NT Agonist Regulates Expression of Nuclear High-affinity Neurotensin Receptors
INSERM U482, Hôpital Saint-Antoine (MTML,PF); Service d'Imagerie Cellulaire, IFR70, Hôpital de la Pitié Salpêtrière (C-MB); EA 3512 Faculté de Médecine Xavier Bichat (DP); and INSERM EMI 0350, Hôpital Saint-Antoine (WR), France
Correspondence to: Dr. P. Forgez, INSERM U482, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine 75571, Paris Cedex 12, France. E-mail: forgez{at}st-antoine.inserm.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: neurotensin NT-1 receptor G-protein-coupled receptor nuclear envelope nuclear soma agonist treatment
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the CNS and in the periphery, the vast majority of NT effects are mediated by the NT-1 receptor. Second-messenger pathways activated by NT-1 receptor have primarily been studied in cultured cell lines expressing the NT-1 receptor. When murine neuroblastoma cells, N1E-115, are challenged with NT, phosphatidyl inositols are hydrolyzed, leading to Ca2+ mobilization and the formation of cGMP (Gilbert and Richelson 1984; Amar et al. 1985
,1987
; Snider et al. 1986
). More recently, it was shown that NT activated the MAP kinases, p44 and p42, leading to immediate or delayed responses to NT involving gene transcription, cell growth, death, or differentiation (PoinotChazel et al. 1996
; Ehlers et al. 1998
,2000
).
The interaction between NT and the NT-1 receptor results in endocytosis of NT-1 receptor through sequestration, internalization, and trafficking of the receptor (Pierce et al. 2002). Several studies using fluorescent NT analogues showed that the ligand was localized within small vesicular organelles in the perinuclear region after its NT-1 receptor internalization (Faure et al. 1995a
,b
). Additional localization studies revealed the presence of the NT-1 receptor in the nuclear compartment (Dana et al. 1989
; Boudin et al. 1998
). This latter phenomenon is not unique because it has already been described for other GPCRs, such as angiotensin type 1, VIP, opioid, prostaglandin, and muscarinic receptors, but the function of these receptor complexes in the nuclear soma or in the nuclear envelope is unknown (Re et al. 1984
; Omary and Kagnoff 1987
; Tang et al. 1992
; Belcheva et al. 1993
; Lind and Cavanagh 1993
; Bhattacharya et al. 1999
). It could also be suggested that the NT-NT-1 receptor interaction is recognized as a nuclear targeting signal, as has been shown for the angiotensin type 1 and opioid receptors (Booz et al. 1992
; Belcheva et al. 1995
).
We demonstrate in this study the presence of NT-1 receptors in the nuclear compartment of the substantia nigra neuron cell bodies, in nuclei isolated from LNM35 pulmonary cells (which endogenously express NT and NT-1 receptor), as well as nuclei from CHO cells stably expressing the NT-1 receptor. Our data suggest a relationship between nuclear expression of the NT-1 receptor and the presence of NT in the extracellular compartment.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electron Microscopy and Quantitative Analysis
We analyzed three rat brains. For each rat, two to four sections from different blocks were systematically scanned. Microscopic fields with cell bodies were photographed at an original magnification of x8300. All silver-enhanced gold granules were classified according to the type of element and the ultrastructure with which they were associated. For perikarya, they were classified as plasma membrane, cytosol (labeling apparently not associated with any organelle), Golgi apparatus/endoplasmic reticulum, vesicle, mitochondria, nuclear envelope, or nuclear soma. We measured the surface of the nucleus and cytoplasm of each cell body on the film negatives using a computer-based image analysis system associated with a CCD camera and the software Historag (Biocom; Les Ulis, France). We analyzed a total of 264 micrographs representing 75 cell bodies and 1119 gold particles.
Cell Culture
Human lung cancer cells (LNM35) were grown in RPMI 1640 (Invitrogen; Cergy Pontoise, France). CHO cells were grown in MEM medium without ribonucleosides and deoxyribonucleosides (Invitrogen). All media were supplemented with 10% fetal calf serum and 2 mM L-glutamine (Invitrogen). For stably transfected CHO cells, 250 µg/ml of geneticin (G418) was added to maintain the transgene. Cells were incubated at 37C in a humidified 5% CO2 atmosphere. At confluence, cells were routinely dispersed in trypsinEDTA 0.25% and subcultured at 1:10 dilution.
Stable Cell Line Establishment
The expression plasmid pNT1-EGFP containing the rat NT-1 receptor coding region and the modified form of EGFP from Aequorea victoria, directed by the CMV promoter, was provided by Dr. LlorensCortes (Lenkei et al. 2000). pNT1-EGFP was stably transfected in CHO cells as described by Boudin et al. (1995)
. Stable transfectants were selected with G418 (1000 µg/ml) and colonies were screened for EGFP expression, using a Nikon Diaphot inverted fluorescence microscope at a x40 magnification, and by [125I]-NT-binding assays (Boudin et al. 1995
).
Isolation of Membrane-depleted Nuclei
Purified nuclei were prepared using a modification of the procedure previously described (Facy et al. 1990). Cells were washed twice in cold PBS and collected by low-speed centrifugation (300 x g for 7 min). The pellet was washed in 20 ml TKCM buffer containing 10 mM Tris-HCl (pH 7.3), 20 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 0.2 mM spermidine, and collected by centrifugation. The pellet was resuspended in 20 ml TKCM buffer and incubated on ice for 1 hr. Cells were homogenized twice with a Potter-Elvehjem tissue grinder in the presence of a mixture of protease inhibitors for mammalian cell and tissue extracts (Sigma-Aldrich; St-Quentin, France), and then centrifuged at 300 x g for 7 min. The cell suspension was layered onto 2 M sucrose, 50 mM Tris-HCl, pH 7.4, and centrifuged at 25,000 x g for 30 min. Nuclei were collected from the bottom of the tubes, and cell membrane homogenates were collected at the interface.
Chromatin Preparation
Nuclei were resuspended in 1 ml of 1 mM Tris-HCl, pH 7.9. The nuclear suspension was incubated on ice for 1 hr before homogenization. The chromatin was separated from the nuclear membranes by centrifugation at 12,000 x g for 30 min.
Control of the Purity of Membrane-depleted Nuclei
CHONT1 cells were labeled with a mix containing 7.5 µCi/ml of L-[35S]-methionine and L-[35S]-cysteine (Amersham Biosciences; Orsay, France) for 16 hr. Membrane homogenates and nuclei were prepared as described above and the radioactivity of each fraction was counted. In parallel, we prepared an unlabeled cell suspension. Before the ultracentrifugation step, we added either 3 x 106 cpm of labeled nuclei or 7 x 107 cpm of labeled membrane. The mixture was layered onto a sucrose layer and centrifuged as described above. The radioactivity was counted in the membrane homogenates and in the nuclear fraction from both samples.
Enzymatic Markers
The purity of the nuclear fraction was assessed by assaying selected enzymatic marker activity for each cell fraction. The activity of 5' nucleotidase was performed using a Sigma kit based on the method developed by Arkesteijn (1976). The ouabain-sensitive Na+-K+-ATPase was assessed by the method of Gache et al. (1976). Ouabain-sensitive Na+-K+-ATPase activity was defined as the activity inhibited by 1 mM ouabain. The K+-EGTA ATPase activity was measured as an enzymatic marker of the endoplasmic reticular membranes (Gache et al. 1976
).
Binding Studies
Nuclei, cell membrane homogenates, and chromatin were washed with 50 mM Tris-HCl, pH 7.4, and were immediately used for [125I]-NT-binding and enzymatic assays. Protein contents were estimated by the method of Bradford using bovine serum albumin as standard (Bradford 1976). Radioligand binding studies were carried out either on cell membrane homogenates or on nuclei. Binding studies were performed as follows: 40 µg of protein from cell membrane homogenates or 80 µg of protein from nuclei was incubated with 0.5 nM [125I]-NT in a final volume of 250 µl of buffer A (50 mM Tris, pH 7.4, 0.2% BSA, and 0.8 mM 1.10-phenantroline, 1.6 mM MgCl2) for 45 min at room temperature (RT). Nonspecific binding was measured in the presence of 1 µM unlabeled NT. The competition binding was carried out under the same conditions, using a range of unlabeled NT concentrations (10-6 to 10-11 M). Binding assays were terminated by addition of ice-cold 50 mM Tris-HCl (pH 7.4) supplemented with 0.2% BSA, followed by filtration through glass microfiber filters (GF/B; Whatman, Maidstone, UK) preincubated in 0.2% polyethylenimine (SigmaAldrich). After washing three times with 5 ml ice-cold buffer, the radioactivity retained on the filters was counted in a
-counter (Wallac model 1470 Wizard). Binding affinities and densities (Kd and Bmax) were estimated by the Ligand EBDA program.
Immunofluorescent Staining of Nuclei for NT-1 Receptor
Fifty µl of purified nuclei was sedimented onto slides with a Cytospin centrifuge. Nuclei were fixed in 2% paraformaldehyde for 30 min. Fixed nuclei were washed three times with PBS at RT before proceeding to immunocytochemistry. Nonspecific binding was blocked with 0.1% Triton X-100/PBS containing 10% normal rabbit serum (NRS) for 1 hr at RT. Nuclei were then washed three times with PBS and incubated for 1 hr at RT with a goat polyclonal anti-NT-1 receptor antibody (Santa Cruz Biotechnology; Santa Cruz, CA) diluted 1:25 in PBS containing 0.1% Triton X-100 and 3% NRS (buffer A). Nuclei were then rinsed three times with PBS and incubated for 45 min at RT with rabbit anti-goatTexas Red secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA) diluted 1:300 in buffer A. After three final rinses in PBS, slides were mounted in Glycergel (DAKO-Cytomation; Trappes, France). Appropriate controls devoid of NT-1 receptor antibody were performed in parallel to determine nonspecific staining.
RNA Extraction and Reverse Transcription-polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from LNM35 cells using the acidic phenol/chloroform guanidine thiocyanate method (Chirgwin et al. 1979). One hundred ng of total LNM35 RNA was reverse-transcribed in a 30-µl reaction mixture containing 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 1 mM each of dNTP, 24 U RNasin (Promega; Charbonnière, France), 1 µg/µl of each oligo (dN) and oligo (dT) (Amersham Biosciences; Orsay, France) for NT transcript, or primer RT-NT-1 (5'-GCTGACGTAGAAGAG-3') for NT-1 receptor transcript, and 200 U Moloney murine leukemia virus reverse transcriptase (Invitrogen; Cergy Pontoise, France) at 37C for 1 hr. The PCR amplification was performed on 1:5 (v/v) dilution of the RT reaction in a mixture containing 16 mM Tris-HCl, pH 8.3, 40 mM KCl, 1 mM MgCl2, 0.2 mM of each dNTP, 25 pmol of the sense primer, 25 pmol of the antisense primer and 1 U Taq polymerase (Applied Biosystems; Les Ulis, France) in a final volume of 50 µl. The PCR primers had the following sequences: S-NT-1 5'-CGTGGAGCTGTACAACTTCA-3' and AS-NT-1 5'-CAGCCAGCAGACCACAAAGG-3' for NT-1 receptor; S-NT 5'-AAGCACATGTTCCCTCTT-3' and AS-NT 5'-CATACAGCTGCCGTTTCAGA-3' for NT (Invitrogen). The amplicon sizes of NT-1 receptor, and NT were 590 and 446 nucleotides, respectively. The amplification profile was divided into 35 cycles of denaturation at 94C for 30 sec, annealing at 57C for 45 sec, and extension at 72C for 45 sec. PCR products were electrophoresed on 1% agarose gels in 90 mM Tris borate and 2 mM EDTA buffer. We routinely introduced a 100 bp DNA ladder (Invitrogen) as a size marker. Gels were stained with ethidium bromide and photographed under a UV lamp.
Confocal Microscopy
Confocal microscope analysis was carried out using the TCS SP Leica microscope (Lasertechnik) equipped with a x63 objective (plan apo; NA 1.4). A focal series was collected for each specimen every 0.5 µm for nuclei from the CHONT1EGFP cell line. Each confocal image shown here corresponded to the middle of seven optical serial sections. For each optical section, double fluorescence was simultaneously acquired using a kryptonargon mixed-gas laser adjusted to 488 nm for GFP and to 568 nm for TRITC. The variable center of spectrophotometers was adjusted to recover green (500550 nm) and red (580630 nm) fluorescence. The signal was treated with line averaging to integrate the signal collected over four lines in order to reduce signal noise. Selected paired sections were then processed to produce a single overlay image (color merged) using a PC computer equipped with Photoshop software (version 6.0) (Adobe; Tucson, AZ). To quantify the intensity of nuclear labeling, Leica TCS-NT software was used. For each experiment, the middle optical sections of 1015 nuclei from control cells and 1015 nuclei from treated cells were studied with the same confocal acquisition parameters. An ellipse was drawn around each nucleus to select the region to analyze and the activated pixel intensity was measured. The results provided include the area and the mean intensity of the selected region.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, in vivo and in vitro cell models were used to demonstrate the physiological relevance of the agonist-induced localization of the NT-1 receptor to the nuclear envelope and the nuclear soma. First, we studied rat SN neuronal cells, which express NT-1 receptors in neural cell bodies and dendrites (Boudin et al. 1998). This brain area is also innervated by a dense network of NT-containing axon terminals, and is consequently exposed to frequent NT stimulation (Jennes et al. 1982
). We observed that NT-1 receptors were found within the nuclear soma of rat SN neuron cell bodies (Figure 1).
In a complementary approach, experiments with CHONT1-EGFP cells showed a direct increase in the nuclear localized NT-1 receptor due to agonist exposure. These results were confirmed in a cell line that endogenously expresses NT and the NT-1 receptor, LNM35. We further demonstrated the effect of an NT antagonist, SR 48692, to extensively inhibit the binding capacity of the NT-1 receptor from LNM35 purified nuclei. These data support the hypothesis that NT acts as the inductive agent for accumulation of the NT-1 receptor in the nuclear compartment.
Agonist-induced targeting of GPCRs at the nuclear envelope and the nuclear soma has been previously observed. In the case of angiotensin type 1 receptor, receptor immunoreactivity was strongly increased in the presence of angiotensin II and in a dose-dependent manner. This effect was blocked by an angiotensin type 1-specific antagonist (Lu et al. 1998). In a similar finding, nuclear upregulation of opioid binding sites in the nuclear envelope and nuclear soma of NG108-15 cells was observed after a long agonist exposure (Belcheva et al. 1995
). The localization of GPCRs to the nucleus in cells exposed to agonist stimulation suggests that the function of the nuclear NT-1 receptor could be related to the cell response evoked by intense and persistent agonist stimuli. In the case of NT, the latter phenomenon may occur in various pathological situations, such as in chronic pancreatitis or in tumors with endocrine differentiation, where NT plasma or tumor tissue concentration is exceedingly increased (TheodorssonNorheim et al. 1983
; Nustede et al. 1991
; Meggiato et al. 1996
).
The preparation of purified nuclei is a delicate task because the nuclei are fragile and difficult to keep intact. Moreover, contamination with plasma membrane is expected during nuclear preparative purification. We obtained nuclei having 95% of purity, and we estimated that the cross-contamination could represent at most 20% of the total specific cpm of the nuclear fraction. Therefore, a large majority of the specific binding was generated by the nucleus-localized receptor. We also observed that the nuclear NT-1 receptor increased in the presence of the agonist and that a specific NT-1 receptor antagonist reduced the nuclear binding capacity in cells endogenously expressing NT. We can rule out that the diminished binding capacity of the nuclear fraction was due to site occupancy by the receptor antagonist, because we have previously demonstrated that SR 48692 does not internalize with the NT-1 receptor. Therefore, downregulation of NT-1 receptors within the nuclei occurred either via blockade of receptor trafficking or blockade of the transduction signal from the cell membrane. However, we did not detect any effect of the NT agonist on the transgene promoter used to generate the stable cell lines (data not shown). These data imply that the accumulated nuclear NT-1 receptor would correspond to internalized receptor, sequestered in the nuclear compartment.
We found that the NT-1 receptor was coupled to G-protein in the nuclear fraction of LNM35 cells. This observation is in agreement with other GPCRs, such as angiotensin type 1, VIP, opioid, prostaglandin, and muscarinic receptors found in the nuclei or in the nuclear envelope (Booz et al. 1992; Belcheva et al. 1993
; Ventura et al. 1998
; Bhattacharya et al. 1999
). At the opposite, nuclear NT-1 receptor from CHONT1 was not sensitive to Gpp(NH)p. In CHONT1, the Bmax of purified nuclei appeared to be equivalent to the Bmax observed in plasma membrane (Boudin et al. 1995
), suggesting an abnormal NT-1 receptor overexpression in the nuclear fraction. In this fraction, coupled NT-1 receptors would probably be undetectable, and they would be hidden by the response of the uncoupled receptors. For the same reason, the high expression of NT-1 receptorEGFP in the nuclei of control CHONT1-EGFP cells is probably abnormally increased.
The activation of renin, angiotensinogen, opoid peptide, or inducible NO synthase gene transcription was previously reported in experiments in which purified nuclei bearing angiotensin type 1, opioid receptors, or the prostaglandin E2 receptor EP3 were exposed to their respective agonists (Eggena et al. 1993; Ventura et al. 1998
; Bhattacharya et al. 1999
). If an additional biological role for nuclear receptor GPCRs is to activate specific genes, the question remains about how the agonists reach the nucleus. In the case of NT, it was previously demonstrated that NT was internalized with its receptor and was retrieved from the perinuclear region within small vesicular organelles (Faure et al. 1995a
,b
). Moreover, further studies indicated that NT was recruited to the trans-Golgi network from late or recycling endosomes (Vandenbulcke et al. 2000
). It would therefore be consistent to suggest that internalized NT could stimulate nuclear NT-1 receptors to activate specific gene transcription.
The present data reveal that nuclear localization of NT-1 receptor is related to agonist exposure. However, further investigations are necessary to define the specific intracrine biological role of receptor NT-1 localized in the nuclear envelope and nuclear soma. These findings, in addition to similar observations made with several receptors from the same family, suggest an additional function for GPCRs related to hormonal regulation.
![]() |
Acknowledgments |
---|
We wish to honor the memory of the late Dr Annie Pierre Sève and to express our appreciation for her assistance. We also wish to express many thanks to Dr Neil Insdorf for his precious help in the writing of the manuscript and for helpful discussions.
We thank Dr Germain Trugnan and Dr Virginie M Pickel for the use of their facilities, Philippe Fontanges, Emmanuel GamelasNagaphaes, and June Chan for expert technical assistance, and Anne Marie Lhiaubet for providing [125I]-NT. We also thank Dr Danielle Gully for providing us with SR 48692 (SanofiSynthelabo).
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amar S, Kitabgi P, Vincent JP (1987) Stimulation of inositol phosphate production by neurotensin in neuroblastoma N1E115 cells: implication of GTP-binding proteins and relationship with the cyclic GMP response. J Neurochem 49:9991006[Medline]
Amar S, Mazella J, Checler F, Kitabgi P, Vincent JP (1985) Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115. Biochem Biophys Res Commun 129:117125[Medline]
Arkesteijn CL (1976) A kinetic method for serum 5'-nucleotidase using stabilised glutamate dehydrogenase. J Clin Chem Clin Biochem 14:155158[Medline]
Belcheva M, Barg J, Rowinski J, Clark WG, Gloeckner CA, Ho A, Gao XM, Chuang DM, et al. (1993) Novel opioid binding sites associated with nuclei of NG10815 neurohybrid cells. J Neurosci 13:104114[Abstract]
Belcheva MM, Gucker S, Chuang DM, Clark WG, Jefcoat LB, McHale RJ, Toth G, et al. (1995) Modulation of opioid binding associated with nuclear matrix and nuclear membranes of NG10815 cells. J Pharmacol Exp Ther 274:15131523[Abstract]
Bhattacharya M, Peri K, Ribeiro-da-Silva A, Almazan G, Shichi H, Hou X, Varma DR, et al. (1999) Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem 274:1571915724
Booz GW, Conrad KM, Hess AL, Singer HA, Baker KM (1992) Angiotensin-II-binding sites on hepatocyte nuclei. Endocrinology 130:36413649[Abstract]
Boudin H, GrauzGuyon A, Faure MP, Forgez P, Lhiaubet AM, Dennis M, Beaudet A, et al. (1995) Immunological recognition of different forms of the neurotensin receptor in transfected cells and rat brain. Biochem J 305:277283[Medline]
Boudin H, Pelaprat D, Rostene W, Pickel VM, Beaudet A (1998) Correlative ultrastructural distribution of neurotensin receptor proteins and binding sites in the rat substantia nigra. J Neurosci 18:84738484
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254[Medline]
Chalon P, Vita N, Kaghad M, Guillemot M, Bonnin J, Delpech B, Le Fur G, et al. (1996) Molecular cloning of a levocabastine-sensitive neurotensin binding site. FEBS Lett 386:9194[Medline]
Chan J, Aoki C, Pickel VM (1990) Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding. J Neurosci Methods 33:113127[Medline]
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945299[Medline]
Dana C, Vial M, Leonard K, Beauregard A, Kitabgi P, Vincent JP, Rostene W, et al. (1989) Electron microscopic localization of neurotensin binding sites in the midbrain tegmentum of the rat. I. Ventral tegmental area and the interfascicular nucleus. J Neurosci 9:22472257[Abstract]
Eggena P, Zhu JH, Clegg K, Barrett JD (1993) Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA. Hypertension 22:496501[Abstract]
Ehlers RA, Bonnor RM, Wang X, Hellmich MR, Evers BM (1998) Signal transduction mechanisms in neurotensin-mediated cellular regulation. Surgery 124:239246[Medline]
Ehlers RA, Zhang Y, Hellmich MR, Evers BM (2000) Neurotensin-mediated activation of MAPK pathways and AP-1 binding in the human pancreatic cancer cell line, MIA PaCa-2. Biochem Biophys Res Commun 269:704708[Medline]
Facy P, Seve AP, Hubert M, Monsigny M, Hubert J (1990) Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60). Exp Cell Res 190:151160[Medline]
Faure MP, Alonso A, Nouel D, Gaudriault G, Dennis M, Vincent JP, Beaudet A (1995a) Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain. J Neurosci 15:41404147[Abstract]
Faure MP, Nouel D, Beaudet A (1995b) Axonal and dendritic transport of internalized neurotensin in rat mesostriatal dopaminergic neurons. Neuroscience 68:519529[Medline]
Gache C, Rossi B, Lazdunski M (1976) (Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis. Eur J Biochem 65:293306[Abstract]
Gilbert JA, Richelson E (1984) Neurotensin stimulates formation of cyclic GMP in murine neuroblastoma clone N1E115. Eur J Pharmacol 99:245246[Medline]
Gully D, Canton M, Boigegrain R, Jeanjean F, Molimard JC, Poncelet M, Gueudet C, et al. (1993) Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor. Proc Natl Acad Sci USA 90:6569[Abstract]
Jennes L, Stumpf WE, Kalivas PW (1982) Neurotensin: topographical distribution in rat brain by immunohistochemistry. J Comp Neurol 210:211224[Medline]
Lenkei Z, Beaudet A, Chartrel N, De Mota N, Irinopoulou T, Braun B, Vaudry H, et al. (2000) A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts. J Histochem Cytochem 48:15531564
Lind GJ, Cavanagh HD (1993) Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit. Invest Ophthalmol Vis Sci 34:29432952[Abstract]
Lu D, Yang H, Shaw G, Raizada MK (1998) Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons. Endocrinology 139:365375
Martin S, Navarro V, Vincent JP, Mazella J (2002) Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line. Gastroenterology 123:11351143[Medline]
Mazella J, Zsurger N, Navarro V, Chabry J, Kaghad M, Caput D, Ferrara P, et al. (1998) The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor. J Biol Chem 273:2627326276
Meggiato T, Ferrara C, Tessari G, Plebani M, De Paoli M, Del Favero G, Naccarato R (1996) Serum neurotensin in human pancreatic cancer. Tumori 82:592595[Medline]
Najimi M, Souaze F, Mendez M, Hermans E, Berbar T, Rostene W, Forgez P (1998) Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Study on neurotensin receptor. J Biol Chem 273:2163421641
Nielsen MS, Jacobsen C, Olivecrona G, Gliemann J, Petersen CM (1999) Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase. J Biol Chem 274:88328836
Nustede R, Kohler H, Folsch UR, Schafmayer A (1991) Plasma concentrations of neurotensin and CCK in patients with chronic pancreatitis with and without enzyme substitution. Pancreas 6:260265[Medline]
Omary MB, Kagnoff MF (1987) Identification of nuclear receptors for VIP on a human colonic adenocarcinoma cell line. Science 238:15781581[Medline]
Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nature Rev Mol Cell Biol 3:639650[Medline]
PoinotChazel C, Portier M, Bouaboula M, Vita N, Pecceu F, Gully D, Monroe JG, et al. (1996) Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24. Biochem J 320:145151[Medline]
Re RN, Vizard DL, Brown J, Bryan SE (1984) Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease. Biochem Biophys Res Commun 119:220227[Medline]
Reinecke M (1985) Neurotensin. Immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone. Prog Histochem Cytochem 16:1172[Medline]
Snider RM, Forray C, Pfenning M, Richelson E (1986) Neurotensin stimulates inositol phospholipid metabolism and calcium mobilization in murine neuroblastoma clone N1E115. J Neurochem 47:12141218[Medline]
Tanaka K, Masu M, Nakanishi S (1990) Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4:847854[Medline]
Tang SS, Rogg H, Schumacher R, Dzau VJ (1992) Characterization of nuclear angiotensin-II-binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology 131:374380[Abstract]
TheodorssonNorheim E, Oberg K, Rosell S, Bostrom H (1983) Neurotensinlike immunoreactivity in plasma and tumor tissue from patients with endocrine tumors of the pancreas and gut. Gastroenterology 85:881889[Medline]
Vandenbulcke F, Nouel D, Vincent J, Mazella J, Beaudet A (2000) Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor [In Process Citation]. J Cell Sci 113:29632975
Ventura C, Maioli M, Pintus G, Posadino AM, Tadolini B (1998) Nuclear opioid receptors activate opioid peptide gene transcription in isolated myocardial nuclei. J Biol Chem 273:1338313386
Vita N, Laurent P, Lefort S, Chalon P, Dumont X, Kaghad M, Gully D, et al. (1993) Cloning and expression of a complementary DNA encoding a high affinity human neurotensin receptor. FEBS Lett 317:139142[Medline]