biological characterization of Trp 11-neurotensin, a precursor of an iodized ligand]. / Analogues radioactifs de la neurotensine. I. Synthèse en phase solide et caractérisation biologique de la [Trp11]-neurotensine, précurseur du ligand iodé.

In order to generate highly labelled neurotensin analogues, synthesis has been performed of two types of precursors, one for iodination and one for tritiation. Iodination of native neurotensin occurs on both tyrosines in position 3 and 11 and thus affects greatly its binding capacities. In this article, synthesis and chemical characterization of [Trp11]-neurotensin are described which can be iodinated without loss of activity. Synthesis was by solid phase procedure on an experimental support, Pab-resin, alpha-(4-chloromethylphenylacetamido)-benzyl copoly (styrene 1 per cent divinylbenzene). After esterification of Boc-Leu by its cesium salt on the Pab-resin, each amino acid was incorporated by a double coupling with dicyclohexylcarbodiimide on a Beckman 990 synthesizer. The trifunctionnal amino acids were protected as follows : Tyr as the 2,6-dichlorobenzyl ether, Glu as benzyl ester, Lys by the benzyloxycarbonyl group, Arg by the tosyl group, and Trp by the formyl group. Boc-Asn was incorporated by the HOBt procedure. The cleavage of peptide-resin bond and the removal of lateral chain protecting groups was realized by hydrofluoric acid with 10 per cent anisol for 1 h at 0 degrees C. The peptide obtained was then treated by NH4HCO3 1 M, pH 9, for 24 h for the removal of tryptophan formyl protecting group. Purification of the crude peptide on Bio-Gel P2 followed by ion exchange chromatography on carboxymethylcellulose (CM 52) and a final desalting on Bio-Gel P2 proved very efficient in removing several shorter contaminants.(ABSTRACT TRUNCATED AT 250 WORDS)

[Radioactive analogs of neurotensin. II. Preparation of tritiated neurotensin from a synthetic multi-unsaturated derivative]. / Analogues radioactifs de la neurotensine. II. Préparation de neurotensine tritiée à partir d'un dérivé multi-insaturé de synthèse.

In this second paper on the synthesis of neurotensin analogues as precursors for radiolabelling, solid phase synthesis of two polyunsaturated peptides, [Dah6, delta Pro7,10]-neurotensin and acetyl-[delta Pro10]-neurotensin-(8-13), are described. The first one contains one triple bond and two double bonds susceptible to tritiation in the same molecule, the second one contains one double bond in the shortest sequence having neurotensin activity. The C-terminal residue, Boc-Leu, was esterified on the chloromethyl-resin by its cesium salt. For the other amino acids a double coupling was carried out, the first one with dicyclohexylcarbodimide and the second one with the amino acid hydroxybenzotriazole ester. Acylation of the second amino acid, on the resin, presented some difficulties to achieve completeness and several acetylations and benzoylations had to be performed in order to block the last 4 per cent of free amines. It seems that these difficulties are related to some batches of chloromethyl-resin. Incorporation of both acetylenic lysine, N alpha-Boc-N epsilon-Z-L-2,6-diamino-4-hexynoic acid, whose synthesis is described, and N alpha-Boc-L-3,4-dehydroproline was without problems in this synthesis. After cleavage by hydrofluoric acid the crude peptides were purified by gel filtration on Bio-Gel P2 and ion exchange chromatography on carboxymethylcellulose (CM 52). [Dah6, delta Pro7,10]-neurotensin so obtained (51 per cent compared to starting Boc-Leu-resin) was in homogeneous form as characterized by amino acid analysis, thin layer chromatography in different systems and high performance liquid chromatography. The hydrogenation or tritiation product was identical with native neurotensin. Unsaturated derivative and neurotensin obtained after catalytic hydrogenation were as active as native neurotensin in inhibition of 125I-[Trp11]-neurotensin binding to rat brain synaptic membranes and in guinea pig ileum contractility test. Substitution of proline and lysine by their dehydro-derivatives did not affect the biological properties of neurotensin. The tritiated neurotensin (160-180 Ci/mmol) should be a good agent for biological characterization of neurotensin receptors and for investigation of the peptide metabolism.

Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin.

Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.

Impaired G protein coupling of the neurotensin receptor 1 by mutations in extracellular loop 3.

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor. We have shown previously that the NTS1 receptor-binding site of the peptide agonist involved residues in extracellular loop 3 and at the extracellular junction of transmembrane domains 4 and 6. Here, we investigated by site-directed mutagenesis residues in extracellular loop 3 that might be involved in agonist-induced activation of the rat NTS1 (rNTS1) receptor. Wild type and mutated receptors were expressed in COS (African green monkey kidney fibroblasts) cells. Labeled agonist and antagonist binding as well as inositol phosphate and cAMP productions were studied. Compared to the wild type NTS1 receptor, the W339A, F344A, H348A and Y349A mutant receptors exhibited (i) decreased proportion of high over low affinity agonist binding sites, (ii) increased sensitivity of high affinity agonist binding to GTP gamma S, and (iii) impaired G protein coupling of high affinity agonist-receptor complexes. The data are consistent with the C-terminal part of extracellular loop 3 being essential for allowing high affinity agonist-NTS1 receptor complexes to couple to G proteins.

Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

An azidonitrophenylaminoacetyl mono[125I]iodoapamin derivative was prepared which showed specific binding to rat neuronal membranes. UV photolysis lead to the irreversible occupation of binding sites. Photo-labeling of intact primary cultured rat neurones followed by membrane solubilization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography revealed the covalent incorporation of radioactivity into 3 main components with Mr = 86,000, 30,000, and 23,000. Labeling was completely prevented by a competing excess of native apamin. Similar studies on purified synaptic membranes from the rat brain showed another labeling pattern with major bands corresponding to Mr = 86,000 and 59,000. Although the reasons for the partial discrepancy between cultured embryonic neurons and an adult brain membrane fraction are not yet clear, we conclude that these proteins are intimately associated with the apamin binding site and are probably components of a type of Ca2+-activated K+ channel.

Agonism, inverse agonism, and neutral antagonism at the constitutively active human neurotensin receptor 2.

Two G protein-coupled neurotensin (NT) receptors, termed NTR1 and NTR2, have been identified so far. In contrast to the NTR1, which has been extensively studied, little is known about the pharmacological and biological properties of the NTR2. In the course of characterizing NT analogs that exhibited binding selectivity for the NTR2, we discovered that this receptor constitutively activated inositol phosphate (IP) production. Here, we report on the constitutive activity of the human NTR2 (hNTR2) transfected in COS cells and on compounds that exhibit agonism, inverse agonism, and neutral antagonism at this receptor. IP levels increased linearly with time, whereas they remained constant in mock-transfected cells. Furthermore, IP production was proportional to the amount of hNTR2 present at the cell membrane. SR 48692, a nonpeptide antagonist of the NTR1, stimulated IP production, whereas levocabastine, a nonpeptide histamine H1 antagonist that binds the NTR2 but not the NTR1, behaved as a weak partial inverse agonist. NT analogs modified at position 11 of the NT molecule, in particular by the introduction of bulky aromatic D amino acids, exhibited binding selectivity at the hNTR2 and also behaved as partial inverse agonists, reversing constitutive IP production up to 50%. Finally, NT barely affected constitutive IP production but antagonized the effects of both agonist and inverse agonist compounds, thus behaving as a neutral antagonist. The unique pharmacological profile of the hNTR2 is discussed in the light of its sequence similarity with the NTR1 and the known binding site topology of NT and SR 48692 in the NTR1.

Binding and internalization of neurotensin in hybrid cells derived from septal cholinergic neurons.

Autoradiographic studies from our laboratory have previously demonstrated a selective association of high affinity neurotensin (NT) binding sites with basal forebrain cholinergic neurons. In search of an in vitro model for further characterization of the role and regulation of these sites, we have examined the binding and internalization of 125I-Tyr3-NT (125I-NT) and fluorescein isothiocyanate (FITC)-conjugated NT (fluo-NT) on SN17 hybrid cells, produced by fusion of embryonic murine septal cells with neuroblastoma. 125I-NT binding to SN17 membrane preparations was specific and saturable. Scatchard analysis of the data was suggestive of an interaction with a single population of sites, the affinity (Kd = 1.7 nM) and pharmacological profile of which were comparable to those of neural NT receptors. No specific binding was observed on the parent neuroblastoma cell line, confirming that the expression of those sites is a neuronal trait. Incubation of whole SN17 cells with 125I-NT resulted in a time- and temperature-dependent internalization of the specifically bound peptide. The t1/2 of this internalization was estimated at 13 min, a value nearly identical to that reported for neurons in culture. Confocal microscopic analyses using fluo-NT indicated that the internalization process was endocytic in nature in that: 1) it was entirely blocked by the endocytosis inhibitor phenylarsine oxide; and 2) it was mediated through small intracytoplasmic particles the size and maturation of which corresponded to that of endosomes. It is proposed that the expression and internalization of NT receptors by SN17 hybrid cells represent a new facet of these cells' cholinergic phenotype that makes them amenable to the study of NT interactions with cholinergic cells.

Stable expression of the cloned rat brain neurotensin receptor into fibroblasts: binding properties, photoaffinity labeling, transduction mechanisms, and internalization.

The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly un-dissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Effect of the nonpeptide neurotensin antagonist, SR 48692, and two enantiomeric analogs, SR 48527 and SR 49711, on neurotensin binding and contractile responses in guinea pig ileum and colon.

The tridecapeptide neurotensin (NT) contracts the guinea pig ileum through a neurogenic process that is mediated in part by acetylcholine and substance P and relaxes the guinea pig colon through a direct action on smooth muscle cells involving the opening of Ca(++)-dependent K+ channels. The non-peptide NT antagonist, SR 48692 (2-[1-(7-chloro-4-quinolinyl)-5-(2,6- dimethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo-(3.3.1.1 .3.7)decan-2- carboxylic acid), potently inhibited NT binding to membranes prepared from the guinea pig ileum and colon with Ki values of approximately 3 nM. SR 48527 ((S)-(+)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3- yl)carbonylamino]cyclohexylacetic acid) and SR 49711 ((R)-(-)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol- 3-yl)carbonylamino]cyclohexylacetic acid), two enantiomers structurally related to SR 48692, were respectively equipotent with and a 100-fold less potent than SR 48692 in inhibiting NT binding in both tissues. In both membrane preparations, NT binding was increased by Mg++ and decreased by Na+ and guanosine 5'-[gamma-thio]triphosphate, whereas SR 48692 binding was not significantly affected by these agents. SR 48692 inhibited NT-induced contraction and relaxation in guinea pig ileum and colon preparations, respectively, with Ki values between 4 and 5 nM. As in binding studies, SR 48527 was as potent, whereas SR 49711 was 100-fold less potent than SR 48692 in antagonizing NT responses in both the guinea pig ileum and colon. Altogether, our results show that NT receptors in the guinea pig ileum and colon, although functionally distinct, are coupled to G-proteins and display similar biochemical and pharmacological properties, in particular with regard to their sensitivity and stereoselectivity toward nonpeptide antagonists related to SR 48692. Because of their high potency to antagonize NT actions in intestinal preparations, SR 48692 and SR 48527 represent useful tools to study the physiological role of NT in the digestive tract.

Identification of residues involved in neurotensin binding and modeling of the agonist binding site in neurotensin receptor 1.

The neurotensin receptor 1 (NTR1) subtype belongs to the family of G protein-coupled receptors and mediates most of the known effects of the neuropeptide including modulation of central dopaminergic transmission. This suggested that nonpeptide agonist mimetics acting at the NTR1 might be helpful in the treatment of Parkinson's disease and schizophrenia. Here, we attempted to define the molecular interactions between neurotensin-(8-13), the pharmacophore of neurotensin, and the rat NTR1. Mutagenesis of the NTR1 identified residues that interact with neurotensin. Structure-activity studies with neurotensin-(8-13) analogs identified the peptide residues that interact with the mutated amino acids in the receptor. By taking these data into account, computer-assisted modeling techniques were used to build a tridimensional model of the neurotensin-(8-13)-binding site in which the N-terminal tetrapeptide of neurotensin-(8-13) fits in the third extracellular loop and the C-terminal dipeptide binds to residues at the junction between the extracellular and transmembrane domains of the receptor. Interestingly, the agonist binding site lies on top of the previously described NTR1-binding site for the nonpeptide neurotensin antagonist SR 48692. Our data provide a basis for understanding at the molecular level the agonist and antagonist binding modes and may help design nonpeptide agonist mimetics of the NTR1.

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis.

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

Binding and toxicity of apamin. Characterization of the active site.

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.

In vivo and in vitro structure-activity studies with peptide and pseudopeptide neurotensin analogs suggest the existence of distinct central neurotensin receptor subtypes.

The present study was designed to compare, with respect to structure-activity relationships, the receptors that subserve the hypothermic and analgesic effects of neurotensin (NT) to the receptor that mediates the effects of NT in mesencephalic dopamine (DA) neurons, and to compare these receptors to the cloned adult rat brain NT receptor and to newborn mouse and rat brain NT receptors. The results show that NT receptors in homogenates from newborn mouse and rat brain and from COS 7 cells transfected with the cloned high-affinity NT receptor from the adult rat brain displayed virtually identical structure-activity relationships toward a series of 12 peptide and pseudopeptide NT analogs, as assessed by the ability of the compounds to inhibit the binding of [125I]NT binding in these systems. Furthermore, when eight of these analogs were tested for their ability to inhibit [125I]NT binding and to potentiate K(+)-evoked DA release in primary cultures of rat mesencephalic neurons, it was found that they all behaved as agonists with binding and biological potencies quite similar to those observed in the other binding assays. Finally and strikingly, when seven of these analogs with checked metabolic stability were tested in vivo for their hypothermic and analgesic (tail-flick test) effects after i.c.v. injection in the mouse, they exhibited relative potencies that were completely different from those obtained in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]SR 48692, the first nonpeptide neurotensin antagonist radioligand: characterization of binding properties and evidence for distinct agonist and antagonist binding domains on the rat neurotensin receptor.

The binding of [3H]SR 48692, a new potent and specific nonpeptide neurotensin (NT) receptor antagonist, was characterized in membranes from mouse fibroblast LTK- cells stably transfected with the G protein-coupled rat NT receptor. The binding of [3H]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [3H]SR 48692 bound to a single population of sites, with a Kd of 3.4 nM and a Bmax value that was 30-40% greater than that observed in saturation experiments with [125I]NT. Two SR 48692-related enantiomers, SR 48527 and SR 49711, were 10 and 1000 times less potent, respectively, than unlabeled SR 48692 in inhibiting [3H]SR 48692. Unlabeled NT inhibited [3H]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (Ki = 0.22 nM) and low (Ki = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 microM), which represented 60, 20, and 20%, respectively, of the total number of [3h]SR 48692 binding sites. Digitonin (10 micrograms/ml) markedly reduced the proportion of NT-insensitive sites without affecting [3H]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [3H]SR 48692 and [125I]NT binding and inverted the proportions of the high and low affinity NT binding sites. A mutant rat NT receptor that contained a deletion in a region (amino acids 45-60) of the amino-terminal extracellular domain near the first transmembrane helix and was expressed in COS M6 cells retained the same affinity for [3H]SR 48692 and the same stereoselectivity for SR 48527 and SR 49711 as the wild-type receptor. In contrast, it bound NT with 3000-fold lower potency. In conclusion, the data indicate that [3H]SR 48692 represents a new, potent, nonpeptide antagonist radioligand of the NT receptor that differentiates between agonist- and antagonist-receptor interactions. Furthermore, the data demonstrate that the peptide agonist and the nonpeptide antagonist bind to distinct regions of the NT receptor.

Mutagenesis and modeling of the neurotensin receptor NTR1. Identification of residues that are critical for binding SR 48692, a nonpeptide neurotensin antagonist.

The two neurotensin receptor subtypes known to date, NTR1 and NTR2, belong to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagonist, is selective for the NTR1. In the present study we attempted, through mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven putative TMs of the NTR1 were defined by sequence comparison and alignment of bovine rhodopsin and G-protein-coupled receptors. Thirty-five amino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for their ability to bind 125I-NT and [3H]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows that the antagonist binding pocket lies near the extracellular side of the transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding. Two of these residues, Arg327 in TM 6 and Tyr351 in TM 7, play a key role in antagonist/receptor interactions. The former appears to form an ionic link with the carboxylic group of SR 48692, as further supported by structure-activity studies using SR 48692 analogs. The data also show that the agonist and antagonist binding sites in the rNTR1 are different and help formulate hypotheses as to the structural basis for the selectivity of SR 48692 toward the NTR1 and NTR2.

Identification of the receptor subtype involved in the analgesic effect of neurotensin.

The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NT-elicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.