title: Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes.
abstract: The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.
authors.affiliation: Institute of Organic and Bioorganic Chemistry, University of Tartu, Jakobi 2, 51014 Tartu, Estonia.
authors.forename: Sergei
authors.initials: S
authors.lastname: Kopanchuk
authors.forename: Santa
authors.initials: S
authors.lastname: Veiksina
authors.forename: Felikss
authors.initials: F
authors.lastname: Mutulis
authors.forename: Ilze
authors.initials: I
authors.lastname: Mutule
authors.forename: Sviatlana
authors.initials: S
authors.lastname: Yahorava
authors.forename: Ilona
authors.initials: I
authors.lastname: Mandrika
authors.forename: Ramona
authors.initials: R
authors.lastname: Petrovska
authors.forename: Ago
authors.initials: A
authors.lastname: Rinken
authors.forename: Jarl E S
authors.initials: JE
authors.lastname: Wikberg
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D019281:Dimerization; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D011869:Radioligand Assay; D044105:Receptor, Melanocortin, Type 4 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007457:Iodine Radioisotopes; D008024:Ligands; D044105:Receptor, Melanocortin, Type 4