Neurotensin Receptors Binding Properties, Transduction Mechanisms, and Purification JEAN-PIERRE VINCENT lnstitut de Phamacologie Molkculaire et Cellulaire UPR 411 CNRS, Sophia Antipolis 06560 klbonne. France
INTRODUCTION Neurotensin is a neuropeptide involved in intercellular communication in the central nervous system and peripheral organs. During the last few years, a primary goal of this laboratory has been to characterize the structural and functional properties of neurotensin receptors in brain and gut. These studies led us to investigate the transduction mechanisms that are triggered by the association of neurotensin with its target cell and finally to purify the neurotensin receptor to homogeneity. This chapter will present an update of our major findings in this area.
BINDING PROPERTIES OF CENTRAL AND PERIPHERAL NEUROTENSIN RECEPTORS Detection and characterization of neurotensin binding sites was carried out with 125I-labeled [monoiodo-Tyr3]neurotensin(2000 Ci/mol) as radioactive ligand. Membranes prepared from brain or gastrointestinal tissues of adult mammals generally contain two different classes of neurotensin binding sites. For example, results illustrated in FIGURE 1A show that the Scatchard plot describing the binding of '251-labeled [Tyr3] neurotensin to adult mouse brain homogenate is curvilinear and can be resolved into two independent linear components.2 Each component represents a single class of noninteracting binding sites. Class 1 sites are characterized by a high affinity (0.13 nM) and a low capacity (TABLE1). By comparison, the affinity of class 2 sites is lower (2.4 nM), and their binding capacity is higher. Class 1 and 2 sites cannot be easily differentiated by their structure-function relationships because they exhibit the same order of affinity for a series of neurotensin analogues. Both types of sites recognize the 8-13 COOH-terminal hexapeptide of the neurotensin sequence.z On the other hand, the affinity of class 1 sites for neurotensin can be selectively decreased by sodium ions or GTP, whereas class 2 sites are much less sensitive to sodium ions and insensitive to GTP. Binding capacities of class 1 and 2 sites remain unchanged under these various conditions. However, the best tool for distinguishing between class 1 and 2 sites is levocabastine. This potent antihistamine-1 drug was introduced by Janssen Pharmaceutica and found to be able to partly inhibit [3H]neurotensin binding to rat brain membranes, although it was devoid of any neurotensin-like ac90
VINCENT NEURmENSIN RECEPTORS
91
tivity.3 Our results indicate that a 1 @ concentration I of levocabastine completely blocks the binding of 1251-labeled[Tyr3]neurotensin to class 2 sites without changing the binding properties of class 1 sites. This "all or none" effect is obviously the simplest way to differentiate class 1 sites from class 2 sites in mouse brain homogenate. Unfortunately levocabastine is active only on class 2 sites of murine species and therefore cannot be used to differentiate class 1 sites from class 2 sites in other mammalian species. The different properties of the two neurotensin-binding sites that are present in mouse brain are summarized in TABLE1. As already mentioned, almost all membrane preparations of central or peripheral origin contain two types of neurotensin binding sites. The amount and the relative proportions of each site vary from one species to the other. Results in FIGURE 2 show that both types of sites appear at different times during development of the mouse brain, the low-affinity class 2 sites being expressed later than the high-affinity class 1 sites. In agreement with results previously obtained from rat,4 brains of 5- to 10-dayold mouse (FIG. lB), rabbit, and rat contain four to six times more high-affinity neurotensin binding sites than brains of adults. Moreover, the low-affinity sites are undetectable in brains of these newborn animals.
TRANSDUCTION MECHANISMS OF NEUROTENSIN RECEPTORS Cell lines of neural or nonneural origin that express neurotensin receptors provide useful models for investigating the biochemical events generated by the association of neurotensin to its target cell. TABLE1 . Compared Properties of Two Different Neurotensin Binding Sites in Brain Homogenate of Adult Mouse Pronertv Dissociation constant (Tris 50 mM, pH 7.5, 25°C) Binding capacity
Site 1
Site 2
Kdl = 0.13 nM
Kd2
=
2.4 nM
Bml = 66 f m o l h g
Bm2 = 160 fmol/mg
Neurotensin sequence recognized
8-13
8-13
Effect of Na+ ions
Affinity decreased (Kdl X 5; ECYt'
Effect of GTP Effect of levocabastine
Affinity decreased (Kdl X 4; ECY;' No effect
=
35 mM)
=
0 . 3 pM)
Affinity decreased (Kd2 X 2; ECYt' = 100 mM) No effect Complete inhibition
Binding activity after CHAPS solubilization
Yes
No
Occurrence in cell cultures (NIE115, HT29, neurons) Involvement in transduction mechanisms
Yes
No
Yes
No
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A 0
-- Levocabastine
0.6
-
B
-- Levocabastine
Kd H = 0.13 nM
Bm H = 65 h U m g
k
I Yb
0
Kd H = 0.185 nM Bm H = 250 h h g
0 +Levocabastinel~M
100
B, fmol/rng
0
100
B, frnol/mg
FIGURE 1. Binding of 'ZSI-labeled[Tyr3]neurotensin to mouse brain homogenate. Freshly prepared homogenate from adult (A) or newborn (B) mouse brain (0.1 mg of protein per assay) were incubated at 2 5 ° C pH 7.5, with increasing concentrations of 1*5I-labeled [Tyr3]neurotensin alone or isotopically diluted with unlabeled neurotensin, in the presence (open symbols) or in the absence (closed symbols) of 1 mM levocabastine. This incubation medium (250 vl) consisted of a 50 mM Tris-HCI buffer containing 0.02% bovine serum albumin, 1 mM 1,lO-phenanthroline and 0.01 mM N-benzyloxycarbonylprolylprolinal to prevent degradation of the ligand. The specific binding was determined by filtration.2 Data are presented under the form of Scatchard plots. A, The resolution of the curvilinear Scatchard plot into two independent linear components is shown by the straight lines. B and F, bound and free concentrations of labeled ligand.
Mouse neuroblastoma cells of clone NlE115 possess high-affinity binding sites for neurotensin that appear during the course of their differentiation in vitro.5 These sites are very similar to class 1 sites of mouse brain homogenates, since their affinity is negatively modulated by sodium ions and GTP without change of the binding cap a ~ i t y Moreover, .~ I251-labeled [Tyr3]neurotensin binding to neuroblastoma cells is totally insensitive to levocabastine. The same type of site is also found in HT29 cells, an adenocarcinoma of human colon,6 and in primary cultured neurons of mouse fetal cortex The association of neurotensin to its receptor in neuroblastoma NlE115 cells triggers three different effects. First, the intracellular cGMP concentration increases according to a rapid and transient mechanism. This effect is calcium-dependent and leads to a maximal cGMP stimulation of 10-fold over basal leve1.8.9 A parallel 20 to 30%decrease of the cAMP basal level is also detected. This inhibitory effect of neurotensin is much easier to study after stimulation of cAMP by prostaglandin El. Under these conditions, neurotensin inhibits cAMP production by 50 to %%.lo A third consequence of the association of neurotensin to its receptor in NlE115 cells is an increase of intracellular inositol phosphate concentrations. In the presence of lithium ions, which block inositol monophosphate hydrolysis, neurotensin produces a rapid and transient stimulation of inositol triphosphate and inositol biphosphate levels and a slower in-
.'
VINCENT NEUROTENSIN RECEPTORS M
E 3 E cc
300 T
\
-
0
93
200
3 m n
3
100
M CI .
V C
G
o
n n
0
5
I
10
15
F60
FIGURE 2. Postnatal ontogeny of high- and low-affinity neurotensin-binding sites in mouse brain. Maximal binding capacities of sites 1 ( 0 )and 2 (0)were calculated from binding experiments as described in FIGURE1. Data are the mean f SEM of three experiments.
crease of the inositol monophosphate concentration. I I We have demonstrated that each one of the three effects described above are direct consequences of neurotensin receptor occupancy and are totally insensitive to levocabastine. Neurotensin is also able to stimulate phosphatidylinositol turnover in HT29 cells without changing the intracellular levels nf CAMP and cGMP.6
PURIFICATION OF NEUROTENSIN RECEPTORS Preliminary Choice The very first choice that should be made concerns the type of receptor to purify. As described above, the properties of the higher affinity class 1 sites from mouse brain homogenate are identical to those of neurotensin receptors that regulate intracellular levels of second messengers in target cells of neurotensin. Because our purpose was to purify functionally relevant neurotensin receptors, we chose to work on these highaffinity sites. Once a given receptor subtype has been chosen, the selection of the preparation used as starting material for solubilization and purification of the receptor is made essentially on the basis of the binding capacity, which must be as high as possible. The availability of a preparation containing only the desired subtype of receptor is obviously a great advantage. The tissue used as the source of receptors should also be readily available fresh and in quantity. The above considerations led us to purify neurotensin receptors from brain homogenates of newborn mice because this preparation is the richest known source of high-affinity neurotensin binding sites (250 fmol/mg protein) and because it does not contain low-affinity sites. Moreover, it is easy to obtain large amounts of newborn mouse brains.
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Solubilization The next step is to devise experimental conditions that would allow us to solubilize the neurotensin receptor in an active state and to monitor its binding activity.
Binding Assay
The choice of a convenient assay for the binding of neurotensin to its soluble receptor is an extremely important step. As far as the binding assay has not been validated, solubilization experiments cannot be properly interpreted. Thus, failure to detect neurotensin binding activity in a soluble extract can be due either to the absence of active receptor in the extract or to the inability of the binding assay to detect it. The necessity of finding simultaneously suitable conditions for solubilization of active receptor and for measurement of the soluble binding activity is one of the main difficulties in studies dealing with receptor solubilization. The best way to solve this problem is probably to use an assay that is as simple as possible. In our case, the separation of IZ5I-labeled[Tyr3]neurotensin bound to the soluble receptor from free ligand was carried out by gel filtration on Sephadex (3-50 (medium). The radioactivity bound to the receptor was eluted in the void volume and counted directly in a y counter.I2
Detergent No general rule or law exists that would permit selection of the most convenient detergent for solubilizing a given receptor without loss of binding activity. Thus, the task of finding the best detergent was accomplished by trial and error. CHAPS (3-[(3-cholamidopropyl)dimethylamonio]-l-propanesulfonicacid) was found to be the only detergent that could solubilize neurotensin receptors in an active form. The soluble binding sites corresponded exclusively to the high-affinity site found in mouse brain homogenates (see below). These sites were quantitatively solubilized by CHAPS concentrations between 0.6 and 0.7%.I2
Stabilizing Agent
In the absence of any stabilizing agent, the half-life (tl12) of the mouse brain receptor solubilized with 0.6% CHAPS was about 3 hr at 0°C. The presence of 0.12% cholesterol hemisuccinate (CHS) in the solubilization buffer largely improved the stability of the receptor ( t ~ 2= 31 hr). Addition of 1 nM neurotensin to mouse brain homogenate prior to the solubilization step further increased the stability at 0°C by a factor of 2 (ti12 = 65 hr). CHAPS-solubilized extracts frozen at -30°C could be stored for weeks without any detectable loss of binding activity, provided they contain 10%glycerol. This additive did not increase the half-life of the receptor at O"C, but protected it against losses of binding activity that occurred upon freezing and thawing.
VINCENT NEUROTENSIN RECEPTORS
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Peptidase Inhibitors In order to purify a receptor protein in its native form, it is necessary to work in the presence of protease inhibitors. Since only four different families of proteolytic enzymes exist, the presence of four specific inhibitors in the purification buffer should be sufficient to protect the receptor completely against degradation by proteases. Neurotensin receptors were solubilized and purified in buffers containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, inhibitor of serine proteases), 1 mM iodoacetamide (inhibitor of thiol proteases), 5 mM EDTA (inhibitor of metalloproteases), and 1 pM pepstatin (inhibitor of acidic proteases). Taking into account all the data described above, solubilization was carried out as follows. Freshly prepared homogenates of newborn mouse brain were incubated at a concentration of 10 mg of protein per ml in a 20 mM Tris-HC1 buffer, pH 7.5, containing 10% glycerol, 0.6%CHAPS, 0.12%CHS, and a mixture of protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 pM pepstatin, 1 mM iodoacetarnide, and 5 mM EDTA). After 30 min at O"C, the incubation medium was centrifuged at 110,000 x g for 15 min. The supernatant, which contained the soluble neurotensin receptor, was either used immediately or stored at -30°C.
Properties of the Soluble Receptor Binding Properties Soluble extracts obtained from either newborn or adult mouse brain contain a single type of high-affinity neurotensin receptors. In both cases, the affinity of the soluble receptor for neurotensin (about 0.3 nM) is decreased by sodium ions and is insensitive to levocabastine. The ability of GTP to negatively modulate the affinity of the receptor for neurotensin is lost on solubilization.12Apart from this difference, CHAPS seems to have solubilized neurotensin receptors that correspond to the membrane-bound class 1 sites (TABLE1). Although the levocabastine-sensitive class 2 sites are more abundant than class 1 sites in adult mouse brain (FIG.lA), class 2 sites are no longer detectable after solubilization. Molecular Structure Gel filtration of the CHAPS-solubilized neurotensin receptor from mouse brain on an Ultrogel AcA34 column calibrated with protein standards gave an approximate molecular mass of 100 kDa.12 The soluble receptor was covalently radiolabeled by photoaffinity and by chemical cross-linking. 12 Both techniques resulted in the specific labeling of the same protein band of molecular mass about 100 kDa.12 To summarize, neurotensin receptors from mouse brain homogenate can be solubilized in an active and stable form using the zwitterionic detergent CHAPS in the presence of CHS. The soluble receptor is a single polypeptide chain of 100 kDa, which binds neurotensin with a high affinity. From these data, it is reasonable to undertake the purification of the receptor.
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ANNALS NEW YORK ACADEMY OF SCIENCES
Purification The soluble neurotensin receptor from newborn mouse brain was purified essentially in a single step of ligand affinity chromatography. However, in order to minimize the total amount of protein loaded on the affinity column, the soluble extract was first prepurified by ion-exchange chromatography. Prepurification Step
The soluble extract was diluted 2.5 times and passed at 4°C through two columns of SP-Sephadex C-25 and hydroxylapatite (100 ml each) connected in series and equilibrated with the Tris-glycerol buffer containing 0.1% CHAPS, 0.02% CHS, and protease inhibitors. About 50% of proteins, including several brain proteases, 13 were retained on the gel, whereas the bulk of the neurotensin-binding activity was collected with the flow-through and used in the next purification step. Afinity Chromatography Preparation of afinity gel: In preliminary experiments, covalent binding of native neurotensin to various chemically activated gels was found to occur in low yields. The reason is probably that the only free amine available for the coupling reaction in the neurotensin sequence is the side chain of Lys6, the NH2-terminal residue being pGlu. To overcome this difficulty, neurotensin (2-13) was used in place of the native peptide to prepare the affinity column (FIG.3). Interestingly, the affinity of the (2-13) sequence for the neurotensin receptor was found to be better than that of native neurotensin in binding competition experiments. The neurotensin (2-13) solution coupled to the gel was radiolabeled by tracer amounts of [3H]neurotensin (2-13). Glutaraldehyde-activated Ultrogel AcA22 (IBF Biotechnics) was chosen as support because it contained a spacer arm and because ligand leakage was low after extensive washing of the affinity gel. The yield of the coupling reaction calculated from the radioactivity incorporated into the gel was between 40 and 70% in seven different experiments. Chromatography of prepurified receptor: The binding activity eluted from SPSephadex C-25 and hydroxylapatite was loaded on the affinity column (FIG.4,fractions 1 to 60), which was then washed with the Tris-glycerol buffer containing 0.1% CHAPS and 0.02% CHS (fractions 61 to 150). When the affinity column was working at its better level of efficiency, pooled fractions 1 to 150 contained almost all the loaded proteins but only 10 to 15% of the total binding activity. Proteins retained nonspecifically on the gel by ionic interactions were eliminated by elution with the washing buffer, which contained 200 mM KC1 (fractions 151 to 170). A small amount (2 to 5%) of specific neurotensin-binding activity was lost during this step. The column was rinsed one more time with 300 ml of washing buffer (fractions 171 to 240), and the neurotensin receptor was eluted with the washing buffer, which contained 1M NaCl (fraction 241 to 280). Under the best conditions, that is, after months of repeated use of the affinity column, the NaCl fraction contained 75 to 80% of the total binding activity loaded on the column. After each run, the column was washed successively with two volumes of 6 M urea (pH 3), two volumes of water, two volumes of 0.5%sodium dodecylsulfate (SDS),
VINCENT NEURUTENSIN RECEPTORS
97
FIGURE 3. Preparation of an affinity column for purification of the neurotensin receptor. The sequence of neurotensinis pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pru-Tyr-Ile-Leu. The COOH-terminal (8-13) sequence is directly involved in the neurotensin-receptor interaction.
and two volumes of water, then reequilibrated with the washing buffer. The affinity gel was either used immediately for a new purification cycle, or stored at 4°C in the presence of 0.02% sodium azide.
Structural and Functional Properties of Punjed Receptor After elimination of NaCl by gel filtration on Sephadex G-50, the purified material eluted from the affinity column bound 1251-labeled[Tyr3]neurotensin specifically and in a saturable manner. Binding parameters calculated from the corresponding linear Scatchard plot were Kd = 0.26 f 0.09 nM and B M =~ 31~ f 6.5 fmol/assay. From the amount of protein estimated by silver staining, the binding capacity of the purified receptor was calculated to be 7 to 8 nmol/mg of protein, corresponding to a purification factor of about 30,000 from the crude soluble receptor. This value is close to the theoretical value of 10 nmol/mg calculated on the basis of one neurotensin binding site per receptor of molecular weight lO0,OOO. The specificity of the neurotensin receptor was not affected during solubilization and purification. The binding of 12SI-labeled[Tyr3]neurotensinto the purified receptor was competitively inhibited by acetylneurotensin(8-13), neurotensin(9-13), and neurotensin(1-12) with relative potencies identical to those observed in membrane homogenate or crude CHAPS-solubilized extracts. The common order of decreasing affinity was neurotensin = acetylneurotensin(8-13) >> neurotensin(9-13) >> neurotensin(1-12).
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protein (rng) binding activity (prnollmg)
Flow through
KCI wash
NaCl eluate
320 f 48
1 f03
0.013 f 0.005
0.06 0.01
5 f 0.6
6130 f 1940
0
m (Y
n
0
Fraction number
FIGURE 4. Purification of the solubilized neurotensin receptor by affinity chromatography. Fractions eluted from the Sephadex C-25 and hydmxylapatite columns were loaded at 4°C on the Ultrogel AcA22neurotensin(2-13) column (3 x 15 cm). The column was washed with the equilibration buffer (fractions 60-150), then eluted with the same buffer containing 0.2 M KCI (fractions 151-170). After a new wash with the equilibration buffer (fractions 171-240). the column was eluted with equilibration buffer containing 1 M NaCl (fractions 241-280). The chromatography was monitored by automatic recording of the absorbance at 280 nm and by measuring the specific binding of 125I-labeled [Tyr3]neurotensin (0.2 nM) to aliquots of each fraction that were previously desalted on Sephadex (3-50. The protein content and binding activity of pooled fractions are indicated at top. Silver-stained gels of pooled fractions are shown. Fraction volume, 5 ml; flow rate, 60 ml/hr.
Affinity chromatography leads to the purification of a major protein band of 100 kDa as determined by silver (FIG.4) or Coomassie blue staining of the protein fraction eluted from the affinity column by 1 M NaCl and analyzed by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Iodination of this fraction with ['251]Nafollowed by SDS-PAGE and autoradiography gave the same result but allowed us to detect some additional minor impurities.14 Finally, the 100-kDa protein was directly identified as the neurotensin receptor by affinity labeling with 1251-labeled [Tyr3]neurotensin in the presence of disuccinimidyl suberate. l4 A value of 100 kDa for the molecular mass of the purified neurotensin receptor is in agreement with data obtained by gel filtration and affinity labeling of the crude soluble receptor.12
CONCLUSION The neurotensin receptor has been solubilized in an active and stable conformation from newborn mouse brain by using the detergent CHAPS in the presence of cho-
VINCENT NEURCYI'ENSIN RECEPTORS
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lesterol hemisuccinate. The soluble receptor was purified essentially in a single step by affinity chromatography on an activated Ultro-gel AcA22-neurotensin(2-13) column without noticeable changes in its binding properties. The technology described above has been applied to neurotensin receptors from brain of other species, including rat, rabbit, horse, bovine, and, more recently, human. The binding characteristics and the molecular structures of the various neurotensin receptors are remarkably similar. Affinities toward neurotensin are between 0.1 and 0.5 nM, and apparent molecular weights are about 100 kDa. The pure preparations are currently being used to obtain partial sequences of the receptor proteins. Our first results indicate that there exists a 50% homology between NHz-terminal sequences of human and mouse receptors. These sequences show no analogy with that deduced from the recently isolated cDNA clone for the adult rat neurotensin receptor. 15 Moreover, molecular masses of receptors purified from newborn mammals, including the rat receptor, are about 100 kDa, whereas the molecular mass of the cloned receptor from adult rat brain is approximately 50 kDa. 15 Therefore, we conclude that the neurotensin receptor expressed early during development of mammalian brain is different in terms of molecular structure from the receptor present in adult brain. Work is in progress in this laboratory to elucidate the structure and function of the neurotensin receptor expressed in newborn brain and to compare it with the receptor found in adult brain.
REFERENCES J. L., J. MAZELLA, S . AMAR,P. KlTABCl& J. P. VINCENT. 1984. Preparation of neuro1. SADOUL, tensin selectively iodinated on the tyrosine-3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem. Biophys. Res. Commun. 120: 812-819. J., C. b U S T l S , C. LABBB,F. CHECLER, P. KITABGI, C. GRANIER, J. VANRIETSCHOTEN 2. MAZELLA, & J. P. VINCENT.1983. [monoiodo-Trp"]Neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity and binding properties to rat brain synaptic membranes. I. Bid. Chem. 258: 3476-3481. 3. SCHOTTE, A., J. E. LEYSEN & P. M. LADURON. 1986. Evidence for a displaceable nonspecific [3H]neurotensin binding site in rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 333: 400-405. 4. SCHOTTE,A. & P. M. LADURON. 1987. Different postnatal ontngeny of two [3H]neurotensin binding sites in rat brain. Brain Res. 408: 326-328. 5 . ~ U S T IcS. , J. MAZELLA, P. KlTABCl& I. P. VINCENT. 1984. High-affinity neurotensin binding sites in differentiated neuroblastoma NlE115 cells. J. Neurochem. 42: 1094-1100. 6. AMAR,S., P. KITABCI& J. P. VINCENT.1986. Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line H'129. FEBS Lett. 201 31-36. 7. CHECLER, F., J. MAZELLA, P. KITABci & J. P. VINCENT.1986. High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons. J. Neurochem. 47: 1742-1748. 8. AMAR,S.,J. MAZELLA, F. CHECLER,P. KITABCI& J. P. VINCENT.1985. Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone NIE115. Biochem. Biophys. Res. Commun. 129: 117-125. J. A,, C. J. MOSES,M. A. PFENNINC & E. RICHELSON.1986. Neurotensin and its 9. GILBERT, analogs-Correlation of specific binding with stimulation of cyclic GMP formation in neuroblastoma clone NlE115. Biochem. Pharmacol. 35: 391-397. 10. Bozou, J. C., S. AMAR,J. P. VINCENT& P. KITABGI. 1986. Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma N1E115 cells: Involvement of the inhibitory GTPbinding component of adenylate cyclase. Mol. Pharmacol. 29: 489-496. 11, AMAR,S., P. KITABCI& J. P. VINCENT.1987. Stimulation of inositol phosphate production by
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12. 13. 14. 15.
ANNALS NEW YORK ACADEMY OF SCIENCES neurotensin in neuroblastoma NlE115 cells: Implication of GTP-binding proteins and relationship with the cyclic GMP response. J. Neurochem. 49: 999-1006. MAZELLA, J., J. CHABRY, P. KITABGI & J. P.VINCENT.1988. Solubilization and characterization of active neurotensin receptors from mouse brain. J. Biol. Chem. 263: 144-149. CHECLER, F., J. P. VINCENT& P. KITABGI.1986. Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes. J. Biol. Chem. 261: 11274-11281. MAZELLA, J., J. CHABRY, N. ZSURGER& J. P. VINCENT.1989. Purification of the neurotensin receptor from mouse brain by affinity chromatography. J. Biol. Chem. 264: 5559-5563. TANAKA, K . , M. MAW& S. NAKANISHI. 1990. Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4: 847-854.
INTRODUCTION Neurotensin is a neuropeptide involved in intercellular communication in the central nervous system and peripheral organs. During the last few years, a primary goal of this laboratory has been to characterize the structural and functional properties of neurotensin receptors in brain and gut. These studies led us to investigate the transduction mechanisms that are triggered by the association of neurotensin with its target cell and finally to purify the neurotensin receptor to homogeneity. This chapter will present an update of our major findings in this area.
BINDING PROPERTIES OF CENTRAL AND PERIPHERAL NEUROTENSIN RECEPTORS Detection and characterization of neurotensin binding sites was carried out with 125I-labeled [monoiodo-Tyr3]neurotensin(2000 Ci/mol) as radioactive ligand. Membranes prepared from brain or gastrointestinal tissues of adult mammals generally contain two different classes of neurotensin binding sites. For example, results illustrated in FIGURE 1A show that the Scatchard plot describing the binding of '251-labeled [Tyr3] neurotensin to adult mouse brain homogenate is curvilinear and can be resolved into two independent linear components.2 Each component represents a single class of noninteracting binding sites. Class 1 sites are characterized by a high affinity (0.13 nM) and a low capacity (TABLE1). By comparison, the affinity of class 2 sites is lower (2.4 nM), and their binding capacity is higher. Class 1 and 2 sites cannot be easily differentiated by their structure-function relationships because they exhibit the same order of affinity for a series of neurotensin analogues. Both types of sites recognize the 8-13 COOH-terminal hexapeptide of the neurotensin sequence.z On the other hand, the affinity of class 1 sites for neurotensin can be selectively decreased by sodium ions or GTP, whereas class 2 sites are much less sensitive to sodium ions and insensitive to GTP. Binding capacities of class 1 and 2 sites remain unchanged under these various conditions. However, the best tool for distinguishing between class 1 and 2 sites is levocabastine. This potent antihistamine-1 drug was introduced by Janssen Pharmaceutica and found to be able to partly inhibit [3H]neurotensin binding to rat brain membranes, although it was devoid of any neurotensin-like ac90
VINCENT NEURmENSIN RECEPTORS
91
tivity.3 Our results indicate that a 1 @ concentration I of levocabastine completely blocks the binding of 1251-labeled[Tyr3]neurotensin to class 2 sites without changing the binding properties of class 1 sites. This "all or none" effect is obviously the simplest way to differentiate class 1 sites from class 2 sites in mouse brain homogenate. Unfortunately levocabastine is active only on class 2 sites of murine species and therefore cannot be used to differentiate class 1 sites from class 2 sites in other mammalian species. The different properties of the two neurotensin-binding sites that are present in mouse brain are summarized in TABLE1. As already mentioned, almost all membrane preparations of central or peripheral origin contain two types of neurotensin binding sites. The amount and the relative proportions of each site vary from one species to the other. Results in FIGURE 2 show that both types of sites appear at different times during development of the mouse brain, the low-affinity class 2 sites being expressed later than the high-affinity class 1 sites. In agreement with results previously obtained from rat,4 brains of 5- to 10-dayold mouse (FIG. lB), rabbit, and rat contain four to six times more high-affinity neurotensin binding sites than brains of adults. Moreover, the low-affinity sites are undetectable in brains of these newborn animals.
TRANSDUCTION MECHANISMS OF NEUROTENSIN RECEPTORS Cell lines of neural or nonneural origin that express neurotensin receptors provide useful models for investigating the biochemical events generated by the association of neurotensin to its target cell. TABLE1 . Compared Properties of Two Different Neurotensin Binding Sites in Brain Homogenate of Adult Mouse Pronertv Dissociation constant (Tris 50 mM, pH 7.5, 25°C) Binding capacity
Site 1
Site 2
Kdl = 0.13 nM
Kd2
=
2.4 nM
Bml = 66 f m o l h g
Bm2 = 160 fmol/mg
Neurotensin sequence recognized
8-13
8-13
Effect of Na+ ions
Affinity decreased (Kdl X 5; ECYt'
Effect of GTP Effect of levocabastine
Affinity decreased (Kdl X 4; ECY;' No effect
=
35 mM)
=
0 . 3 pM)
Affinity decreased (Kd2 X 2; ECYt' = 100 mM) No effect Complete inhibition
Binding activity after CHAPS solubilization
Yes
No
Occurrence in cell cultures (NIE115, HT29, neurons) Involvement in transduction mechanisms
Yes
No
Yes
No
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A 0
-- Levocabastine
0.6
-
B
-- Levocabastine
Kd H = 0.13 nM
Bm H = 65 h U m g
k
I Yb
0
Kd H = 0.185 nM Bm H = 250 h h g
0 +Levocabastinel~M
100
B, fmol/rng
0
100
B, frnol/mg
FIGURE 1. Binding of 'ZSI-labeled[Tyr3]neurotensin to mouse brain homogenate. Freshly prepared homogenate from adult (A) or newborn (B) mouse brain (0.1 mg of protein per assay) were incubated at 2 5 ° C pH 7.5, with increasing concentrations of 1*5I-labeled [Tyr3]neurotensin alone or isotopically diluted with unlabeled neurotensin, in the presence (open symbols) or in the absence (closed symbols) of 1 mM levocabastine. This incubation medium (250 vl) consisted of a 50 mM Tris-HCI buffer containing 0.02% bovine serum albumin, 1 mM 1,lO-phenanthroline and 0.01 mM N-benzyloxycarbonylprolylprolinal to prevent degradation of the ligand. The specific binding was determined by filtration.2 Data are presented under the form of Scatchard plots. A, The resolution of the curvilinear Scatchard plot into two independent linear components is shown by the straight lines. B and F, bound and free concentrations of labeled ligand.
Mouse neuroblastoma cells of clone NlE115 possess high-affinity binding sites for neurotensin that appear during the course of their differentiation in vitro.5 These sites are very similar to class 1 sites of mouse brain homogenates, since their affinity is negatively modulated by sodium ions and GTP without change of the binding cap a ~ i t y Moreover, .~ I251-labeled [Tyr3]neurotensin binding to neuroblastoma cells is totally insensitive to levocabastine. The same type of site is also found in HT29 cells, an adenocarcinoma of human colon,6 and in primary cultured neurons of mouse fetal cortex The association of neurotensin to its receptor in neuroblastoma NlE115 cells triggers three different effects. First, the intracellular cGMP concentration increases according to a rapid and transient mechanism. This effect is calcium-dependent and leads to a maximal cGMP stimulation of 10-fold over basal leve1.8.9 A parallel 20 to 30%decrease of the cAMP basal level is also detected. This inhibitory effect of neurotensin is much easier to study after stimulation of cAMP by prostaglandin El. Under these conditions, neurotensin inhibits cAMP production by 50 to %%.lo A third consequence of the association of neurotensin to its receptor in NlE115 cells is an increase of intracellular inositol phosphate concentrations. In the presence of lithium ions, which block inositol monophosphate hydrolysis, neurotensin produces a rapid and transient stimulation of inositol triphosphate and inositol biphosphate levels and a slower in-
.'
VINCENT NEUROTENSIN RECEPTORS M
E 3 E cc
300 T
\
-
0
93
200
3 m n
3
100
M CI .
V C
G
o
n n
0
5
I
10
15
F60
FIGURE 2. Postnatal ontogeny of high- and low-affinity neurotensin-binding sites in mouse brain. Maximal binding capacities of sites 1 ( 0 )and 2 (0)were calculated from binding experiments as described in FIGURE1. Data are the mean f SEM of three experiments.
crease of the inositol monophosphate concentration. I I We have demonstrated that each one of the three effects described above are direct consequences of neurotensin receptor occupancy and are totally insensitive to levocabastine. Neurotensin is also able to stimulate phosphatidylinositol turnover in HT29 cells without changing the intracellular levels nf CAMP and cGMP.6
PURIFICATION OF NEUROTENSIN RECEPTORS Preliminary Choice The very first choice that should be made concerns the type of receptor to purify. As described above, the properties of the higher affinity class 1 sites from mouse brain homogenate are identical to those of neurotensin receptors that regulate intracellular levels of second messengers in target cells of neurotensin. Because our purpose was to purify functionally relevant neurotensin receptors, we chose to work on these highaffinity sites. Once a given receptor subtype has been chosen, the selection of the preparation used as starting material for solubilization and purification of the receptor is made essentially on the basis of the binding capacity, which must be as high as possible. The availability of a preparation containing only the desired subtype of receptor is obviously a great advantage. The tissue used as the source of receptors should also be readily available fresh and in quantity. The above considerations led us to purify neurotensin receptors from brain homogenates of newborn mice because this preparation is the richest known source of high-affinity neurotensin binding sites (250 fmol/mg protein) and because it does not contain low-affinity sites. Moreover, it is easy to obtain large amounts of newborn mouse brains.
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Solubilization The next step is to devise experimental conditions that would allow us to solubilize the neurotensin receptor in an active state and to monitor its binding activity.
Binding Assay
The choice of a convenient assay for the binding of neurotensin to its soluble receptor is an extremely important step. As far as the binding assay has not been validated, solubilization experiments cannot be properly interpreted. Thus, failure to detect neurotensin binding activity in a soluble extract can be due either to the absence of active receptor in the extract or to the inability of the binding assay to detect it. The necessity of finding simultaneously suitable conditions for solubilization of active receptor and for measurement of the soluble binding activity is one of the main difficulties in studies dealing with receptor solubilization. The best way to solve this problem is probably to use an assay that is as simple as possible. In our case, the separation of IZ5I-labeled[Tyr3]neurotensin bound to the soluble receptor from free ligand was carried out by gel filtration on Sephadex (3-50 (medium). The radioactivity bound to the receptor was eluted in the void volume and counted directly in a y counter.I2
Detergent No general rule or law exists that would permit selection of the most convenient detergent for solubilizing a given receptor without loss of binding activity. Thus, the task of finding the best detergent was accomplished by trial and error. CHAPS (3-[(3-cholamidopropyl)dimethylamonio]-l-propanesulfonicacid) was found to be the only detergent that could solubilize neurotensin receptors in an active form. The soluble binding sites corresponded exclusively to the high-affinity site found in mouse brain homogenates (see below). These sites were quantitatively solubilized by CHAPS concentrations between 0.6 and 0.7%.I2
Stabilizing Agent
In the absence of any stabilizing agent, the half-life (tl12) of the mouse brain receptor solubilized with 0.6% CHAPS was about 3 hr at 0°C. The presence of 0.12% cholesterol hemisuccinate (CHS) in the solubilization buffer largely improved the stability of the receptor ( t ~ 2= 31 hr). Addition of 1 nM neurotensin to mouse brain homogenate prior to the solubilization step further increased the stability at 0°C by a factor of 2 (ti12 = 65 hr). CHAPS-solubilized extracts frozen at -30°C could be stored for weeks without any detectable loss of binding activity, provided they contain 10%glycerol. This additive did not increase the half-life of the receptor at O"C, but protected it against losses of binding activity that occurred upon freezing and thawing.
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Peptidase Inhibitors In order to purify a receptor protein in its native form, it is necessary to work in the presence of protease inhibitors. Since only four different families of proteolytic enzymes exist, the presence of four specific inhibitors in the purification buffer should be sufficient to protect the receptor completely against degradation by proteases. Neurotensin receptors were solubilized and purified in buffers containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, inhibitor of serine proteases), 1 mM iodoacetamide (inhibitor of thiol proteases), 5 mM EDTA (inhibitor of metalloproteases), and 1 pM pepstatin (inhibitor of acidic proteases). Taking into account all the data described above, solubilization was carried out as follows. Freshly prepared homogenates of newborn mouse brain were incubated at a concentration of 10 mg of protein per ml in a 20 mM Tris-HC1 buffer, pH 7.5, containing 10% glycerol, 0.6%CHAPS, 0.12%CHS, and a mixture of protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 pM pepstatin, 1 mM iodoacetarnide, and 5 mM EDTA). After 30 min at O"C, the incubation medium was centrifuged at 110,000 x g for 15 min. The supernatant, which contained the soluble neurotensin receptor, was either used immediately or stored at -30°C.
Properties of the Soluble Receptor Binding Properties Soluble extracts obtained from either newborn or adult mouse brain contain a single type of high-affinity neurotensin receptors. In both cases, the affinity of the soluble receptor for neurotensin (about 0.3 nM) is decreased by sodium ions and is insensitive to levocabastine. The ability of GTP to negatively modulate the affinity of the receptor for neurotensin is lost on solubilization.12Apart from this difference, CHAPS seems to have solubilized neurotensin receptors that correspond to the membrane-bound class 1 sites (TABLE1). Although the levocabastine-sensitive class 2 sites are more abundant than class 1 sites in adult mouse brain (FIG.lA), class 2 sites are no longer detectable after solubilization. Molecular Structure Gel filtration of the CHAPS-solubilized neurotensin receptor from mouse brain on an Ultrogel AcA34 column calibrated with protein standards gave an approximate molecular mass of 100 kDa.12 The soluble receptor was covalently radiolabeled by photoaffinity and by chemical cross-linking. 12 Both techniques resulted in the specific labeling of the same protein band of molecular mass about 100 kDa.12 To summarize, neurotensin receptors from mouse brain homogenate can be solubilized in an active and stable form using the zwitterionic detergent CHAPS in the presence of CHS. The soluble receptor is a single polypeptide chain of 100 kDa, which binds neurotensin with a high affinity. From these data, it is reasonable to undertake the purification of the receptor.
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ANNALS NEW YORK ACADEMY OF SCIENCES
Purification The soluble neurotensin receptor from newborn mouse brain was purified essentially in a single step of ligand affinity chromatography. However, in order to minimize the total amount of protein loaded on the affinity column, the soluble extract was first prepurified by ion-exchange chromatography. Prepurification Step
The soluble extract was diluted 2.5 times and passed at 4°C through two columns of SP-Sephadex C-25 and hydroxylapatite (100 ml each) connected in series and equilibrated with the Tris-glycerol buffer containing 0.1% CHAPS, 0.02% CHS, and protease inhibitors. About 50% of proteins, including several brain proteases, 13 were retained on the gel, whereas the bulk of the neurotensin-binding activity was collected with the flow-through and used in the next purification step. Afinity Chromatography Preparation of afinity gel: In preliminary experiments, covalent binding of native neurotensin to various chemically activated gels was found to occur in low yields. The reason is probably that the only free amine available for the coupling reaction in the neurotensin sequence is the side chain of Lys6, the NH2-terminal residue being pGlu. To overcome this difficulty, neurotensin (2-13) was used in place of the native peptide to prepare the affinity column (FIG.3). Interestingly, the affinity of the (2-13) sequence for the neurotensin receptor was found to be better than that of native neurotensin in binding competition experiments. The neurotensin (2-13) solution coupled to the gel was radiolabeled by tracer amounts of [3H]neurotensin (2-13). Glutaraldehyde-activated Ultrogel AcA22 (IBF Biotechnics) was chosen as support because it contained a spacer arm and because ligand leakage was low after extensive washing of the affinity gel. The yield of the coupling reaction calculated from the radioactivity incorporated into the gel was between 40 and 70% in seven different experiments. Chromatography of prepurified receptor: The binding activity eluted from SPSephadex C-25 and hydroxylapatite was loaded on the affinity column (FIG.4,fractions 1 to 60), which was then washed with the Tris-glycerol buffer containing 0.1% CHAPS and 0.02% CHS (fractions 61 to 150). When the affinity column was working at its better level of efficiency, pooled fractions 1 to 150 contained almost all the loaded proteins but only 10 to 15% of the total binding activity. Proteins retained nonspecifically on the gel by ionic interactions were eliminated by elution with the washing buffer, which contained 200 mM KC1 (fractions 151 to 170). A small amount (2 to 5%) of specific neurotensin-binding activity was lost during this step. The column was rinsed one more time with 300 ml of washing buffer (fractions 171 to 240), and the neurotensin receptor was eluted with the washing buffer, which contained 1M NaCl (fraction 241 to 280). Under the best conditions, that is, after months of repeated use of the affinity column, the NaCl fraction contained 75 to 80% of the total binding activity loaded on the column. After each run, the column was washed successively with two volumes of 6 M urea (pH 3), two volumes of water, two volumes of 0.5%sodium dodecylsulfate (SDS),
VINCENT NEURUTENSIN RECEPTORS
97
FIGURE 3. Preparation of an affinity column for purification of the neurotensin receptor. The sequence of neurotensinis pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pru-Tyr-Ile-Leu. The COOH-terminal (8-13) sequence is directly involved in the neurotensin-receptor interaction.
and two volumes of water, then reequilibrated with the washing buffer. The affinity gel was either used immediately for a new purification cycle, or stored at 4°C in the presence of 0.02% sodium azide.
Structural and Functional Properties of Punjed Receptor After elimination of NaCl by gel filtration on Sephadex G-50, the purified material eluted from the affinity column bound 1251-labeled[Tyr3]neurotensin specifically and in a saturable manner. Binding parameters calculated from the corresponding linear Scatchard plot were Kd = 0.26 f 0.09 nM and B M =~ 31~ f 6.5 fmol/assay. From the amount of protein estimated by silver staining, the binding capacity of the purified receptor was calculated to be 7 to 8 nmol/mg of protein, corresponding to a purification factor of about 30,000 from the crude soluble receptor. This value is close to the theoretical value of 10 nmol/mg calculated on the basis of one neurotensin binding site per receptor of molecular weight lO0,OOO. The specificity of the neurotensin receptor was not affected during solubilization and purification. The binding of 12SI-labeled[Tyr3]neurotensinto the purified receptor was competitively inhibited by acetylneurotensin(8-13), neurotensin(9-13), and neurotensin(1-12) with relative potencies identical to those observed in membrane homogenate or crude CHAPS-solubilized extracts. The common order of decreasing affinity was neurotensin = acetylneurotensin(8-13) >> neurotensin(9-13) >> neurotensin(1-12).
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protein (rng) binding activity (prnollmg)
Flow through
KCI wash
NaCl eluate
320 f 48
1 f03
0.013 f 0.005
0.06 0.01
5 f 0.6
6130 f 1940
0
m (Y
n
0
Fraction number
FIGURE 4. Purification of the solubilized neurotensin receptor by affinity chromatography. Fractions eluted from the Sephadex C-25 and hydmxylapatite columns were loaded at 4°C on the Ultrogel AcA22neurotensin(2-13) column (3 x 15 cm). The column was washed with the equilibration buffer (fractions 60-150), then eluted with the same buffer containing 0.2 M KCI (fractions 151-170). After a new wash with the equilibration buffer (fractions 171-240). the column was eluted with equilibration buffer containing 1 M NaCl (fractions 241-280). The chromatography was monitored by automatic recording of the absorbance at 280 nm and by measuring the specific binding of 125I-labeled [Tyr3]neurotensin (0.2 nM) to aliquots of each fraction that were previously desalted on Sephadex (3-50. The protein content and binding activity of pooled fractions are indicated at top. Silver-stained gels of pooled fractions are shown. Fraction volume, 5 ml; flow rate, 60 ml/hr.
Affinity chromatography leads to the purification of a major protein band of 100 kDa as determined by silver (FIG.4) or Coomassie blue staining of the protein fraction eluted from the affinity column by 1 M NaCl and analyzed by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Iodination of this fraction with ['251]Nafollowed by SDS-PAGE and autoradiography gave the same result but allowed us to detect some additional minor impurities.14 Finally, the 100-kDa protein was directly identified as the neurotensin receptor by affinity labeling with 1251-labeled [Tyr3]neurotensin in the presence of disuccinimidyl suberate. l4 A value of 100 kDa for the molecular mass of the purified neurotensin receptor is in agreement with data obtained by gel filtration and affinity labeling of the crude soluble receptor.12
CONCLUSION The neurotensin receptor has been solubilized in an active and stable conformation from newborn mouse brain by using the detergent CHAPS in the presence of cho-
VINCENT NEURCYI'ENSIN RECEPTORS
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lesterol hemisuccinate. The soluble receptor was purified essentially in a single step by affinity chromatography on an activated Ultro-gel AcA22-neurotensin(2-13) column without noticeable changes in its binding properties. The technology described above has been applied to neurotensin receptors from brain of other species, including rat, rabbit, horse, bovine, and, more recently, human. The binding characteristics and the molecular structures of the various neurotensin receptors are remarkably similar. Affinities toward neurotensin are between 0.1 and 0.5 nM, and apparent molecular weights are about 100 kDa. The pure preparations are currently being used to obtain partial sequences of the receptor proteins. Our first results indicate that there exists a 50% homology between NHz-terminal sequences of human and mouse receptors. These sequences show no analogy with that deduced from the recently isolated cDNA clone for the adult rat neurotensin receptor. 15 Moreover, molecular masses of receptors purified from newborn mammals, including the rat receptor, are about 100 kDa, whereas the molecular mass of the cloned receptor from adult rat brain is approximately 50 kDa. 15 Therefore, we conclude that the neurotensin receptor expressed early during development of mammalian brain is different in terms of molecular structure from the receptor present in adult brain. Work is in progress in this laboratory to elucidate the structure and function of the neurotensin receptor expressed in newborn brain and to compare it with the receptor found in adult brain.
REFERENCES J. L., J. MAZELLA, S . AMAR,P. KlTABCl& J. P. VINCENT. 1984. Preparation of neuro1. SADOUL, tensin selectively iodinated on the tyrosine-3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem. Biophys. Res. Commun. 120: 812-819. J., C. b U S T l S , C. LABBB,F. CHECLER, P. KITABGI, C. GRANIER, J. VANRIETSCHOTEN 2. MAZELLA, & J. P. VINCENT.1983. [monoiodo-Trp"]Neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity and binding properties to rat brain synaptic membranes. I. Bid. Chem. 258: 3476-3481. 3. SCHOTTE, A., J. E. LEYSEN & P. M. LADURON. 1986. Evidence for a displaceable nonspecific [3H]neurotensin binding site in rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 333: 400-405. 4. SCHOTTE,A. & P. M. LADURON. 1987. Different postnatal ontngeny of two [3H]neurotensin binding sites in rat brain. Brain Res. 408: 326-328. 5 . ~ U S T IcS. , J. MAZELLA, P. KlTABCl& I. P. VINCENT. 1984. High-affinity neurotensin binding sites in differentiated neuroblastoma NlE115 cells. J. Neurochem. 42: 1094-1100. 6. AMAR,S., P. KITABCI& J. P. VINCENT.1986. Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line H'129. FEBS Lett. 201 31-36. 7. CHECLER, F., J. MAZELLA, P. KITABci & J. P. VINCENT.1986. High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons. J. Neurochem. 47: 1742-1748. 8. AMAR,S.,J. MAZELLA, F. CHECLER,P. KITABCI& J. P. VINCENT.1985. Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone NIE115. Biochem. Biophys. Res. Commun. 129: 117-125. J. A,, C. J. MOSES,M. A. PFENNINC & E. RICHELSON.1986. Neurotensin and its 9. GILBERT, analogs-Correlation of specific binding with stimulation of cyclic GMP formation in neuroblastoma clone NlE115. Biochem. Pharmacol. 35: 391-397. 10. Bozou, J. C., S. AMAR,J. P. VINCENT& P. KITABGI. 1986. Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma N1E115 cells: Involvement of the inhibitory GTPbinding component of adenylate cyclase. Mol. Pharmacol. 29: 489-496. 11, AMAR,S., P. KITABCI& J. P. VINCENT.1987. Stimulation of inositol phosphate production by
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ANNALS NEW YORK ACADEMY OF SCIENCES neurotensin in neuroblastoma NlE115 cells: Implication of GTP-binding proteins and relationship with the cyclic GMP response. J. Neurochem. 49: 999-1006. MAZELLA, J., J. CHABRY, P. KITABGI & J. P.VINCENT.1988. Solubilization and characterization of active neurotensin receptors from mouse brain. J. Biol. Chem. 263: 144-149. CHECLER, F., J. P. VINCENT& P. KITABGI.1986. Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes. J. Biol. Chem. 261: 11274-11281. MAZELLA, J., J. CHABRY, N. ZSURGER& J. P. VINCENT.1989. Purification of the neurotensin receptor from mouse brain by affinity chromatography. J. Biol. Chem. 264: 5559-5563. TANAKA, K . , M. MAW& S. NAKANISHI. 1990. Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4: 847-854.
