The Two NK-1 Binding Sites Correspond to Distinct, Independent, and Non-Interconvertible Receptor Conformational States As Confirmed by Plasmon-Waveguide Resonance Spectroscopy
| Autor: | Bernard Mouillac, Sandrine Sagan, Victor J. Hruby, Diane Delaroche, Isabel D. Alves, Solange Lavielle, Gordon Tollin, Zdzislaw Salamon |
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| Přispěvatelé: | Synthèse, Structure et Fonction de Molécules Bioactives (SSFMB), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2006 |
| Předmět: |
Protein Conformation
Stereochemistry Gene Expression Substance P CHO Cells Ligands Biochemistry Article 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Protein structure Cricetinae Animals Humans Surface plasmon resonance Binding site Receptor 030304 developmental biology 0303 health sciences Binding Sites Receptors Neurokinin-1 Surface Plasmon Resonance Ligand (biochemistry) chemistry cAMP-dependent pathway Neurokinin A 030217 neurology & neurosurgery |
| Zdroj: | Biochemistry Biochemistry, 2006, 45, pp.5309-5318 Biochemistry, American Chemical Society, 2006, 45, pp.5309-5318 HAL |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi052586d |
| Popis: | Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand–receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (Kd = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h). |
| Databáze: | OpenAIRE |
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