Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies

Abstract

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys6,(p-Bz) Phe8,-Pro9,Met(O2)11]SP-(7-11) and [BAPA0,(p-Bz)Phe8]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, 173TMPSR177, for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of 173TMP175 in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.