The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by I-125-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEW-PEHPNR). We have previously shown that I-125-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M-181 within this same receptor sequence. Importantly, both of these peptides (I-125-iodohistidyl(1)-Bpa(7)NKA and I-125-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carhoxyl-terminal tail. In this report, me also show that site-directed mutagenesis of M-181 to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.