title: The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy.
abstract: 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(O(2))(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 (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(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).
authors.affiliation: Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA. alves@ccr.jussieu.fr
authors.forename: Isabel D
authors.initials: ID
authors.lastname: Alves
authors.forename: Diane
authors.initials: D
authors.lastname: Delaroche
authors.forename: Bernard
authors.initials: B
authors.lastname: Mouillac
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Salamon
authors.forename: Gordon
authors.initials: G
authors.lastname: Tollin
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Hruby
authors.forename: Solange
authors.initials: S
authors.lastname: Lavielle
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D011487:Protein Conformation; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D008024:Ligands; D018040:Receptors, Neurokinin-1