MAPPING PEPTIDE-BINDING DOMAINS OF THE SUBSTANCE-P (NK-1) RECEPTOR FROM P388D(1) CELLS WITH PHOTOLABILE AGONISTS

The tachykinin substance P (SP) is a peptide trans mitter of primary afferents, Its actions on both central and peripheral targets are mediated by a G-protein-coupled receptor of known primary structure. To identify contact sites between the undecapeptide SP and its receptor, we prepared radiolabeled photoreactive analogs of SP (H-RPKPQQFFGLM-NH2) by replacing amino acids in the peptide with p-benzoyl-L-phenylalanine (BPA). SP, EPA(3)-SP, and BPA(8)-SP bind with high affinity (K-d < 3 nM) to SP receptors on the murine cell line P388D(1), triggering intracellular calcium responses. Both binding and calcium responses are blocked by the specific SP receptor antagonist CP-96345. On photolysis, radioiodinated BPA(3)-SP and BPA(8)-SP covalently label a heterogeneously glycosylated protein of about 75 kDa; labeling is abolished by excess unlabeled SP or CP-96345. The labeled receptors were digested with V8 protease and/or trypsin, and the resulting fragments were analyzed by electrophore sis, high pressure liquid chromatography, and chemical or enzymatic modification. BPA(3)-SP and BPA(8)-SP photoincorporate into different regions of the murine SP receptor. The results establish that the third and the eighth positions of SP, respectively, interact with the NH2-terminal extracellular tail (residues 1-21) and second extracellular loop (residues 173-183) of the SP receptor. A model for the agonist peptide binding sites of the SP receptor is proposed based on photoaffinity labeling and mutagenesis studies.