1. Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies.
- Author
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Lequin O, Bolbach G, Frank F, Convert O, Girault-Lagrange S, Chassaing G, Lavielle S, and Sagan S
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, Carboxypeptidases chemistry, Carboxypeptidases metabolism, Cathepsin A, Cattle, Cell Membrane metabolism, Cricetinae, Humans, Kinetics, Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Sequence Data, Peptide Biosynthesis, Peptides chemistry, Phenylalanine chemistry, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Rats, Receptors, Neurokinin-1 metabolism, Rhodopsin chemistry, Salicylates, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Streptavidin pharmacology, Substance P metabolism, Trypsin pharmacology, Phenylalanine analogs & derivatives, Receptors, Neurokinin-1 chemistry, Substance P chemistry
- 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-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]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, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) 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.
- Published
- 2002
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