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• W2048495619 abstract "Based on the conformationally constrained d-Trp-Phe-d-Trp (wFw) core of the prototype inverse agonist [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the d-Trp-Phe-d-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH2, where Isn is isonipecotic acid) ∼80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH2 was found to be a functionally biased agonist. Thus, wFw-Isn-NH2 mediated potent and efficacious signaling through the Gαq and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα12/13 pathway. The recognition pattern of wFw-Isn-NH2 with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH2 was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH2 binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH2 is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III. Based on the conformationally constrained d-Trp-Phe-d-Trp (wFw) core of the prototype inverse agonist [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the d-Trp-Phe-d-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH2, where Isn is isonipecotic acid) ∼80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH2 was found to be a functionally biased agonist. Thus, wFw-Isn-NH2 mediated potent and efficacious signaling through the Gαq and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα12/13 pathway. The recognition pattern of wFw-Isn-NH2 with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH2 was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH2 binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH2 is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III. Ghrelin is a neuroendocrine hormone that differs from other peptide hormones by a fatty acid modification, which is crucial for both the binding and activation of its receptor (1Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7260) Google Scholar). Ghrelin is synthesized mainly in the gastrointestinal tract, where the gene coding for the peptide sequence is expressed together with the enzyme responsible for the acylation of the fatty acid to the ghrelin peptide sequence (2Kirchner H. Gutierrez J.A. Solenberg P.J. Pfluger P.T. Czyzyk T.A. Willency J.A. Schürmann A. Joost H.G. Jandacek R.J. Hale J.E. Heiman M.L. Tschöp M.H. Nat. Med. 2009; 15: 741-745Crossref PubMed Scopus (333) Google Scholar, 3Yang J. Zhao T.J. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10750-10755Crossref PubMed Scopus (151) Google Scholar). Multiple functions have been described for ghrelin since it was discovered. Initially it was believed that growth hormone secretion induced by ghrelin receptors in the hypothalamus and the pituitary was the primary function of ghrelin (4Kojima M. Hosoda H. Matsuo H. Kangawa K. Trends Endocrinol. Metab. 2001; 12: 118-122Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). However, the function of ghrelin in the hypothalamus, and in particular in the arcuate nucleus, has become the focus of attention over the last decade. In the arcuate nucleus, ghrelin is responsible for increased activity in the NPY (neuropeptide Y) and AGRP (Agouti-related protein) neurones leading to increased appetite, decreased energy expenditure, and fat accumulation (5Tschöp M. Smiley D.L. Heiman M.L. Nature. 2000; 407: 908-913Crossref PubMed Scopus (3392) Google Scholar, 6Nakazato M. Murakami N. Date Y. Kojima M. Matsuo H. Kangawa K. Matsukura S. Nature. 2001; 409: 194-198Crossref PubMed Scopus (2953) Google Scholar). High receptor expression is also observed in the ventromedial nucleus of the hypothalamus, and the function in this area has been proposed to be orexigenic based on the regulation of fatty acid metabolism (7López M. Lage R. Saha A.K. Pérez-Tilve D. Vázquez M.J. Varela L. Sangiao-Alvarellos S. Tovar S. Raghay K. Rodríguez-Cuenca S. Deoliveira R.M. Castañeda T. Datta R. Dong J.Z. Culler M. Sleeman M.W. Alvarez C.V. Gallego R. Lelliott C.J. Carling D. Tschöp M.H. Diéguez C. Vidal-Puig A. Cell Metab. 2008; 7: 389-399Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). More recently it has been demonstrated that ghrelin is also involved in reward-seeking behavior such as alcohol and cocaine abuse or intake of palatable food through interaction with the dopaminergic system in the ventral tegmental area and the substantia niagra (8Egecioglu E. Jerlhag E. Salome N. Skibicka K.P. Haage D. Bohlooly Y. Andersson D. Bjursell M. Perrissoud D. Engel J.A. Dickson S.L. Addict. Biol. 2010; 15: 304-311Crossref PubMed Scopus (261) Google Scholar, 9Jerlhag E. Egecioglu E. Landgren S. Salomé N. Heilig M. Moechars D. Datta R. Perrissoud D. Dickson S.L. Engel J.A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11318-11323Crossref PubMed Scopus (320) Google Scholar, 10Diano S. Farr S.A. Benoit S.C. McNay E.C. da Silva I. Horvath B. Gaskin F.S. Nonaka N. Jaeger L.B. Banks W.A. Morley J.E. Pinto S. Sherwin R.S. Xu L. Yamada K.A. Sleeman M.W. Tschöp M.H. Horvath T.L. Nat. Neurosci. 2006; 9: 381-388Crossref PubMed Scopus (694) Google Scholar). The development of drugs that modulate the signaling of the ghrelin receptor system has been pursued by the pharmaceutical industry for the last three decades. Agonist compounds were developed as so-called growth hormone secretagogues even before it was realized that they worked through the ghrelin receptor and its cloning. Such compounds, for example MK-677, were used in clinical trials both for the treatment of growth hormone deficiency and for the treatment of the frail elderly. However, these compounds never reached the market, mainly because of a lack of efficacy (11Murphy M.G. Weiss S. McClung M. Schnitzer T. Cerchio K. Connor J. Krupa D. Gertz B.J. J. Clin. Endocrinol. Metab. 2001; 86: 1116-1125Crossref PubMed Scopus (86) Google Scholar, 12Bach M.A. Rockwood K. Zetterberg C. Thamsborg G. Hébert R. Devogelaer J.P. Christiansen J.S. Rizzoli R. Ochsner J.L. Beisaw N. Gluck O. Yu L. Schwab T. Farrington J. Taylor A.M. Ng J. Fuh V. J. Am. Geriatr. Soc. 2004; 52: 516-523Crossref PubMed Scopus (73) Google Scholar). Today non-peptide ghrelin receptor agonists and ghrelin analogues are being developed in attempts to treat, for example, various forms of cachexia and malnourishment in hospitalized patients (13Lundholm K. Gunnebo L. Körner U. Iresjö B.M. Engström C. Hyltander A. Smedh U. Bosaeus I. Cancer. 2010; 116: 2044-2052Crossref PubMed Scopus (84) Google Scholar). Antagonists or inverse agonists of the ghrelin receptor have been proposed as a potential treatment for obesity, diabetes, and also more recently alcohol abuse; but these are still in early development (14Holst B. Schwartz T.W. Trends Pharmacol. Sci. 2004; 25: 113-117Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Interestingly, functionally biased agonists, i.e. agonists with the ability to induce selective receptor conformations responsible for interaction with only a limited selection of the downstream signaling pathways, have been described for many 7TM 3The abbreviations used are: 7TMseven-transmembraneTMtransmembraneSREserum response elementAbuγ-amino butyric acidAbzaminobenzoic acidAcp(1R,4S)-(+)-4-amino-2-cyclopentene-1-carboxylic acidAep4-(2-aminoethyl)piperazine-1-ylacetic acidAmbaminomethylbenzoic acidIsnisonipecotic acidAhxaminohexanoic acidECL-2bextracellular loop IIbFmocN-(9-fluorenyl)methoxycarbonylbis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPDBProtein Data Bank. receptors (15Galandrin S. Oligny-Longpré G. Bouvier M. Trends Pharmacol. Sci. 2007; 28: 423-430Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 16Michel M.C. Alewijnse A.E. Mol. Pharmacol. 2007; 72: 1097-1099Crossref PubMed Scopus (35) Google Scholar). One therapeutic potential of such biased ligands would be to avoid unwanted side effects mediated through a particular pathway, as described for the niacin receptor GPR109A agonists (17Richman J.G. Kanemitsu-Parks M. Gaidarov I. Cameron J.S. Griffin P. Zheng H. Guerra N.C. Cham L. Maciejewski-Lenoir D. Behan D.P. Boatman D. Chen R. Skinner P. Ornelas P. Waters M.G. Wright S.D. Semple G. Connolly D.T. J. Biol. Chem. 2007; 282: 18028-18036Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In the case of the ghrelin receptor, a biased agonist could potentially act as a functionally specific agonist, i.e. able to modulate energy expenditure and food intake without affecting growth hormone secretion or vice versa. The structural understanding of biased signaling has been addressed only to a limited degree, and information is primarily based on mutations that selectively decouple one signaling pathway and not the other (18Holst B. Hastrup H. Raffetseder U. Martini L. Schwartz T.W. J. Biol. Chem. 2001; 276: 19793-19799Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 19Rajagopal S. Rajagopal K. Lefkowitz R.J. Nat. Rev. Drug. Discov. 2010; 9: 373-386Crossref PubMed Scopus (667) Google Scholar). seven-transmembrane transmembrane serum response element γ-amino butyric acid aminobenzoic acid (1R,4S)-(+)-4-amino-2-cyclopentene-1-carboxylic acid 4-(2-aminoethyl)piperazine-1-ylacetic acid aminomethylbenzoic acid isonipecotic acid aminohexanoic acid extracellular loop IIb N-(9-fluorenyl)methoxycarbonyl 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol Protein Data Bank. Through structure-function analysis of the first inverse agonist for the ghrelin receptor, [d-Arg1,d-Phe5,d-Trp7,9,Leu11] substance P (20Holst B. Cygankiewicz A. Jensen T.H. Ankersen M. Schwartz T.W. Mol. Endocrinol. 2003; 17: 2201-2210Crossref PubMed Scopus (422) Google Scholar), we have previously identified the carboxyamidated, C-terminal pentapeptide (d-Trp-Phe-d-Trp-Leu-Leu) as the essential core peptide, which by itself displays a characteristic biphasic dose-response curve, i.e. combined agonism and inverse agonism (Fig. 1) (21Holst B. Lang M. Brandt E. Bach A. Howard A. Frimurer T.M. Beck-Sickinger A. Schwartz T.W. Mol. Pharmacol. 2006; 70: 936-946Crossref PubMed Scopus (83) Google Scholar). By the addition of a single amino acid at its N terminus this pentapeptide could, depending on the type of N-terminal residue, be converted into either a pure agonist or a pure inverse agonist (21Holst B. Lang M. Brandt E. Bach A. Howard A. Frimurer T.M. Beck-Sickinger A. Schwartz T.W. Mol. Pharmacol. 2006; 70: 936-946Crossref PubMed Scopus (83) Google Scholar, 22Holst B. Mokrosinski J. Lang M. Brandt E. Nygaard R. Frimurer T.M. Beck-Sickinger A.G. Schwartz T.W. J. Biol. Chem. 2007; 282: 15799-15811Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Importantly, the characteristic essential d-Trp-Phe-d-Trp (wFw) motif strongly restricts the conformational freedom of this type of peptide ligand because essentially the wFw motif is found in only two different dominating conformations (22Holst B. Mokrosinski J. Lang M. Brandt E. Nygaard R. Frimurer T.M. Beck-Sickinger A.G. Schwartz T.W. J. Biol. Chem. 2007; 282: 15799-15811Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The structure-activity relationship analysis indicated that the more flexible C-terminal Leu-Leu dipeptide functions mainly as a spacer or linker between the wFw core and the functionally highly important C-terminal carboxyamide moiety. Thus, one aim of the present study was to try to exchange the Leu-Leu part of wFw ligands with a non-peptide linker structure (Fig. 1). In this way novel, potent, ghrelin receptor ligands could in fact be generated. However, in contrast to all previously characterized peptide and non-peptide ligands, these novel wFw peptide-mimetic ligands were found to be biased agonists in respect to their ability to activate signaling through the classical Gαq pathway but not through the Gα12/13 SRE signaling pathway. Importantly, mutational mapping of important receptor residues for the function of the most efficacious biased agonists, wFw-Isn-amide, demonstrated a novel receptor interaction mode. This lacked a crucial, charged anchor point in TM III, GluIII:09, which is shared by all of the unbiased ghrelin receptor agonists. Ghrelin was purchased from Bachem (Bubendorf, Swicherland). The non-peptide compound MK-677 (L-163,191) was kindly provided by Andrew Howard (Merck Research Laboratories). γ-Amino butyric acid (Abu), 3-aminobenzoic acid (3Abz), 4-aminobenzoic acid (4Abz), (1R,4S)-(+)-4-amino-2-cyclopentene-1-carboxylic acid (Acp), 4-(2-aminoethyl)piperazine-1-ylacetic acid (Aep), β-alanine (βAla), 3-aminomethylbenzoic acid (3Amb), 4-aminomethylbenzoic acid (4Amb), isonipecotic acid (Isn), and aminohexanoic acid (Ahx) were purchased from Fluka or Sigma-Aldrich. The peptides were synthesized by solid-phase technique on an automated multiple peptide synthesizer (Syro; MultiSynTech, Bochum, Germany) by using Rink amide resin (30 mg, resin loading 0.6 mmol/g) as described recently (23Lang M. Söll R.M. Dürrenberger F. Dautzenberg F.M. Beck-Sickinger A.G. J. Med. Chem. 2004; 47: 1153-1160Crossref PubMed Scopus (62) Google Scholar). The non-natural amino acids Abu, 3Abz, 4Abz, Acp, Aep, βAla, 3Amb, 4Amb, Isn, and Ahx were coupled manually directly at the Rink amide resin (30 mg, resin loading 0.6 mmol/g) after removal of the Fmoc group with 30% piperidine in N,N-dimethylformamide twice for 20 min. The coupling reaction was performed twice with four equivalents of the Fmoc-protected un-natural amino acids, which were activated by 4 equivalents of 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium and 8 equivalents of N,N-diisopropyl-ethylamin for 1 h. Completeness of the reaction was analyzed by a ninhydrin assay (24Kaiser E. Colescott R.L. Bossinger C.D. Cook P.I. Anal. Biochem. 1970; 34: 595-598Crossref PubMed Scopus (3510) Google Scholar). The core segment wFw was synthesized as described previously (23Lang M. Söll R.M. Dürrenberger F. Dautzenberg F.M. Beck-Sickinger A.G. J. Med. Chem. 2004; 47: 1153-1160Crossref PubMed Scopus (62) Google Scholar). The peptide mimetics were cleaved from the resin in one step using trifluoroacetic acid, precipitated from ice-cold diethyl ether, washed, and finally lyophilized. Purification of the peptides was achieved by preparative HPLC on an RP C18 column (Vydac, 250 × 25 mm, 10 μm) with a gradient of 20–60% B in A (A = 0.1% trifluoroacetic acid in water; B = 0.08% trifluoroacetic acid in acetonitrile) over a 60-min time span at a flow rate of 10 ml/min (λ = 220 nm). The peptides were analyzed by MALDI mass spectrometry on an Voyager-DE RP work station (Applied Biosystems, Darmstadt, Germany) and by analytical reversed-phase HPLC on a Vydac RP-18 column (4.6 × 250 mm, 5 μm, 300 Å) using linear gradients of 10–60% B in A over 30 min and a flow rate of 0.6 ml/min (λ = 220 nm). The observed masses were in full agreement with the calculated masses, and the purity of all peptides was >95% accordingly to analytical HPLC. The human ghrelin/receptor cDNA was cloned by PCR from a human brain cDNA library. The cDNA was cloned into the eukaryotic expression vector pCMV-Tag2B made by Stratagene (La Jolla, CA) for epitope tagging of proteins. Mutations were constructed by PCR using the overlap expression method (25Holst B. Zoffmann S. Elling C.E. Hjorth S.A. Schwartz T.W. Mol. Pharmacol. 1998; 53: 166-175Crossref PubMed Scopus (64) Google Scholar). The PCR products were digested with the appropriate restriction endonucleases (BamHI and EcoRI), purified, and cloned into the pCMV-Tag2B vector. All PCR experiments were performed using Pfu polymerase (Stratagene) according to the instructions of the manufacturer. All mutations were verified by restriction endonuclease mapping and subsequent DNA sequence analysis using an automated sequencer (ABI PRISM 310; Applied Biosystems, Foster City, CA). COS-7 cells were grown in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal calf serum, 2 mm glutamine, 180 units/ml penicillin, and 45 μg/ml streptomycin. Cells were transfected using the calcium phosphate precipitation method with chloroquine added. The amount of cDNA (20 μg/75 cm2) resulting in maximal basal signaling was used for the dose-response curves. HEK-293 cells were grown in Dulbecco's modified Eagle's medium adjusted to contain 4500 mg/liter glucose (Invitrogen), 10% fetal bovine serum, 180 units/ml penicillin, and 45 μg/ml streptomycin at 10% CO2 and 37 °C. Stably transfected HEK-293 cells were grown in the same medium. One day after transfection, COS-7 cells were incubated for 24 h with 5 μCi of myo-[3H]inositol (Amersham Biosciences) in 0.3 ml of medium supplemented with 10% fetal calf serum, 2 mm glutamine, 180 units/ml penicillin, and 45 μg/ml streptomycin/well. Cells were washed twice in buffer (20 mm HEPES, pH 7.4, supplemented with 140 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 10 mm glucose, and 0.05% (w/v) bovine serum) and incubated in 0.5 ml of buffer supplemented with 10 mm LiCl at 37 °C for 30 min. After stimulation with various concentrations of peptide and/or non-peptides for 45 min at 37 °C, cells were extracted with 10 mm formic acid followed by incubation on ice for 30 min. The resulting supernatant was purified on anion exchange resin (AG 1-X8; Bio-Rad) to isolate the negatively charged inositol phosphates. After application of the cell extract to the column, the content was washed twice with washing buffer (60 mm sodium formate and 5 mm sodium tetraborate decahydrate) to remove glycerophosphoinositol. Inositol phosphates were eluded by the addition of elution buffer (1 m ammonium formate and 100 mm formic acid), and eluates were added to 10 ml of Wallac Optiphase HiSafe 3 (PerkinElmer Life Sciences). Determinations were made in duplicates. The columns containing AG 1-X8 anion exchange resin were regenerated by the addition of 3 ml of regeneration buffer (3 m ammonium formate and 100 mm formic acid) and 10 ml of water. Cells were transfected and seeded out in parallel with those used for inositol phosphate accumulation assay. The cells were washed twice, fixed, and incubated in blocking solution (phosphate-buffered saline and 3% dry milk) for 60 min at room temperature. Cells were kept at room temperature for all subsequent steps. Cells were incubated for 2 h with anti-FLAG (M2) antibody (Sigma) at a 1:300 dilution. After three washes, cells were incubated for 2 h with anti-mouse horseradish peroxidase (Amersham Biosciences)-conjugated antibody at a dilution of 1:4000. After extensive washing, the immunoreactivity was revealed by the addition of horseradish peroxidase substrate according to the manufacturer's instructions. EC50 values were determined by nonlinear regression using Prism version 3.0 software (GraphPad Software, San Diego, CA). The basal constitutive activity is expressed as a percentage of the ghrelin-induced activation for each mutant construct of the ghrelin receptor. In TABLE 1, TABLE 2, Fmut indicates the -fold shift in potency induced by the structural change in the mutated receptor compared with the wild-type receptor.TABLE 1Characterization of nine small peptide compounds with their respective structures Open table in a new tab TABLE 2Characterization of a library of 23 mutant versions of the ghrelin receptor with substitutions systematically placed throughout the main ligand-binding crevice and in the extracellular part of the receptorMutationBallesteros-Weinstein numberingGhrelinConstitutive activitywFw-Isn-NH2Expression leveln%a100% = maximal ghrelin stimulation of WT receptor.n% EmaxEC50nFmutnmWT1346 ± 15210042 ± 5211,0AspII:20Asn2.600,62 ± 0,15369 ± 2857 ± 1136 ± 540,9PheIII:04Ser3.281,20 ± 0,21339 ± 22177 ± 1828 ± 540,7GlnIII:05Ala3.290,37 ± 0,11349 ± 31133 ± 926 ± 630,6SerIII:08Ala3.321,20 ± 0,08336 ± 21647 ± 10100 ± 3042,4GluIII:09Gln3.330,75 ± 0,18344 ± 21030 ± 254 ± 1741,3ThrIII:12Ala3.360,73 ± 0,17364 ± 54110 ± 98,3 ± 350,2SerIV:16Ala4.560,78 ± 0,12341 ± 31072 ± 15120 ± 1032,9IleIV:20Ala4.601,00 ± 0,3346 ± 21072 ± 18350 ± 1348,3R198L0,94 ± 0,08331 ± 37190 ± 42140 ± 1733,3E196Q0,99 ± 0,04349 ± 28130 ± 2847 ± 1441,1MetV:05Ala5.361,30 ± 0,4349 ± 21381 ± 1974 ± 1641,8VaIV:08Ala5.420,53 ± 0,06353 ± 11151 ± 15110 ± 1032,6SerV:09Ala5.430,73 ± 0,12341 ± 2754 ± 1283 ± 1532,0PheV:12Ala5.460,68 ± 0,07322 ± 21260 ± 19110 ± 2032,6PheVI:16Ala6.510,72 ± 0,2330 ± 11353 ± 5260 ± 5046,2ArgVI20:Gln6.550,67 ± 0,26320 ± 21233 ± 15110 ± 8042,6SerVI:24Ala6.581,00 ± 0,20345 ± 2789 ± 2662 ± 2441,5PheVI:23Ala6.591,10 ± 0,20350 ± 27NA>10003>24GlnVII: −02Ala7.320,49 ± 0,06343 ± 4692 ± 3062 ± 2341,5AsnVII:02Ala7.351,10 ± 0,2326 ± 21467 ± 1175 ± 2831,8PheVII:06Leu7.391,20 ± 0,5338 ± 3887 ± 171000 ± 200524PheVII:09Ala7.420,64 ± 0,14324 ± 5817 ± 491 ± 1032,2a 100% = maximal ghrelin stimulation of WT receptor. Open table in a new tab HEK-293 cells (30,000 cells/well) seeded in 96-well plates were transiently transfected with a mixture of SRE-Luc (PathDetect SRE Cis reporting system; Stratagene) and the indicated amounts of receptor DNA. After transfection, cells were maintained in low serum (2.5%) throughout the experiments and treated with the respective inhibitor of the intracellular signaling pathways. One day after transfection, cells were treated with the respective ligands in an assay volume of 100 μl of medium for 5 h. The assay was terminated by washing the cells twice with PBS and adding 100 μl of luciferase assay reagent (LucLite; PerkinElmer Life Sciences). Luminescence was measured in a TopCount NXT (PerkinElmer Life Sciences) microplate scintillation and luminescence counter for 5 s. Luminescence values are given as relative light units. COS-7 cells (seeding density, 150,000 cells/well) were transfected in the assay plates. Two days after transfection, the indicated concentrations of ligand were added to the assay medium without any serum and incubated for 10 min at 37 °C. The reactions were stopped by removal of the medium and two washing steps with ice-cold PBS. The cells were lysed in sample buffer and separated on SDS-10% PAGE according to the method of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Proteins were transferred onto nitrocellulose, and Western blot analysis was carried out using a 1:5000 dilution of mouse monoclonal anti-phospho-ERK1/2 antibody (Santa Cruz Biotechnology). Total ERK protein was determined using a 1:10,000 dilution of anti-ERK antibody (Santa Cruz Biotechnology). Blots were probed using anti-mouse horseradish peroxidase-conjugated secondary antibodies, visualized using enhanced chemiluminescence reagent (Amersham Biosciences), and quantified by densitometric analysis. ERK1/2 phosphorylation was normalized according to the loading of protein by expressing the data as a ratio of phospho-ERK1/2 over total ERK1/2. Results were expressed as a percentage of the value obtained in nonstimulated, mock-transfected cells. Transfected COS-7 cells were transferred to culture plates 1 day after transfection at a density of ∼5000 cells/well, aiming at 5–8% binding of the radioactive ligand. Two days after transfection competition binding experiments were performed for 3 h at 4 °C using ∼25 pm 35S-labeled MK-677 (provided by Andrew Howard, Merck). Binding assays were performed in 0.1 ml of a 50 mm HEPES buffer, pH 7.4, supplemented with 1 mm CaCl2, 5 mm MgCl2, 0.1% (w/v) bovine serum albumin, and 40 μg/ml bacitracin. Nonspecific binding was determined as the binding in the presence of 1 μm unlabeled ghrelin. Cells were washed twice in 0.1 ml of ice-cold buffer; 50 μl of lysis buffer/scintillation fluid (30% ethoxylated alkylphenol and 70% diisopropylnaphthalene isomers) was added, and the bound radioactivity was counted. Determinations were made in triplicate. Initial experiments showed that steady state binding was reached with the radioactive ligand under these conditions. Stably transfected HEK-293TR cells (Invitrogen) overexpressed the ghrelin receptor cDNA modified with an N-terminal SNAP tag (New England Biolabs) and under the control of a tetracycline-inducible promoter. Cells were seeded into poly-d-lysine-coated 96-well imaging plates (Greiner 655090; Greiner Bio-One, Gloucester, UK), and receptor expression was initiated by tetracycline treatment (100 ng/ml) for 18–21 h. Cell surface ghrelin receptors were first labeled with membrane-impermeant SNAP-Surface AF488 (0.1 μm in DMEM, New England Biolabs) for 30 min at 37 °C, washed, and treated with ligands at 37 °C in Hanks' balanced salt solution containing 0.1% BSA and 5 μg/ml Alexa Fluor 633-conjugated transferrin (Invitrogen). Incubations were terminated by fixation (3% paraformaldehyde), and cell nuclei were also labeled (H33342, 1 μg/ml in phosphate-buffered saline). Images at four sites/well were then acquired using the IX Ultra confocal plate reader (Molecular Devices, Sunnyvale, CA; 40× ELWD objective) with the appropriate excitation and emission filters for nuclei labeling (405 nm excitation), SNAP-Surface AF488-labeled ghrelin receptors (488 nm), and transferrin (633 nm). Automated translocation analysis of plate reader images (MetaXpress 2.0, Molecular Devices) quantified the fluorescence intensity of labeled ghrelin receptors within 3-μm-diameter internal compartments identified by transferrin labeling, which the predominant destination of internalized ghrelin receptors. Individual concentration response curves performed in triplicate were normalized to vehicle (0%) and 1 μm ghrelin (100%) controls. Pooled data were used to obtain EC50 values with GraphPad Prism (sigmoidal fit, nH 0.9–1.0). GTP-Rho and activated Rho of RC-4B/C cell lysates were assessed by a pulldown assay according to the manufacturer's description (catalog No. BK036, Cytoskeleton, Inc., Denver, CO). In short, cells were grown to 60–80% confluency and incubated in serum-free media. The cells were subjected to 10 min of stimulation by ghrelin, Isn-wFw-NH2, or solute. The cells were washed in PBS and lysed. After protein quantification, 500–800 μg of total protein in the lysates was precipitated by rhotekin beads. Precipitates were loaded onto a NuPAGE 10% bis-tris gel (Invitrogen), transferred to a PVDF membrane (Invitrogen) in a transfer buffer (40 mm glycine, 50 mm Trizma base, 1.3 mm SDS, and 20% ethanol (v/v)), blocked with TBST (150 mm NaCl, 50 mm Trizma base, and 0.1% Tween 20) supplemented with 5% BSA, and immunoblotted using anti-RhoA monoclonal antibody (Cytoskeleton, Inc., primary) and secondary antibody (goat anti-mouse IgG horseradish peroxidase-conjugated antibody, Thermo Scientific), both in TBST. The membranes were washed, and SuperSignal (Thermo Scientific) was added for visualization. The PVDF membranes were analyzed on a FluorChem HD2 (Alpha Innotech). 12 Sprague-Dawley rats (Taconic, Ejby, Denmark) were stereotaxically implanted with a stainless steel cannula (Holm Finmekanik AS, Copenhagen, Denmark) aimed at the right lateral ventricle (1 mm caudal, 1.5 mm lateral to the bregma, and 4 mm ventral to the cranium externa). The cannula and supporting bolts were secured with dental cement (Poly-F Plus, Dentsply). The animals were anesthetized with Hypnorm/Dormicum, 0.2 ml/kg body weight (fentanyl citrate, 0.07875 mg/ml; fluanisone, 2.5 mg/ml; and midazolam, 1.25 mg/ml). Pre- and post-surgery rats received analgesic treatment (Rimadyl (Pfizer), 5 mg/kg). Rats were handled daily during the recovery week and were housed in feeding cages for adaptation. After recovery, cannula placement was confirmed by measuring the drinking response to administration of angiotensin II (100 nmol/rat in 4 μl of saline; data not shown). Rats that showed a positive drinking response were used in the study. Injection with ghrelin, Isn-wFw-NH2, or ve" @default.
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• W2048495619 date "2011-06-01" @default.
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• W2048495619 title "Unique Interaction Pattern for a Functionally Biased Ghrelin Receptor Agonist" @default.
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• W2048495619 doi "https://doi.org/10.1074/jbc.m110.173237" @default.
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