Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m412914200
ISSN1083-351X
AutoresStephen J. Perry, Sachiko Junger, Trudy A. Kohout, Sam R.J. Hoare, R. Scott Struthers, Dimitri E. Grigoriadis, Richard A. Maki,
Tópico(s)Hormonal Regulation and Hypertension
ResumoThe corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit β-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or β-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated. The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit β-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or β-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated. The 41-amino acid neuropeptide corticotropin releasing factor (CRF) 1The abbreviations used are: CRF, corticotropin releasing factor; ACTH, adrenocorticotrophic hormone; Nle, norleucine; HEK, human embryonic kidney; CHO, Chinese hamster ovary; ECD, extracellular domain; GPCR, G protein-coupled receptor; PTH, parathyroid hormone; GRK, GPCR kinase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCR, C-C chemokine receptor; PDZ, PSD-95/Dlg/ZO-1; CXCL, C-X-C chemokine ligand; GTPγS, guanosine 5′-O-(3-thiotriphosphate). is the principal regulator of the hypothalamic-pituitary-adrenal axis, and as such plays a critical role in mediating the response to stress in the body (1Rivier C. Vale W. Nature. 1983; 305: 325-327Crossref PubMed Scopus (479) Google Scholar, 2Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (4029) Google Scholar). In mammals, CRF and the related urocortins 1, 2, and 3 bind to and activate two distinct G protein-coupled receptors (GPCRs), termed CRF1 and CRF2 (3Dautzenberg F.M. Hauger R.L. Trends Pharmacol. Sci. 2002; 23: 71-77Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The CRF1 receptor is expressed mainly in the pituitary and central nervous system, where it is responsible for most of the central functions of CRF and urocortin 1, including integration of endocrine, autonomic and behavioral responses to stress, and adrenocorticotrophic hormone (ACTH) release from corticotrope cells of the anterior pituitary (4Koob G.F. Heinrichs S.C. Pich E.M. Menzaghi F. Baldwin H. Miczek K. Britton K.T. Ciba Found. Symp. 1993; 172 (290-275): 277-289PubMed Google Scholar). Furthermore, there is strong evidence that alterations in the CRF1 receptor system occur in many anxiety and depressive disorders (5Holsboer F. J. Psychiatr. Res. 1999; 33: 181-214Crossref PubMed Scopus (585) Google Scholar, 6Gilligan P.J. Robertson D.W. Zaczek R. J. Med. Chem. 2000; 43: 1641-1660Crossref PubMed Scopus (158) Google Scholar, 7Grigoriadis D.E. Haddach M. Ling N. Saunders J. Curr. Med. Chem. Cent. Nerv. Syst. Agents. 2001; 1: 63-97Crossref Google Scholar). CRF2 receptors bind all three urocortins with high affinity and CRF with lower affinity (3Dautzenberg F.M. Hauger R.L. Trends Pharmacol. Sci. 2002; 23: 71-77Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). These receptors exist as three independent isoforms (CRF2(a), CRF2(b), and CRF2(c)) and are expressed both in the central nervous system and the periphery, including in the heart, skeletal muscle, gastrointestinal tract, and epididymis (3Dautzenberg F.M. Hauger R.L. Trends Pharmacol. Sci. 2002; 23: 71-77Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The functions performed by the various isoforms of the CRF2 receptor are currently being elucidated (8Bale T.L. Vale W.W. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 525-557Crossref PubMed Scopus (1066) Google Scholar). Both CRF1 and CRF2 receptors belong to the Class B family of G protein-coupled receptors, which includes (but is not limited to) the receptors for glucagon, parathyroid hormone (PTH), secretin, and vasoactive intestinal peptide. All class B receptors possess a large extracellular domain (ECD) with which they bind with high affinity to the carboxyl-terminal regions of their peptide ligands (9Harmar A.J. Genome Biol. 2001; 2 (3013.3011-3013.3010)Crossref PubMed Google Scholar). This interaction alone is not sufficient to stimulate coupling of the receptor to G proteins, however, and a second interaction must occur between the juxtamembrane domain of the receptor (the transmembrane helices and intervening loops) and the first few residues within the amino-terminal portion of the peptide ligand (7Grigoriadis D.E. Haddach M. Ling N. Saunders J. Curr. Med. Chem. Cent. Nerv. Syst. Agents. 2001; 1: 63-97Crossref Google Scholar, 10Perrin M.H. Vale W.W. Ann. N. Y. Acad. Sci. 1999; 885: 312-328Crossref PubMed Scopus (393) Google Scholar). Because discrete regions of class B ligands perform high affinity binding and receptor stimulation, truncating the endogenous peptides at their amino termini produces high affinity competitive antagonists for class B receptors. Further modifications made to CRF truncated in this manner have produced a number of different antagonist peptides including astressin (cyclo(30–33)-[d-Phe12,Nle21,38,Glu30,Lys33]CRF-(12–41)), a high affinity antagonist for CRF1 receptors that also possesses enhanced biological stability, allowing its extensive use in vivo to dissect the functions of the CRF system (11Gulyas J. Rivier C. Perrin M. Koerber S.C. Sutton S. Corrigan A. Lahrichi S.L. Craig A.G. Vale W. Rivier J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10575-10579Crossref PubMed Scopus (229) Google Scholar, 12Rivier C. Rivier J. Lee S. Brain Res. 1996; 721: 83-90Crossref PubMed Scopus (39) Google Scholar). Astressin has no detectable agonist activity at the CRF1 receptor and thus it is believed to act as a neutral competitive antagonist. In addition to binding to the ECD of CRF1 receptors, a recent report has suggested that astressin may form a second low affinity contact with the juxtamembrane domain because astressin retains the ability to inhibit CRF activation of a CRF1 receptor fragment that lacks the ECD (13Hoare S.R. Sullivan S.K. Schwarz D.A. Ling N. Vale W.W. Crowe P.D. Grigoriadis D.E. Biochemistry. 2004; 43: 3996-4011Crossref PubMed Scopus (75) Google Scholar). Following activation by agonists, almost all GPCRs undergo a series of modifications to prevent continuous signaling of the receptor, and to enable the cells on which they are expressed to regulate their sensitivity to future exposures to agonist. This is achieved first by preventing the activated receptors from further interacting with G proteins (desensitization), and then by internalizing the receptors into intracellular compartments (also called sequestration or endocytosis) (14Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1072) Google Scholar, 15Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Desensitization occurs through phosphorylation of intracellular domains of the receptor by GPCR kinases (GRKs) that specifically recognize agonist-occupied receptor molecules, followed by the recruitment and binding of β-arrestins, which sterically hinder further receptor-G protein coupling. The subsequent internalization of the receptors can occur through multiple pathways, the most common of which utilize clathrin-coated pits and caveolae, although some less well defined pathways have also been described, including those that use non-coated vesicles and macropinosomes (16Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (454) Google Scholar). Internalization can result in either short or long term reductions in sensitivity to further agonist stimulation depending on whether the receptors become resensitized and recycle back to the cell surface, or are targeted for degradation (down-regulated) (17von Zastrow M. Life Sci. 2003; 74: 217-224Crossref PubMed Scopus (153) Google Scholar). A few examples of GPCRs undergoing regulation by antagonists have also been described, including internalization and down-regulation of the 5-hydroxytryptamine type 2A receptor by several atypical antipsychotics (18Roth B.L. Berry S.A. Kroeze W.K. Willins D.L. Kristiansen K. Crit. Rev. Neurobiol. 1998; 12: 319-338Crossref PubMed Scopus (98) Google Scholar, 19Gray J.A. Roth B.L. Brain Res. Bull. 2001; 56: 441-451Crossref PubMed Scopus (243) Google Scholar); down-regulation of the gonadotropin releasing hormone receptor in pituitary gonadotrophs by the gonadotropin releasing hormone analogue cetrorelix (20Halmos G. Schally A.V. Pinski J. Vadillo-Buenfil M. Groot K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2398-2402Crossref PubMed Scopus (80) Google Scholar); and phosphorylation and internalization of angiotensin II type 1A receptor by several antagonist peptide analogs of angiotensin II (21Thomas W.G. Qian H. Chang C.S. Karnik S. J. Biol. Chem. 2000; 275: 2893-2900Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Crossref PubMed Scopus (203) Google Scholar). Furthermore, it has recently been reported that the class B PTH1 receptor also undergoes internalization following binding to the truncated antagonist peptide PTH-(7–34), a process that is independent of receptor activation (23Sneddon W.B. Syme C.A. Bisello A. Magyar C.E. Rochdi M.D. Parent J.L. Weinman E.J. Abou-Samra A.B. Friedman P.A. J. Biol. Chem. 2003; 278: 43787-43796Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In light of these discoveries, we investigated whether this phenomenon of peptide antagonist-induced internalization also occurs with CRF1 receptors, and to probe the mechanism(s) underlying this process. Materials—Peptides were synthesized by solid-phase methodology on a Beckman Coulter 990 peptide synthesizer (Fulton, CA) and purified as previously described (13Hoare S.R. Sullivan S.K. Schwarz D.A. Ling N. Vale W.W. Crowe P.D. Grigoriadis D.E. Biochemistry. 2004; 43: 3996-4011Crossref PubMed Scopus (75) Google Scholar). All chemicals were purchased from Sigma unless otherwise stated. Tissue culture medium and reagents were from Mediatech (Herndon, VA), except fetal bovine serum was from HyClone (Logan, UT) and horse serum from Invitrogen. Renilla mulleri GFP was licensed from Prolume Inc. (Pinetop, AZ). Membranes prepared from Ltk- cells expressing human CRF1 (hCRF1) receptors and from human embryonic kidney (HEK-293) cells expressing hCRF1, rat CRF1 (rCRF1), and rCRF1-ECD/activin IIB chimera receptors have been described previously (13Hoare S.R. Sullivan S.K. Schwarz D.A. Ling N. Vale W.W. Crowe P.D. Grigoriadis D.E. Biochemistry. 2004; 43: 3996-4011Crossref PubMed Scopus (75) Google Scholar, 24Hoare S.R. Sullivan S.K. Ling N. Crowe P.D. Grigoriadis D.E. Mol. Pharmacol. 2003; 63: 751-765Crossref PubMed Scopus (70) Google Scholar). Mammalian Expression Constructs—Construction of hCRF1 receptor tagged with the hemagglutinin signal sequence and FLAG epitope (HA-FL-CRF1) in pcDNA5/FRT/V5-His®TOPO®, its stable expression in CHO-K1 Flp-In cells (designated CHO-CRF1 cells), and its indistinguishable pharmacology from the wild-type hCRF1 receptor are described previously (25Hoare S.J.R. Sullivan S.K. Fan J. Khongsaly K. Grigoriadis D.E. Peptides. 2005; 26: 457-470Crossref PubMed Scopus (51) Google Scholar). Complementary DNAs for dynamin 1, caveolin 1, and β-arrestin2 were amplified from a human brain cDNA library, and inserted into the pcDNA3.1/V5-His®TOPO® vector following the manufacturer's instructions. Mutations were made in dynamin 1 (K44A) and caveolin 1 (S80A, S80E) (26Shigematsu S. Watson R.T. Khan A.H. Pessin J.E. J. Biol. Chem. 2003; 278: 10683-10690Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) using the QuikChange® site-directed mutagenesis kit following the manufacturer's instructions (Stratagene, La Jolla, CA). The β-arrestin2-R. mulleri GFP construct was made by adding EcoRI restriction sites to the β-arrestin2 and R. mulleri GFP coding sequences by PCR with the primer pairs: 5′-AAAGAATTCACCATGGGGGAGAAACCC-3′ and 5′-AAAGAATTCGCAGAGTTGATCATCATAGTC-3′; and 5′-AGAATTCGGAAGCAAGCAGATCCTGAAGAAC-3′ and 5′-TCACGATGCGGCCGCTACA-3′, respectively. Both products were cloned into pcDNA3.1/V5-His®TOPO® and subsequently digested with EcoRI. The released β-arrestin2 fragment was purified and subsequently ligated into the linearized R. mulleri GFP construct. All plasmid DNA constructs were amplified in Escherichia coli using standard molecular biology procedures, harvested using Qiagen® DNA preparation kits, and their correct sequences confirmed by DNA sequence analysis using an ABI377 automated DNA sequencer and Big-Dye™ Terminator version 3.0 sequencing kits (Applied Biosystems, Foster City, CA). Cell Culture and Transfection—HEK-293 cells, Chinese hamster ovary Flp-In (CHO-K1 Flp-In) cells, and mouse pituitary corticotrope adenoma AtT-20/D16v-F2 cells (AtT20, purchased from ATCC) were maintained at 37 °C and 5% CO2, in Dulbecco's modified Eagle's medium supplemented with 10 mm HEPES, pH 7.4, 0.2 mm glutamine, 1 mm sodium pyruvate, and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively), and either 10% (v/v) heat-inactivated fetal bovine serum for HEK-293 and CHO-K1 Flp-In, or 10% (v/v) heat-inactivated horse serum for AtT20. Stable expression of receptor in CHO-CRF1 cells was maintained by selection with 500 μg/ml hygromycin B. Transient transfections into HEK-293 cells were performed using FuGENE 6® (Roche Diagnostics) at a ratio of 3 μl FuGENE 6® to every 1 μg of plasmid DNA. Experiments were performed on these cells 48 h after transfection. Overexpressed caveolin 1 mutants and dynamin 1 K44A were detected in cell lysates separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin 1 and anti-dynamin 1 antibodies (Upstate, Charlottesville, VA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and detection by chemiluminescence (Pierce, Rockford, IL). Measurement of Receptor Internalization by Flow Cytometry—Cells transiently or stably expressing FL-CRF1 receptor were seeded at 4 × 105 cells/well in poly-d-lysine-coated 6-well dishes (BIOCOAT™, Fort Washington, PA). The following day the cells were subjected to the appropriate drug treatments, washed twice with ice-cold internalization medium (Dulbecco's modified Eagle's medium containing 25 mm HEPES, pH 7.4, 0.2 mm glutamine, 1 mm sodium pyruvate, 50 IU/ml penicillin, and 50 μg/ml streptomycin) and then incubated for 1 h at 4 °C with anti-FLAG M2 antibody (Sigma) diluted 1:500 in internalization medium. The cells were then washed three times with ice-cold internalization medium and incubated at 4 °C in the dark for a further 30 min with goat anti-mouse IgG antibody conjugated to Alexa Fluor™ 488 dye (Molecular Probes, Eugene, OR) diluted to 1:250 in internalization medium. Cells were subsequently washed with ice-cold PBS three times, detached from the dishes with PBS containing 5 mm EDTA, and fixed by the addition of formaldehyde to 0.8% (w/v). The fluorescence intensity of 104 cells from each well was then measured on a FACScan™ flow cytometer (BD Biosciences). Concanavalin A (Sigma) was added to the cells at 0.25 mg/ml 1 h prior to stimulation. Hypertonic medium treatment, potassium depletion, and disruption of caveolae with filipin III were carried out using previously described methods (27Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar, 28Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 29Schnitzer J.E. Oh P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (778) Google Scholar). Examination of Receptor Internalization by Fluorescence Microscopy—CRF1 receptor internalization was visualized using a previously described method with a minor modification (30Gage R.M. Kim K.A. Cao T.T. von Zastrow M. J. Biol. Chem. 2001; 276: 44712-44720Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Briefly, transiently transfected AtT20 or HEK-293 cells expressing FL-CRF1 receptor with or without β-arrestin2-R. mulleri GFP were grown on Nunc® Lab-Tek® II CC2® multichamber glass slides. Surface receptors on AtT20 cells were labeled (20 min at 37 °C) with Alexa Fluor 488-conjugated M1 anti-FLAG antibody, or on HEK-293 cells with M1 anti-FLAG-Cy3 conjugate (prepared according to the manufacturer's instructions using either Alexa Fluor 488 Monoclonal Antibody Labeling Kit, (Molecular Probes), or Cy3 mono-Reactive Dye Pack (Amersham Biosciences)). Cells were washed once and exposed to 10 μm CRF or 10 μm astressin (2 h at 37 °C) to induce internalization. Cells were washed twice with PBS and immediately fixed with 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature. Cells were washed three times at 10-min intervals with PBS before mounting using ProLong® Gold antifade reagent (Molecular Probes). Fluorescence was visualized on an Olympus IX70 inverted microscope equipped with the CARV confocal module (Kinetic Imaging, Nottingham, UK) using appropriate dichroic filter sets. Images were acquired with a MicroMAX cooled CCD camera (Princeton Instruments, Trenton, NJ) and processed using the Meta-Morph® Imaging System (Universal Imaging Corporation, Downingtown, PA). [32P]Orthophosphate (32Pi) Labeling and Receptor Immunoprecipitation—CHO-CRF1 or CHO-K1 Flp-In cells were serum starved for 1 h in phosphate-free Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine and 25 mm HEPES, pH 7.4. Cells were labeled with 100 μCi/ml of 32Pi (PerkinElmer Life Sciences) for 1 h, then 100 nm microcystin-LF (Calbiochem, San Diego, CA) was added and the cells were incubated for a further 15 min before stimulation with 100 nm astressin or CRF at 37 °C, or left untreated. Cells were washed twice with PBS and extracts were prepared by lysing cells in 500 μl of glycerol lysis buffer (50 mm HEPES, pH 7.4, 0.5% (v/v) Nonidet P-40, 250 mm NaCl, 2 mm EDTA, 10% (v/v) glycerol, 100 μm Na3VO4, 10 mm NaF, 100 nm microcystin-LF, and Complete® EDTA-free protease inhibitor mixture tablet (Roche)). The samples were clarified by centrifugation, and FL-CRF1 receptor was immunoprecipitated from equal amounts of cell lysate with 30 μl of M2 anti-FLAG-agarose conjugate (Sigma) for 16 h with constant mixing. Immunoprecipitates were washed 5 times with glycerol buffer and eluted with 40 μl of 2 × SDS sample buffer (Invitrogen) supplemented with 200 mm dl-1,4-dithiothreitol. Half the volume (20 μl) of the immunoprecipitates was resolved on a 4–20% Tris glycine SDS-polyacrylamide gel (Invitrogen). The gel was dried and incorporation of 32Pi measured using the VersaDoc3000 phosphorimager (Bio-Rad). Measurement of Receptor Down-regulation by ELISA—CHO-CRF1 cells were seeded at 7.5 × 104 cells/well in poly-d-lysine-coated 96-well dishes (BIOCOAT). The following day the cells were subjected to the appropriate treatments, washed once with ice-cold PBS and lysed in 200 μl/well of ice-cold lysis buffer (1% (v/v) Nonidet P-40 in PBS supplemented with Complete EDTA-free protease inhibitor mixture, Roche) for 30 min at 4 °C with constant agitation. Detergent-insoluble fractions were sedimented by 10 min centrifugation at 2,000 × g, and the amount of CRF1 receptor present in the clarified cell lysates was quantified by ELISA. Briefly, protein concentration was measured using the BCA protein assay method (Pierce). Equal amounts of protein (12 μg/well) were transferred to anti-FLAG M2-coated 96-well plates (Sigma) and incubated overnight at 4 °C. Each well was washed 4 times with ELISA wash buffer (0.05% (v/v) Tween 20 in PBS) and incubated for 2 h at room temperature with the previously described anti-CRF1 receptor antiserum 4467a-CRF1 (31Chatzaki E. Murphy B.J. Wang L. Million M. Ohning G.V. Crowe P.D. Petroski R. Tache Y. Grigoriadis D.E. J. Neurochem. 2004; 88: 1-11Crossref PubMed Scopus (76) Google Scholar) diluted 1:10,000 in antibody dilution buffer (PBS containing 1% (w/v) bovine serum albumin). The plates were washed, incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) diluted 1:2,000 in antibody dilution buffer, followed by a final four washes. Plates were incubated with 200 μl/well of ready-to-use horseradish peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (Sigma) for 30 min at room temperature, followed by addition of 100 μl/well 0.5 m H2SO4 to stop the reaction. The optical density of each well was read at 450 nm using an EMax microplate reader (Molecular Devices, Sunnyvale, CA). To determine whether CRF and the antagonist astressin are both capable of inducing the internalization of the CRF1 receptor, CHO-CRF1 cells (CHO-K1 cells stably expressing the hemagglutinin-FLAG-tagged hCRF1 receptor) were treated with 100 nm CRF or astressin for 30 min to 24 h, and the loss of receptors from the cell surface was measured by flow cytometry. Time-matched vehicle-treated controls were also performed to allow all data points to be normalized to the appropriate level of cell surface expression. Fig. 1A shows that both CRF and astressin induced substantial internalization over this period, however, the total amount of internalization following 24 h stimulation with CRF was greater than with astressin (71 ± 7.4 and 51 ± 8.1%, respectively). This difference was the result of a higher rate of receptor sequestration by CRF in the first hour of stimulation (63% with CRF, 17% with astressin), after which sequestration by both peptides proceeded at similar rates (evident from the similar slopes of the graphs between 2 and 24 h in Fig. 1A). Full dose-response relationships were then produced for CRF and astressin to allow the EC50 values to be calculated (Fig. 1B). Both peptides internalized CRF1 receptor in a dose-dependent manner, however, despite the fact that astressin possessed only partial efficacy in the internalization assay, astressin and CRF promoted internalization with almost identical potencies (EC50 = 7.6 and 7.8 nm, respectively). Taken together, these data indicate that the agonist CRF possesses high efficacy for internalizing CRF1 receptors, whereas the antagonist astressin appears to possess only partial efficacy. Despite this difference in internalization efficacy, astressin and CRF were equipotent for this effect. We next determined whether the phenomenon of astressin-induced internalization also occurred in a cell line that expresses the CRF1 receptor endogenously. Corticotropes of the anterior pituitary are major sites of CRF1 receptor expression, where it stimulates the secretion of ACTH into the blood in response to CRF released from the hypothalamus (2Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (4029) Google Scholar, 32Chen R. Lewis K.A. Perrin M.H. Vale W.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8967-8971Crossref PubMed Scopus (905) Google Scholar). The mouse pituitary corticotrope adenoma AtT20 cell line expresses CRF1 receptors (∼100 fmol/mg of membrane protein, data not shown), produces cyclic AMP (25Hoare S.J.R. Sullivan S.K. Fan J. Khongsaly K. Grigoriadis D.E. Peptides. 2005; 26: 457-470Crossref PubMed Scopus (51) Google Scholar), and secretes ACTH (33Richardson U.I. Life Sci. 1983; 33: 1981-1988Crossref PubMed Scopus (12) Google Scholar) when challenged with CRF peptide. To measure CRF1 receptor internalization, AtT20 cells were transfected with FLAG-tagged CRF1 receptor to allow its movement to be tracked both by immunocytochemistry and flow cytometry (Fig. 2). Prior to treatment, immunostaining of live AtT20 cells with anti-FLAG M1 antibody conjugated to Alexa Fluor 488 dye revealed substantial cell surface expression and no visible staining of intracellular receptors (not shown). The cells were then treated for 2 h with vehicle, or with a maximal dose of CRF or astressin (10 μm), and the redistribution of the immunofluorescence-stained receptors was monitored. No internalization of CRF1 receptors was observed in cells treated with vehicle for 2 h (Fig. 2A, i), demonstrating that the conjugated M1 antibody alone did not induce receptor internalization. Following treatment with CRF, however, many of the labeled receptor molecules had redistributed from the cell surface into compartments within the cytosol (Fig. 2A, ii). A less robust but similar pattern of CRF1 receptor redistribution to intracellular compartments was observed following astressin treatment, with substantial levels of receptor remaining at the cell surface (Fig. 2A, iii). This reduced level of receptor redistribution with astressin correlated well with the amount of CRF1 receptor internalization measured in AtT20 cells using flow cytometry (Fig. 2B), where CRF induced internalization of 39% after 2 h, increasing to 45% at 4 h, while astressin internalized 9% after 2 h and 16% after 4 h. The majority of GPCRs are internalized via clathrin-mediated endocytosis (16Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (454) Google Scholar). This process normally requires the receptors to be bound to agonist, phosphorylated by GRKs, and to recruit cytosolic arrestins, however, clathrin-mediated internalization following antagonist binding has also been reported (34Sneddon W.B. Magyar C.E. Willick G.E. Syme C.A. Galbiati F. Bisello A. Friedman P.A. Endocrinology. 2004; 145: 2815-2823Crossref PubMed Scopus (80) Google Scholar). Furthermore, some peptide antagonists are known to cause receptor phosphorylation without activating G protein coupling (21Thomas W.G. Qian H. Chang C.S. Karnik S. J. Biol. Chem. 2000; 275: 2893-2900Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Crossref PubMed Scopus (203) Google Scholar, 34Sneddon W.B. Magyar C.E. Willick G.E. Syme C.A. Galbiati F. Bisello A. Friedman P.A. Endocrinology. 2004; 145: 2815-2823Crossref PubMed Scopus (80) Google Scholar). To determine whether such mechanisms could underlie astressin-induced CRF1 receptor internalization, we compared the ability of astressin and CRF to promote CRF1 receptor phosphorylation and the recruitment of β-arrestin2 (Fig. 3, A and B). CHO-CRF1 cells were metabolically labeled with 32Pi, stimulated with 100 nm astressin or CRF between 5 min and 1 h, and the amount of radioactivity incorporated into the CRF1 receptor was assessed by autoradiography of receptor immunoprecipitates. Fig. 3A shows that stimulation of cells with astressin caused no phosphorylation of the rece
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