Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies.
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.
Lequin
Olivier
O
Unité Mixte de Recherches 7613 CNRS, Université Paul et Marie Curie, 4 place Jussieu, 75252 Paris cedex 05, France.
Bolbach
Gérard
G
Frank
Fabrice
F
Convert
Odile
O
Girault-Lagrange
Sophie
S
Chassaing
Gérard
G
Lavielle
Solange
S
Sagan
Sandrine
S
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism; D043402:Cathepsin A; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry; D012459:Salicylates; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019809:Streptavidin / Q000494:pharmacology; D013373:Substance P / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D010455:Peptides; D018040:Receptors, Neurokinin-1; D012459:Salicylates; D013373:Substance P; D010649:Phenylalanine; C006715:4-benzoylsalicylic acid; D012243:Rhodopsin; D019809:Streptavidin; C053764:benzoylphenylalanine; D002268:Carboxypeptidases; C468629:CTSA protein, human; D043402:Cathepsin A; D014357:Trypsin