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• W1984893089 abstract "Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine γ-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-γ2-MSH for Gly10 from [Nle4]α-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-γ2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-γ2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [d-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket. Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine γ-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-γ2-MSH for Gly10 from [Nle4]α-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-γ2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-γ2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [d-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket. The melanocortins, adrenocorticotropic hormone, α-, β-, and γ-melanocyte-stimulating hormone (MSH), 1The abbreviations used are: γ-MSH, γ-melanocyte-stimulating hormone; MC, melanocortin; TM, transmembrane; CRE, cAMP response element; MT, melanotan 1The abbreviations used are: γ-MSH, γ-melanocyte-stimulating hormone; MC, melanocortin; TM, transmembrane; CRE, cAMP response element; MT, melanotan are derived from the precursor protein pro-opiomelanocortin. Pro-opiomelanocortin is expressed in the pituitary gland, in two brain nuclei (1Gee C.E. Chen C.L.C. Roberts J.L. Thompson R. Watson S.J. Nature. 1983; 306: 374-375Crossref PubMed Scopus (201) Google Scholar), and in several peripheral tissues (2Saito E. Odell W.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3792-3796Crossref PubMed Scopus (26) Google Scholar). Effects of melanocortins have been described on behavior (3Eberle A.N. The Melanotropins. S. Karger A. G., Basel1988Google Scholar), metabolism (4Fan W. Boston B.A. Kesterson R.A. Hruby V.J. Cone R.D. Nature. 1997; 385: 165-168Crossref PubMed Scopus (1649) Google Scholar), fever, inflammation (5Tatro J.B. Neuroimmunomodulation. 1996; 3: 259-284Crossref PubMed Scopus (127) Google Scholar), analgesia (6Contreras P.C. Takemori A.E. J. Pharmacol. Exp. Ther. 1984; 229: 21-26PubMed Google Scholar), addiction (7Alvaro J.D. Tatro J.B. Duman R.S. Life Sci. 1997; 61: 1-9Crossref PubMed Scopus (50) Google Scholar), nerve regeneration (8Strand F.L. Rose K.J. Zuccarelli L.A. Kume J. Alves S.E. Antonawich F.J. Garrett L.Y. Physiol. Rev. 1991; 71: 1017-1046Crossref PubMed Scopus (143) Google Scholar), and the cardiovascular system (9Van Bergen P. Janssen P.M. Hoogerhout P. De Wildt D.J. Versteeg D.H. Eur. J. Pharmacol. 1995; 294: 795-803Crossref PubMed Scopus (37) Google Scholar, 10Gruber K.A. Callahan M.F. Am. J. Physiol. 1989; 257: r681-r694PubMed Google Scholar). Since virtually all [125I]NDP-α-MSH-binding sites overlap with expression of either MC3R and/or MC4R mRNA, these are the main MC receptors in the brain mediating the variety of effects of melanocortin peptides (11Tatro J.B. Entwistle M.L. Brain Res. 1994; 635: 148-158Crossref PubMed Scopus (42) Google Scholar, 12Low M.J. Simerly R.B. Cone R.D. Curr. Opin. Endocrinol. Diab. 1994; 1: 79-88Crossref Google Scholar, 13Adan R.A. Gispen W.H. Peptides. 1997; 18: 1279-1287Crossref PubMed Scopus (116) Google Scholar). The melanocortin (MC) receptor subtypes, MC1R, MC2R, MC3R, MC4R, and MC5R (14Gantz I. Miwa H. Konda Y. Shimoto Y. Tashiro T. Watson S.J. DelValle J. Yamada T. J. Biol. Chem. 1993; 268: 15174-15179Abstract Full Text PDF PubMed Google Scholar, 15Gantz I. Konda T. Tashiro T. Shimoto Y. Miwa H. Munzert G. Watson S.J. DelValle J. Yamada T. J. Biol. Chem. 1993; 268: 8246-8250Abstract Full Text PDF PubMed Google Scholar, 16Gantz I. Shimoto Y. Konda Y. Miwa H. Dickinson C.J. Yamada T. Biochem. Biophys. Res. Commun. 1994; 200: 1214-1220Crossref PubMed Scopus (285) Google Scholar, 17Roselli-Rehfuss L. Mountjoy K.G. Robbins L.S. Mortrud M.T. Low M.J. Tatro J.B. Entwistle M.L. Simerly R.B. Cone R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8856-8860Crossref PubMed Scopus (659) Google Scholar, 18Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1443) Google Scholar, 19Chhajlani V. Wikberg J.E.S. FEBS Lett. 1992; 309: 417-420Crossref PubMed Scopus (575) Google Scholar, 20Chhajlani V. Muceniece R. Wikberg J.E.S. Biochem. Biophys. Res. Commun. 1993; 195: 866-873Crossref PubMed Scopus (317) Google Scholar) constitute a subfamily within G-protein-coupled receptor superfamily. MC receptors differ in ligand binding specificity as well as in tissue distribution. Insight into these two aspects is essential to understand the physiological functions of the pro-opiomelanocortin/MC receptor system. Therefore, it is essential to identify ligands that can discriminate between the MC3R and the MC4R. The development of selective ligands for the MC receptors has been hampered by the absence of detailed knowledge about the structural requirements of peptide ligands for selective MC receptor binding and activation. Nevertheless, it has been demonstrated that HFRW (MSH (6Contreras P.C. Takemori A.E. J. Pharmacol. Exp. Ther. 1984; 229: 21-26PubMed Google Scholar, 7Alvaro J.D. Tatro J.B. Duman R.S. Life Sci. 1997; 61: 1-9Crossref PubMed Scopus (50) Google Scholar, 8Strand F.L. Rose K.J. Zuccarelli L.A. Kume J. Alves S.E. Antonawich F.J. Garrett L.Y. Physiol. Rev. 1991; 71: 1017-1046Crossref PubMed Scopus (143) Google Scholar, 9Van Bergen P. Janssen P.M. Hoogerhout P. De Wildt D.J. Versteeg D.H. Eur. J. Pharmacol. 1995; 294: 795-803Crossref PubMed Scopus (37) Google Scholar)) forms the core sequence of melanocortins, which is necessary to bind to all MC receptors (21Haskell-Luevano C. Sawyer T.K. Trumpp-Kallmeyer S. Bikker J.A. Humblet C. Gantz I. Hruby V.J. Drug Des. Discov. 1996; 14: 197-211PubMed Google Scholar, 22Sahm U.G. Qarawi M.A. Olivier G.W. Ahmed A.R. Branch S.K. Moss S.H. Pouton C.W. FEBS Lett. 1994; 350: 29-32Crossref PubMed Scopus (36) Google Scholar, 23Sahm U.G. Olivier G.W. Branch S.K. Moss S.H. Pouton C.W. Peptides. 1994; 15: 1297-1302Crossref PubMed Scopus (77) Google Scholar). Ligand selectivity may therefore be determined by residues outside the core region either through a selective interaction with different receptor subtypes, by altering folding of the core sequence, or by a combination of both. Although the MC3R and MC4R both recognize α-MSH, the affinity of γ-MSH is 50-fold higher for the MC3R. Of the three forms of γ-MSH, γ1- and γ2-MSH (11 and 12 amino acid residues, respectively) are most related, while γ3-MSH has an extended C terminus. In vivo amidation of the C-terminal Gly12 residue of γ2-MSH results in the formation of γ1-MSH with an C-terminal Phe11-amide. In mammals, the natural forms of γ-MSH contain an additional N-terminal Lys residue (3Eberle A.N. The Melanotropins. S. Karger A. G., Basel1988Google Scholar). α-MSH and Lys-γ2-MSH both contain the core sequence, HFRW, a Tyr residue at position 2 and a Met residue at position 4, while N- and C-terminal residues and the residue at position 5 differ (Fig.1). Recently it was shown that Asp10 in Lys-γ2-MSH determined MC3R selective activation (24Oosterom J. Burbach J.P. Gispen W.H. Adan R.A. Eur. J. Pharmacol. 1998; 354: R9-11Crossref PubMed Scopus (12) Google Scholar). However, it is not clear whether replacement of Asp10 in Lys-γ2-MSH increases binding affinity or only increases efficacy for the MC4R. Moreover, it remains to be determined whether there exists a direct selective interaction of Asp10 with the MC3R, or whether Asp10 induces a peptide structure that is favorable for the MC3R. To solve this problem a more detailed analysis is required regarding the contribution of each individual amino acid in the ligand to receptor binding and activation. Therefore, the aim of this study was to gain insight into the molecular mechanism of the selectivity of γ2-MSH for the MC3Rversus the MC4R. Using a gain of function approach, we tested [Nle4]α-MSH and [Nle4]Lys-γ2-MSH and derivatives with exchanged amino acid residues on in vitro binding and activation of wild type and chimeric MC3R/MC4R and mutant MC4Rs. We demonstrate here that the selectivity of γ2-MSH for the MC3R versus the MC4R is determined by a single amino acid residue in the ligand and, to a large extend, a single residue in the receptor. A new concept is proposed in which MC3R/MC4R selectivity is determined by how the melanocortin core sequence is presented to the receptor-binding pocket. NDP-α-MSH ([Nle4,d-Phe7]α-MSH), α-MSH, and γ2-MSH were purchased from Bachem Feinchemicalien, Bubendorf, Switzerland. Lys-γ2-MSH was synthesized by the National Institute of Public Health and Environmental Protection (RIVM), Bilthoven, The Netherlands (9Van Bergen P. Janssen P.M. Hoogerhout P. De Wildt D.J. Versteeg D.H. Eur. J. Pharmacol. 1995; 294: 795-803Crossref PubMed Scopus (37) Google Scholar). γ1-MSH was a gift from Organon, Oss, The Netherlands. MT-II was a gift from V. Hruby (Tucson, AZ). [d-Tyr7]MT-II, [Nle4]α-MSH, [Nle4,Gly5,Asp10]α-MSH, [Nle4,Phe12]α-MSH, [Nle3]γ2-MSH, [Nle4]Lys-γ2-MSH, [Nle4,Gly10]Lys-γ2-MSH, [Nle4,Pro12]Lys-γ2-MSH, and [Nle4,Gly10,Pro12]Lys-γ2-MSH were synthesized using solid phase N-(9-fluorenyl)methoxycarbonyl chemistry and purified as described in Schaaper et al. (25Schaaper W.M.M. Adan R.A.H. Posthuma T.A. Oosterom J. Gispen W.H. Meloen R.H. Lett. Peptide Sci. 1998; 5: 1-4Google Scholar). The products were analyzed using liquid chromatography mass spectrometry. Ion spray mass spectrometry performed on a Micromass Quattro sq confirmed the expected molecular weights. All other peptides mentioned in Table II were synthesized using pepscan and their concentration and purity were verified by high pressure liquid chromatography (26Meloen R.H. Puijk W.C. Langeveld J.P.M. Langedijk J.P.M. van Amerongen A. Schaaper W.M.M. Anonymous Immunological Recognition of Peptides in Medicine and Biology. CRC Press Inc., Boca Raton, FL1995: 15-31Google Scholar). All peptides in Table II were N-terminal acetylated and C-terminal amidated. Peptides were dissolved in 1 mm hydrogen chloride and diluted in phosphate-buffered saline or binding buffer (see below).Table IIInhibition constants (Ki in nm) and EC50values (in nm) of synthetic α- and γ-MSH analogues for the MC3R and MC4RPeptideEC50(nm)K i (nm)MC3RMC4RMC3RMC4RNDP-α-MSH––0.94aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.8.1aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.α-MSH3.01bData published previously in (24) but were reproduced for this study.9.1bData published previously in (24) but were reproduced for this study.2.9319.4[Lys1]α-MSH2.711.22.0723.1[Val3]α-MSH2.7517.74.8936.3[Gly5]α-MSH3.99bData published previously in (24) but were reproduced for this study.7.22bData published previously in (24) but were reproduced for this study.5.0919.0[Asp10]α-MSH>100aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.>500aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.212aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.3000aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.[Gly5, Asp10]α-MSH5.79aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.,bData published previously in (24) but were reproduced for this study.28.7aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.,bData published previously in (24) but were reproduced for this study.3.6365.9aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.[Arg11]α-MSH14.8aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.39aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.30.2aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.79.5aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.[Phe12]α-MSH3.518aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.1.54aValues significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.16.8[Gly13]α-MSH2.374.901.6917.3Lys-γ2-MSH5.03bData published previously in (24) but were reproduced for this study.109bData published previously in (24) but were reproduced for this study.0.8638.4[Ser1]Lys-γ2-MSH3.811282.74cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.228cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.[Ser3]Lys-γ2-MSH3.561631.73120cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.[Gly10]Lys-γ2-MSH9.90bData published previously in (24) but were reproduced for this study.16bData published previously in (24) but were reproduced for this study.,cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.0.497.57cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.[Lys11]Lys-γ2-MSH2.18cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.57.8cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.1.1256.5[Pro12]Lys-γ2-MSH3.2725.2cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.1.3311.8cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.[Val13]Lys-γ2-MSH4.591380.86131cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.[Gly10, Pro12]Lys-γ2-MSH3.9514.5cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.0.672.99cValues significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2.All peptides were N-terminal and C-terminal amidated and contain a Nle4. The rat MC3R and the human MC4R were transiently transfected in 293 cells. K i values were determined by displacement of [125I]NDP-α-MSH and calculated with 95% confidence interval derived from 11 duplicate data points. EC50values were determined in the β-galactosidase assay in the same batch of transfected cells to exclude influence of receptor expression levels. For each peptide 12 data points were measured in quadruplicate and EC50 values were calculated with a 95% confidence interval. Both K i and EC50 values are representative of at least two independent experiments.a Values significantly different (p < 0.05) from reference peptide [Nle4]α-MSH.b Data published previously in (24Oosterom J. Burbach J.P. Gispen W.H. Adan R.A. Eur. J. Pharmacol. 1998; 354: R9-11Crossref PubMed Scopus (12) Google Scholar) but were reproduced for this study.c Values significantly different from reference peptide Ac-[Nle4]Lys-γ2-MSH-NH2. Open table in a new tab All peptides were N-terminal and C-terminal amidated and contain a Nle4. The rat MC3R and the human MC4R were transiently transfected in 293 cells. K i values were determined by displacement of [125I]NDP-α-MSH and calculated with 95% confidence interval derived from 11 duplicate data points. EC50values were determined in the β-galactosidase assay in the same batch of transfected cells to exclude influence of receptor expression levels. For each peptide 12 data points were measured in quadruplicate and EC50 values were calculated with a 95% confidence interval. Both K i and EC50 values are representative of at least two independent experiments. Iodination was performed as described by Tatro et al. (27Huang Q.H. Entwistle M.L. Alvaro J.D. Duman R.S. Hruby V.J. Tatro J.B. J. Neurosci. 1997; 17: 3343-3351Crossref PubMed Google Scholar). In short, 4 μg of NDP-α-MSH was mixed with 1.2 IU of bovine lactoperoxidase (Calbiochem) and 1 mCi of Na125I (ICN) in a final volume of 100 μl of 0.05 m phosphate buffer (pH 6.5). Then 5 μl of 0.003% H2O2 was added every 60 s. After 4 min 50 μl of 1 mm dithiothreitol was added to stop the reaction. Then, the sample was high pressure liquid chromatography purified with a μBondapak C18 column 3.9 × 300 mm (Waters, Division of Millipore) by elution with a 22–52% acetonitrile gradient in 10 mm ammonium acetate (pH 5.5) in 40 min. Specific activity of the [125I]NDP-α-MSH was determined in the β-galactosidase activation assay and was approximately 0.4 pmol/100,000 cpm. Chimeric receptors were made with polymerase chain reaction using overlapping primers directed against the region coding for identical residues in the rat MC3R and human MC4R in TM1, the junction of TM3-IC2, and in TM5 (Fig. 2). Thus, within the junction sites of the chimeric receptor the amino acid sequence was not changed. Both chimeric and mutant receptors were made with a similar stategy. In the first polymerase chain reaction (Cloned Pfu DNA Polymerase, Stratagene) separate fragments were obtained with primer T7 combined with the 3′ mutagenic/chimeric primer and with SP6 combined with the 5′ mutagenic/chimeric primer (complete overlap with the 3′ primer). Fragments were isolated and purified from agarose gel (QIAEX Gel Extraction Kit, QIAGEN) and a second polymerase chain reaction (Taq DNA polymerase, Perkin-Elmer) was performed with the two purified bands as template for primers SP6 and T7. The polymerase chain reaction products were digested with BamHI andXhoI, separated through agarose gel electrophoresis, and purified. Chimera 3B, MC4(267–282 MC3), MC4(267–273 MC3), MC4(278–282 MC3), and MC4R mutants, F267L/Y268I (double mutant), F267L, Y268I, S270T, Q273T, M281T were cloned into pcDNA3.1. Chimera 3AB, 3C, 3D, and 4D were cloned into pcDNA1/Neo). The inserts of the clones were sequenced completely (T7 sequencing kit, Pharmacia Biotech) and only mutations introduced by the mutagenic/chimeric primers were found. HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Optimal amounts of receptor cDNA used for transfection of cells for the activation assay were determined empirically. Approximately 7 × 106 cells were transiently transfected with 15–200 ng of the rat MC3R, the human MC4R, chimeric or mutant receptor cDNA constructs, combined with 7 μg of the pCRELacZ for the β-galactosidase activation assay using the calcium phosphate precipitation method. For the receptor binding assay, cells were transfected with 7 μg of the receptor cDNA constructs. The assay makes use of the β-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein resulting from increased intracellular cAMP and Ca2+ (28Chen W. Shields T.S. Stork P.J.S. Cone R.D. Anal. Biochem. 1995; 226: 349-354Crossref PubMed Scopus (181) Google Scholar). Twenty-four hours after transfection 293 HEK cells were distributed into 96-well plates (Primaria). The next day the cells were treated for 6 h with various concentrations of peptides in Dulbecco's modified Eagle's medium supplemented with 0.5% bovine serum albumin, 25 mm Hepes (pH 7.4), and 50 μg/ml (150 KIU/ml) aprotinin (Sigma). After treatment the cells were lysed, frozen, thawed, and assayed for β-galactosidase activity. For each peptide 12 data points were measured in quadruplicate. EC50values were then calculated with a 95% confidence interval using GraphPad Prism software (sigmoidal dose-response curve fitting, variable slope). Experiments were repeated at least twice with the same results. 293 HEK cells stably expressing human MC4R, rat MC3R, mutant MC4(Y268I), and chimera MC4(267–282 MC3) were grown in poly-l-lysine-coated (Sigma) 24-well Costar plates. Agonist stimulated adenylate cyclase activity was measured as described by Salomon (29Salomon Y. Adv. Cyclic Nucleotide. Res. 1979; 10: 35-55PubMed Google Scholar, 30Gerst J.E. Sole J. Mather J.P. Salomon Y. Mol. Cell. Endocrinol. 1986; 46: 137-147Crossref PubMed Scopus (44) Google Scholar). In short, after prelabeling with 500 μl of [3H]adenine (NEN Life Science Products Inc.) in a concentration of 2 μCi/ml, the 293 cells were incubated for 20 min at 37 °C in phosphate-buffered saline containing 0.1 mmisobutylmethylxanthine (IBMX), 1 μm forskolin, and agonist in a concentration ranging from 10−11 to 10−5m. The cells were harvested and [3H]cAMP formation was determined. For each peptide 12 duplicate data points were measured. EC50 values were then calculated with a 95% confidence interval using GraphPad Prism software (sigmoidal dose-response curve fitting, variable slope). Transfected HEK 293 cells were grown in poly-l-lysine-coated 24-well Costar plates. Two days after transfection, the cells were incubated with 100,000 cpm of [125I]NDP-α-MSH and various concentrations of peptides diluted in binding buffer consisting of Ham's F-10 medium (Life Technologies, Inc.) (pH 7.4) containing 2.5 mm calcium chloride, 0.25% bovine serum albumin, 10 mm Hepes, and 50 μg/ml (150 KIU/ml) aprotinin. After incubation for 30 min at room temperature, the cells were washed twice with ice-cold Tris-buffered saline containing 2.5 mm calcium chloride and lysed in 1m sodium hydroxide. Radioactivity of the lysates was counted in a Packard Cobra γ-counter. Competition curves were fitted from 11 duplicate data points with GraphPad Prism software, nonlinear regression, one site competition. K i values were calculated using the Cheng and Prusoff equation with 95% confidence interval. Experiments were repeated at least twice with the same results. In order to delineate α/γ-MSH selectivity we first excluded influence of peptide length, N- and C-terminal modifications, and oxidation of the Met residue, and therefore used synthetic [Nle4]α-MSH and [Nle4]Ac-Lys-γ2-MSH-NH2 as reference peptides (the latter will be referred to as [Nle4]Lys-γ2-MSH). Table I shows that, using displacement of [125I]NDP-α-MSH, the affinity for [Nle4]α-MSH, as compared with α-MSH, was increased about 2-fold for both the MC3R and the MC4R. Also [Nle4]Lys-γ2-MSH showed increased affinity for both the MC3R and the MC4R as compared with γ1-MSH, γ2-MSH, Lys-γ2-MSH, and [Nle3]γ2-MSH. On the MC4R, [Nle4]Lys-γ2-MSH displayed a 2-fold lower affinity and 20-fold lower activity of cAMP-dependent β-galactosidase activity than [Nle4]α-MSH (TableII). On the MC3R, however, [Nle4]Lys-γ-MSH exhibited a 3.5-fold higher affinity and similar activity as compared with [Nle4]α-MSH. Even though [Nle4]α-MSH had a 6-fold lower affinity for the MC4R than for the MC3R, [Nle4]Lys-γ2-MSH maintained selectivity for the MC3R, since the affinity is almost 50 times higher for the MC3R than for the MC4R. Thus, [Nle4]α-MSH and [Nle4]Lys-γ2-MSH appeared to be valid as reference peptides in studies aimed at delineating amino acid residues important for MC3R and MC4R selectivity.Table IInhibition constants (Ki in nm) of natural occurring and synthetic MSH peptides for the MC3R and MC4RMC3RMC4Rα-MSHaN-terminal acetylated and C-terminal amidated peptides.4.437[Nle4]α-MSHaN-terminal acetylated and C-terminal amidated peptides.,bReference peptides used in this study.2.919γ2-MSH5.9175γ1-MSH3.295Lys-γ2-MSH3.575[Nle3]γ2-MSHaN-terminal acetylated and C-terminal amidated peptides.3.385[Nle4]Lys-γ2-MSHaN-terminal acetylated and C-terminal amidated peptides.,bReference peptides used in this study.0.938The rat MC3R and the human MC4R were transiently transfected in 293 cells. The K i values were determined by displacement of [125I]NDP-α-MSH. Data are given as K ivalues derived from 11 duplicate data points. Values are representative of at least two independent experiments.a N-terminal acetylated and C-terminal amidated peptides.b Reference peptides used in this study. Open table in a new tab The rat MC3R and the human MC4R were transiently transfected in 293 cells. The K i values were determined by displacement of [125I]NDP-α-MSH. Data are given as K ivalues derived from 11 duplicate data points. Values are representative of at least two independent experiments. First, each amino acid residue in [Nle4]α-MSH was substituted separately for the corresponding residues of [Nle4]Lys-γ2-MSH. When Ser1, Ser3, Glu5, Pro12, or Val13 were substituted for Lys1, Val3, Gly5, Phe12, and Gly13, respectively, no significant effect was observed on affinity or activity for the MC3R nor the MC4R as compared with reference peptide [Nle4]-α-MSH (upper panel of Table II). The only exception was [Nle4,Phe12]α-MSH, which exhibited a 2-fold decrease in activity for the MC4R and a 2-fold increase in affinity for the MC3R. However, when Gly10 or Lys11 of [Nle4]α-MSH were substituted for Asp10 or Arg11 a clear loss of affinity and activity was observed for both the MC3R and MC4R. When the Glu5 to Gly5 substitution was combined with the Gly10to Asp10 substitution in [Nle4]α-MSH, the affinity and activity for the MC4R decreased more than 3-fold, whereas the affinity for the MC3R remained the same as for [Nle4]α-MSH. Next, each amino acid residue in [Nle4]Lys-γ2-MSH was substituted for corresponding residues of [Nle4]α-MSH (lower panel of Table II). When Lys1 was substituted for Ser1 a slight decrease in affinity was observed for both MC3R and MC4R but the activity remained unaffected. Substitution of Arg11 for Lys11 slightly increased activity for both receptors. Substitution of Val3 for Ser3 or Gly13 for Val13 decreased affinity for only the MC4R, while the activity remained unaffected. With respect to binding affinity, [Val13]Lys-γ2-MSH displayed the largest difference between MC3 and MC4 as did [Ser3]Lys-γ2-MSH in activation. Interestingly, when Asp10 was substituted for Gly10, a 5-fold increase in both affinity and activity for the MC4R was observed, while the MC3R was unaffected. [Nle4,Gly10]Lys-γ2-MSH had an even higher affinity for the MC4R than [Nle4]α-MSH. The Phe12 for Pro12 substitution also gave an increase in affinity and activity of more than 3-fold for the MC4R, but not for the MC3R. Strikingly, the Asp10 to Gly10 substitution combined with the Phe12 to Pro12 substitution further increased the affinity to almost 13-fold for the MC4R, but there was no additive effect of these two substitutions on MC4R activation. The MC3R and the MC4R share 58% overall amino acid identity and 76% similarity. The transmembrane regions (TM) show the highest degree of homology while the intra- and extracellular loops (IC and EC) have lower homology (Fig. 2). To identify regions of the MC3R responsible for [Nle4]Lys-γ2-MSH selectivity a series of MC3R and MC4R chimeric receptors were generated with boundaries in stretches of amino acid residues with complete homology in order to minimize effects on receptor folding. These chimeric receptors were designed to determine the contribution of each of the extracellular domains to ligand recognition. Thus, the first extracellular loop with or without the N-terminal domain (named 3B and 3AB, respectively), the second (3C) and third (3D) extracellular loop of the MC4R were swapped individually with the corresponding domain of the MC3R. These chimeric receptors were transfected into 293 HEK cells and the affinities of NDP-α-MSH, [Nle4]α-MSH, [Nle4]Lys-γ2-MSH, and [Nle4,Gly5,Asp10]α-MSH were determined (the latter two peptides displayed MC3R selectivity). Fig. 3 summarizes ligand affinity for the MC3R, MC4R, and five chimeric receptors. All chimera bound [125I]NDP-α-MSH, demonstrating that they were all expressed on the plasma membrane. In general, the affinities of MSH peptides for MC3R and chimera 3AB, 3B, and 4D were higher than for MC4R and chimeras 3C and 3D. For example, the affinity of NDP-α-MSH on these chimera was 6–20-fold higher than for the MC4R, but the same as for the MC3R. All chimeric receptors, except 3D, had the same affinity profile as the MC4R, in which the affinity of NDP-α-MSH ≥ [Nle4]α-MSH > [Nle4]Lys-γ2-MSH = [Nle4,Gly5,Asp10]α-MSH. TheK i values of [Nle4,Gly5,Asp10]α-MSH (data not shown in Fig. 3) for 3AB (1.9 nm), 3B (5.0 nm), 3C (131 nm)," @default.
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• W1984893089 title "Conformation of the Core Sequence in Melanocortin Peptides Directs Selectivity for the Melanocortin MC3 and MC4 Receptors" @default.
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• W1984893089 doi "https://doi.org/10.1074/jbc.274.24.16853" @default.
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