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• W2025502308 abstract "Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y1 receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y1 receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y1 receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y1 receptors. Upon agonist challenge, the EGFP signal decreased rapidly ( t12 = 107 ± 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y1 receptor antagonist, or by pertussis toxin, in agreement with Y1 receptor activation. Internalization of EGFP-hNPY Y1 receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y1 receptors. Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y1 receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y1 receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y1 receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y1 receptors. Upon agonist challenge, the EGFP signal decreased rapidly ( t12 = 107 ± 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y1 receptor antagonist, or by pertussis toxin, in agreement with Y1 receptor activation. Internalization of EGFP-hNPY Y1 receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y1 receptors. Neuropeptide Y (NPY), 1NPYneuropeptide YhNPYhuman neuropeptide YhPYYhuman peptide YYhPPhuman pancreatic polypeptideEGFPenhanced green fluorescent proteinGPCRG protein-coupled receptorHEKhuman embryonic kidneyCHOChinese hamster ovaryGiinhibitory GTP-binding protein of adenylyl cyclasePEphycoerythrinPTXpertussis toxinBIBP3226{(R)-N 2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide}GFPgreen fluorescent proteinPBSphosphate-buffered salineBSAbovine serum albumin[Ca2+] iintracellular calcium a 36-amino acid residue peptide isolated from porcine brain (1Tatemoto K. Carlquist M. Mutt V. Nature. 1982; 296: 659-660Crossref PubMed Scopus (2020) Google Scholar, 2Tatemoto K. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5485-5489Crossref PubMed Scopus (1287) Google Scholar), is one of the most abundant and widely distributed neuropeptides in the central and peripheral nervous systems where it is co-localized with noradrenaline and ATP (3Lundberg J.M. Hokfelt T. Prog. Brain Res. 1986; 68: 241-262Crossref PubMed Scopus (239) Google Scholar). NPY modulates numerous physiological processes including regulation of cardiovascular and renal functions, intestinal motility, memory, anxiety, seizures, feeding, circadian rhythms, and nociception (4Blomqvist A.G. Herzog H. Trends Neurosci. 1997; 20: 294-298Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 5Balasubramaniam A.A. Peptides. 1997; 18: 445-457Crossref PubMed Scopus (198) Google Scholar, 6Michel M.C. Beck-Sickinger A. Cox H. Doods H.N. Herzog H. Larhammar D. Quirion R. Schwartz T. Westfall T. Pharmacol. Rev. 1998; 50: 143-150PubMed Google Scholar). From expression cloning and pharmacological studies with various truncated peptide fragments or with endogenously produced peptide YY (PYY) and pancreatic polypeptide (PP), 5 distinct NPY receptor subtypes have been so far identified: Y1, Y2, Y4, Y5, and y6 (6Michel M.C. Beck-Sickinger A. Cox H. Doods H.N. Herzog H. Larhammar D. Quirion R. Schwartz T. Westfall T. Pharmacol. Rev. 1998; 50: 143-150PubMed Google Scholar). The most common actions of these receptor subtypes are inhibition of adenylyl cyclase via pertussis toxin-sensitive GTP-binding proteins Gi/Go and/or phospholipase C activation and mobilization of Ca2+ from intracellular stores. neuropeptide Y human neuropeptide Y human peptide YY human pancreatic polypeptide enhanced green fluorescent protein G protein-coupled receptor human embryonic kidney Chinese hamster ovary inhibitory GTP-binding protein of adenylyl cyclase phycoerythrin pertussis toxin {(R)-N 2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine amide} green fluorescent protein phosphate-buffered saline bovine serum albumin intracellular calcium The wide spectrum of expression of the NPY Y1 receptor subtype and data from knock-out studies suggests that the Y1 receptor mediates many of the physiological and pathophysiological actions of NPY (7Kushi A. Sasai H. Koizumi H. Takeda N. Yokoyama M. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15659-15664Crossref PubMed Scopus (188) Google Scholar, 8Pedrazzini T. Seydoux J. Kunstner P. Aubert J.F. Grouzmann E. Beermann F. Brunner H.R. Nat. Med. 1998; 4: 722-726Crossref PubMed Scopus (330) Google Scholar, 9Naveilhan P. Hassani H. Lucas G. Blakeman K.H. Hao J.X., Xu, X.J. Wiesenfeld-Hallin Z. Thoren P. Ernfors P. Nature. 2001; 409: 513-517Crossref PubMed Scopus (157) Google Scholar). However, the physiological role and potential therapeutic implications of NPY and the Y1 receptor have been difficult to establish due to a lack of selective pharmacological tools. Thus, elucidation of the mechanisms involved in the regulation of Y1 receptors should contribute to a better understanding of the physiological roles of NPY. Prolonged activation of G protein-coupled receptors (GPCRs) leads to a greatly decreased sensitivity of the receptor to a subsequent agonist challenge (10Bohm S.K. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (466) Google Scholar). This phenomenon, termed desensitization, is a general physiological mechanism by which receptors adapt to a changing environment. The molecular mechanisms that lead to agonist-dependent desensitization of G protein-coupled receptors are not fully defined, but several distinct events are involved, including uncoupling of receptors from their heterotrimeric G proteins and reduction in the number of receptors at the cell surface by either internalization or down-regulation which may be associated with degradation (11Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (858) Google Scholar, 12Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 13Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Internalization is thought to be initiated by receptor phosphorylation by G protein-coupled receptor kinases and binding of β-arrestin to phosphorylated receptors, leading to targeting to clathrin-coated pits (14Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar, 15Goodman O.B., Jr. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1179) Google Scholar). For example, internalization of receptors has been suggested to be the mechanism for somatostatin receptor desensitization (16Beaumont V. Hepworth M.B. Luty J.S. Kelly E. Henderson G. J. Biol. Chem. 1998; 273: 33174-33183Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, the dynamic relationships between internalization, desensitization, and down-regulation are far from being well understood. Desensitization of NPY receptor responsiveness occurs after NPY exposure in isolated organ preparations (17Van Riper D.A. Bevan J.A. Circ. Res. 1991; 68: 568-577Crossref PubMed Scopus (13) Google Scholar, 18Xia J. Neild T.O. Kotecha N. Br. J. Pharmacol. 1992; 107: 771-776Crossref PubMed Scopus (23) Google Scholar, 19Moriarty M. Potter E.K. McCloskey D.I. J. Auton. Nerv. Syst. 1993; 45: 21-28Abstract Full Text PDF PubMed Scopus (4) Google Scholar, 20Cox H.M. Tough I.R. Br. J. Pharmacol. 1995; 116: 2673-2678Crossref PubMed Scopus (26) Google Scholar, 21Sawa T. Mameya S. Yoshimura M. Itsuno M. Makiyama K. Niwa M. Taniyama K. Eur. J. Pharmacol. 1995; 276: 223-230Crossref PubMed Scopus (22) Google Scholar). Rapid desensitization of NPY-stimulated Ca2+ mobilization occurs in HEL cells and involves multiple effectors including PKC and tyrosine kinase (22Michel M.C. Br. J. Pharmacol. 1994; 112: 499-504Crossref PubMed Scopus (13) Google Scholar). However, neither the underlying molecular mechanisms nor the intracellular trafficking pathways involved are well known. Recent studies indicate that endogenous Y1 receptors in SK-N-MC neuroblastoma (23Fabry M. Langer M. Rothen-Rutishauser B. Wunderli-Allenspach H. Hocker H. Beck-Sickinger A.G. Eur. J. Biochem. 2000; 267: 5631-5637Crossref PubMed Scopus (40) Google Scholar) and guinea pig Y1 but not Y2receptors expressed in CHO cells (24Parker S.L. Kane J.K. Parker M.S. Berglund M.M. Lundell I.A. Li M.D. Eur. J. Biochem. 2001; 268: 877-886Crossref PubMed Scopus (64) Google Scholar) are internalized. On the other hand, in pharmacological studies, hNPY Y4 receptors expressed in CHO cells were resistant to desensitization and internalization (25Voisin T. Goumain M. Lorinet A.M. Maoret J.J. Laburthe M. J. Pharmacol. Exp. Ther. 2000; 292: 638-646PubMed Google Scholar), although in radioligand studies, rat Y4 receptors expressed in CHO cells were found to be internalized (24Parker S.L. Kane J.K. Parker M.S. Berglund M.M. Lundell I.A. Li M.D. Eur. J. Biochem. 2001; 268: 877-886Crossref PubMed Scopus (64) Google Scholar). The development of fusion proteins tagged with green fluorescent protein (GFP) allows real time analysis of membrane receptor trafficking, including synthesis and degradation, in single cells (26Kallal L. Benovic J.L. Trends Pharmacol. Sci. 2000; 21: 175-180Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In the present work, EGFP was fused to the NH2-terminal of the full-length human NPY Y1 and Y2 receptors and these chimeric constructs were transfected into HEK293 cells. The functional properties of fluorescent hNPY Y1 receptors were similar to those of wild-type hNPY Y1 receptors after transfection in these cells, and were the same as found for NPY Y1 receptor-mediated responses in SK-N-MC neuroblastoma cells (27Fuhlendorff J. Gether U. Aakerlund L. Langeland-Johansen N. Thogersen H. Melberg S.G. Olsen U.B. Thastrup O. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 182-186Crossref PubMed Scopus (376) Google Scholar, 28Wahlestedt C. Regunathan S. Reis D.J. Life Sci. 1992; 50: L7-L12Crossref PubMed Scopus (175) Google Scholar). By measuring the EGFP fluorescence of chimeric receptors after challenge with hNPY, we report that human NPY Y1 but not Y2 receptors undergo rapid clathrin-dependent internalization in an endosomal recycling compartment. Control HEK293 cells and HEK293 cells expressing hNPY Y1, EGFP-hNPY Y1 and EGFP-hNPY Y2receptors were cultured to ∼80% confluence in T-75 flasks in minimal essential medium with Earle's salt supplemented with 10% fetal calf serum, 2 mm glutamine, and 1% antibiotics (penicillin/streptomycin). cDNA of human NPY Y1 and Y2 receptors (29Herzog H. Hort Y.J. Ball H.J. Hayes G. Shine J. Selbie L.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5794-5798Crossref PubMed Scopus (395) Google Scholar,30Rose P.M. Fernandes P. Lynch J.S. Frazier S.T. Fisher S.M. Kodukula K. Kienzle B. Seethala R. J. Biol. Chem. 1995; 270: 22661-22664Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) were kindly provided by Prof. H. Herzog (Garvan Institut of Medical Research, Sydney, Australia). The hNPY Y1 receptor sequence was subcloned in the expression vector pCEP4 (Invitrogen) between theHindIII and NotI sites to yield pCEP4-hNPY Y1. The sp-EGFP sequence was excised from pCEP4-sp-EGFP-NK2R (31Vollmer J.Y. Alix P. Chollet A. Takeda K. Galzi J.L. J. Biol. Chem. 1999; 274: 37915-37922Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) by BglII andHindIII restriction enzymes. The hNPY Y1receptor sequence was excised from pCEP4-hNPY Y1 usingBglII and BamHI. The two sequences were subcloned in pCEP4 between the HindIII and BamHI sites to yield pCEP4-sp-EGFP-hNPY Y1. HEK293 cells were transfected by calcium phosphate precipitation (32Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar) and selected with 4000 μg/ml hygromycin B. EGFP-hNPY Y1 and hNPY Y1receptors were expressed stably for about 2 months (10 cell passages). EGFP-hNPY Y2 receptors were constructed and transfected in HEK293 cells using analogous procedures. NPY receptor coupling to adenylyl cyclase was investigated by measuring the dose-dependent inhibitory effects of NPY and related peptide analogues on forskolin-stimulated cAMP accumulation. Near confluent HEK293 cultures expressing wild type hNPY Y1 or EGFP-hNPY Y1 receptors were harvested and seeded in 24-well plates coated with collagen (60 μg/ml) at an initial density of 50,000. After 3–4 days of culture (∼80% confluency), the cells were treated as previously described for CHP 234 neuroblastoma cells (33Lynch J.W. Lemos V.S. Bucher B. Stoclet J.C. Takeda K. J. Biol. Chem. 1994; 269: 8226-8233Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were washed with 0.5 ml of PBS (in mm: 137 NaCl, 2.7 KCl, 0.9 CaCl2, 0.5 MgCl2, 6.5 Na2HPO4, 1.5 KH2PO4, pH 7.2), and then incubated at 37 °C for 10 min with 0.5 ml assay buffer (in mm: 150 NaCl, 5 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 10 HEPES, 10 mg/ml BSA, pH 7.4). After removal of buffer, 0.45 ml of fresh buffer containing 0.5 mm3-isobutyl-1-methyl-xanthine (IBMX) was added for 10 min and cells were exposed to 10 μm forskolin without and with peptides (10−12-10−6m) for 20 min at 37 °C. We found that at this submaximal forskolin concentration, cAMP levels reach a maximum within 20 min (Fig. 1 E). The reaction was stopped by addition of 1 volume of ice-cold 0.2m HCl. Cells were further disrupted by sonication and suspensions were centrifuged at 14,000 × g for 15 min. The resulting supernatants were stored at −20 °C until determination of cAMP by radioimmunoassay (34Cailla H.L. Racine-Weisbuch M.S. Delaage M.A. Anal. Biochem. 1973; 56: 394-407Crossref PubMed Scopus (267) Google Scholar). To study agonist-dependent desensitization of the inhibition of adenylyl cyclase activity, cells were preincubated for 10 min at 37 °C without or with either 1 or 100 nm hNPY. The cells were then washed with incubation medium to remove unbound hNPY during 10 min and thereafter assayed as above using different concentrations of hNPY for inhibition of forskolin-stimulated cAMP accumulation. For imaging studies, HEK293 cells transfected with EGFP-hNPY Y1 receptors were plated at low density (5–8 × 105 cells/dish) in culture dishes in which a 2-cm diameter hole had been cut in the base and replaced by a thin (0.17-mm) glass coverslip. Measurements of [Ca2+] i were made as previously described (33Lynch J.W. Lemos V.S. Bucher B. Stoclet J.C. Takeda K. J. Biol. Chem. 1994; 269: 8226-8233Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were incubated for 60 min at 37 °C in complete medium containing in addition 2.5 μm fura 2/acetoxymethyl ester (fura 2/AM), then washed and left equilibrated for 10 min in normal external bath solution (in mm: NaCl, 140; KCl, 5; CaCl2, 2; MgCl2, 2; glucose, 11; HEPES, 10; pH 7.3 with NaOH). Single-cell fluorescence measurements were made at 37 °C on an inverted microscope (Nikon Diaphot) using a ×40 oil immersion objective (Nikon UV-Fluor, numerical aperture 1.3) and an imaging system (Imstar, Paris, France). Fura-2 was excited alternately at 340 and 380 nm using a 100-watt Hg lamp. Emitted fluorescence was filtered at 510 ± 20 nm using a bandpass filter (Nikon) and measured with a Darkstar-800 intensified CCD camera (Photonics Sciences, Millham, United Kingdom). Images were digitized onto a computer and analyzed using Imstar software. Ratiometric Ca2+ images were generated at 3-s intervals, using 4 averaged images at each wavelength. After background subtraction, [Ca2+] i was averaged from pixels within manually outlined cell areas. Values of [Ca2+] i were calculated as reported before (33Lynch J.W. Lemos V.S. Bucher B. Stoclet J.C. Takeda K. J. Biol. Chem. 1994; 269: 8226-8233Abstract Full Text PDF PubMed Google Scholar). NPY or carbachol was locally microperfused during 1 min by pressure-ejection (10 kPa) from fine diameter (2 μm) puffer pipettes positioned ∼100–20 μm from the cell. For washout between successive agonist applications, the bath (volume 1.5 ml) was superfused at ∼1 ml/min. Transfected HEK293 cells were grown on 35-mm plastic dishes and when needed, preincubated with different drugs in complete medium. Cells were then washed with PBS, harvested after incubation in a PBS-EDTA, 5 mm buffer, pH 7.4, and resuspended in Krebs-Ringer buffer containing or not drugs used for preincubation. Fluorescence acquisitions were made from cell suspensions using a spectrofluorimeter (Fluorolog, SPEX) equipped with a 450-watt Xe lamp, a double grating excitation monochromator, and a single grating emission monochromator. Slits were set to 4 mm yielding bandwidths of 7.2 nm at excitation and 14.4 nm at emission. Data were acquired with a photon counting photomultiplier (linear up to 107 counts/s). Unless otherwise stated, cell suspensions (106 cells/ml) were placed in a 1-ml cuvette with magnetic stirring and maintained at 37 °C in a thermostatted cuvette handler. Time base recordings were typically made every 1 s. For endosome/EGFP-hNPY Y1 receptor co-localization, cells were split and grown for 2 days in 24-well plates on 12-mm glass coverslips coated with rat type I collagen. On the day of experiment, cells were washed twice with Krebs-Ringer buffer (in mm: 136 NaCl, 1.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 5 NaHCO3, 1.2 CaCl2, 0.21 EGTA, 5.5 glucose, 20 Hepes, 1 mg/ml BSA, pH 7.4). Cells were then starved by 1 h incubation in serum-free medium. The cells were then incubated for periods ranging from 0 to 60 min in Krebs-Ringer buffer containing 20 μg/ml transferrin-Texas Red with or without 100 nm hNPY at 37 °C. Transferrin uptake was stopped by placing cells on ice and washing them immediately with ice-cold Krebs-Ringer buffer. The cells were then fixed in 4% paraformaldehyde/Krebs-Ringer buffer (without glucose and BSA) for 15 min at 4 °C and then incubated for 15 min in NH4Cl 50 mm toquench any remaining paraformaldehyde and remove cellular autofluorescence. Coverslips were mounted onto microscope slides using an anti-fading agent, Mowiol (Calbiochem). Cells were observed with an inverted microscope (Nikon Eclipse TE300) and a laser scanning confocal imaging system (Bio-Rad MRC 1024 ES) using a Plan Apo 60 × 1.20 numerical aperture water immersion objective (Nikon). Electronic zoom was 2, the pinhole was 2.7, and the pixel size was 0.275 μm. Excitation was from a 30 mW Kr/Ar laser at 10% power. Each stack of two-dimensional images was acquired sequentially in the green channel (PMT2, excitation 488, emission 522 nm) and in the red channel (PMT1, excitation 568 nm, emission 605 nm), before stepping (0.3 μm) the objective in theZ axis. For lysosome/EGFP-hNPY Y1 receptor co-localization, images were acquired from living cells. Cells were split and grown on collagen-coated glass coverslips which formed the base of 35-mm plastic dishes. On the day of experiment, cells were washed twice with Krebs-Ringer buffer, and then incubated for 0 to 60 min in buffer containing 50 nm LysoTracker Red (Molecular Probes) with or without 100 nm hNPY at 37 °C. The reaction was stopped by placing cells on ice, and washing them with ice-cold Krebs-Ringer buffer. Images were acquired as described for transferrin-Texas Red-EGFP-hNPY Y1 receptor co-localization. Co-localization analysis was done using AnalyzeAVW 3.0 software installed on a Silicon Graphics work station. The product of red (transferrin-Texas Red or LysoTracker Red) and green (EGFP) images was calculated, and co-localized yellow regions of interest were defined using a threshold of 10% of maximum product intensity. Volume measurements were made from image stacks using in-house developed View3d software in the PV Wave environment. After segmentation and suppression of isolated voxels using a binary filter, the volume and intensity of co-localized voxels were calculated. HEK293 cells transfected or not with EGFP-hNPY Y1 receptors were plated on 6-well plates (Falcon) pre-coated with collagen in 3 ml of complete medium and grown for 2 days. Cells were then preincubated for 1 h at 37 °C in 1 ml/well labeling medium without fetal calf serum (minimal essential medium without methionine and without cysteine, Invitrogen) and, unless otherwise stated, radiolabeled for 90 min in 1 ml/well labeling medium + 1% fetal calf serum + 0.1 mCi/well [35S]methionine-cysteine (Promix, Amersham Biosciences, Inc.) and then washed for 15 min in chase medium (complete minimal essential medium supplemented with 1 × cold methionine + cysteine and 20 μm cycloheximide). Cells were exposed or not to 400 nm hNPY in 1 ml of Krebs-Ringer buffer for 10 min in the incubator. Thereafter, they were incubated in 1 ml of chase medium containing 100 μm cycloheximide for 0 to 7 h. At the end of the chase, cells were detached from the substratum with 1.6 ml of Versene buffer (PBS + 5 mm EDTA) and centrifuged in 2-ml tubes at low speed (825 × g, 3 min, 4 °C). The cell pellet was then processed for immunoprecipitation. Each cell pellet was resuspended in 500 μl of cold lysis buffer (150 mm NaCl, 1.5 mmMgCl2, 10% glycerol, 5 mm EDTA, 50 mm Hepes, pH 7.5) supplemented with 1 mmdithiothreitol and 1 tablet of complete EDTA-free protease inhibitor (for 10 ml of buffer, Roche Molecular Biochemicals). A post-nuclear supernatant was prepared by passing the cells 100 times through a 22-guage syringe and discarding the pelleted material obtained after 10 min centrifugation at 367 × g. The membranes of the supernatant were solubilized by adding 1% Triton X-100 and 0.2% SDS and vortexing for 30 min at room temperature. Unsolubilized material was pelleted by centrifugation (15,000 × g, 15 min, 4 °C) and discarded. The supernatant was incubated overnight at 4 °C with a monoclonal mouse anti-GFP antibody (1 μg/ml, Roche Molecular Biochemicals). Protein A-Sepharose (45 mg) prewashed in H2O was equilibrated in washing buffer (lysis buffer + 1% Triton X-100) and incubated overnight with 800 μl of washing buffer + 1% BSA + 0.2% SDS + 10% cold HEK lysate. The next day, the EGFP-hNPY Y1-anti-GFP complex was bound to Protein A-Sepharose (7.5 mg/1 ml) for 1 h at 4 °C and sedimented by 1 min centrifugation at 367 × g. The immunoprecipitated complex was washed 3 times and resuspended twice in 30 μl of Laemmli buffer (100 mm Tris-HCl, pH 6.8, 5 mm EDTA, 100 mm dithiothreitol, 4% SDS, 10% glycerol). Samples were vortexed for 30 min at room temperature and heated at 55 °C for 10 min prior to loading on a 10% SDS-PAGE gel. For quantification, gels were blotted on polyvinylidene difluoride membranes and analyzed for radioactivity after 48 h exposition in a PhosphorImager cassette. For immunoblotting, blots were incubated with an affinity purified polyclonal antibody against EGFP made in our laboratory, followed by incubation with a goat anti-rabbit secondary antibody conjugated to horseradish peroxydase (Amersham Biosciences Inc.). Detection was carried out using enhanced chemiluminescence (SuperSignalTMWestPico, PerboScience, France). Cells expressing EGFP-hNPY Y1 receptors were suspended in 20 ml of Krebs-Ringer buffer at a concentration of 106 cells/ml and the suspension was separated into 2 tubes of 10 ml. 100 nm NPY was added to one of the tubes and at times 0, 1, 2, 3, 5, 10, 20, and 30 min later, 900 μl of the cell suspension were collected from both tubes and fixed by transfer to a 2-ml tube containing 900 μl of 8% paraformaldehyde and 0.2% sodium azide in PBS for 10 min at room temperature. After fixation, cells were pelleted by centifugation (3 min, 2000 × g) at 4 °C and resuspended in 1 ml of PBS + 1% BSA on ice for 10 min to 2 h to block nonspecific antibody labeling. Immunolabeling of EGFP-hNPY Y1 receptors expressed at the cell surface was done using a monoclonal mouse anti-GFP (Roche Molecular Biochemicals; 1/1000 dilution) for 30 min at room temperature. Cell were then washed 3 times with 1 ml of PBS + 1% BSA and secondary labeling done using a R-phycoerythrin-conjugated AffiniPure F(ab′)2 fragment goat anti-mouse IgG (Immunotech, Marseille; 1/1000). Samples were washed 3 times in 1 ml of PBS, fixed for 3 min in PBS + 4% paraformaldehyde, resuspended in 1 ml of PBS, and stored overnight. Phycoerythrin (PE) staining was quantified by flow cytometric analysis (10,000 cells per sample) on a cytometer (FACStar, Becton-Dickinson). Mean PE fluorescence intensity was calculated only from cells having high EGFP intensities using FACStar data software, after subtraction of nonspecific staining by the two antibodies measured on nontransfected cells and correction for baseline variations using EGFP-hNPY Y1 cells not exposed to agonist. hNPY, hNPY-(13–36), hPYY, and hPP were from Neosystem (Strasbourg, France). All other chemicals were from Sigma. BIBP3226 and BIIE0246 were kindly supplied by Dr. H. Doods (Boehringer Ingelheim, Biberach, Germany). HEK293 cells, which lack endogenous Y1 receptors, were transfected with native hNPY Y1 or fluorescent EGFP-hNPY Y1 receptors. Expression of fluorescent receptors was confirmed by the characteristic excitation and emission spectra of EGFP (data not shown), and plasma membrane localization was verified in confocal micrographs (see Fig.7). To determine whether the transfected receptors were functional, coupling to adenylyl cyclase was assessed by measuring the inhibition of forskolin-stimulated cAMP accumulation following receptor activation (Fig. 1). Responses were obtained with NPY and related peptide analogues and in the presence of BIBP3226, a selective Y1 antagonist. In control HEK293 cells, hNPY (10−12-10−6m) had no effect, confirming the absence of endogenously expressed functional NPY receptors (not shown). Both hNPY and hPYY inhibited cAMP accumulation in cells expressing hNPY Y1 and EGFP-hNPY Y1receptors (Fig. 1, A and C; EC50values for hNPY were 0.21 ± 0.09 and 0.09 ± 0.03 nm for Y1 and EGFP Y1 cells, respectively; p > 0.05; EC50 values for hPYY were 0.40 ± 0.14 and 0.03 ± 0.01 nm for Y1 and EGFP Y1 cells, respectively;p < 0.05; differences between means were evaluated using the Mann-Whitney U test). We obtained comparable EC50 values for Y1 receptors in SK-N-MC neuroblastoma cells (0.09 ± 0.02 and 0.10 ± 0.02 nm for hNPY and hPYY, respectively; not shown), in agreement with previous reports (27Fuhlendorff J. Gether U. Aakerlund L. Langeland-Johansen N. Thogersen H. Melberg S.G. Olsen U.B. Thastrup O. Schwartz T.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 182-186Crossref PubMed Scopus (376) Google Scholar, 28Wahlestedt C. Regunathan S. Reis D.J. Life Sci. 1992; 50: L7-L12Crossref PubMed Scopus (175) Google Scholar). Compared with hNPY and hPYY, the partial Y1 agonist hNPY-(13–36) was much less potent (EC50 values of 17.50 ± 1.65 and 3.27 ± 0.77 nm for Y1 and EGFP Y1 cells respectively), and the Y4 agonist hPP was ineffective. This rank order of potency is as expected for Y1 receptors. The Y1 antagonist BIBP3226 induced a parallel rightwards shift in the hNPY dose-response curve with no change in the maximal effect for both hNPY Y1 and EGFP-hNPY Y1 expressing cells (Fig. 1, B and D)," @default.
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