Mapping Peptide-binding Domains of the Substance P (NK-1) Receptor from P388D1 Cells with Photolabile Agonists

Abstract

The tachykinin substance P (SP) is a peptide transmitter 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, BPA3-SP, and BPA8-SP bind with high affinity (Kd < 3 nM) to SP receptors on the murine cell line P388D1, triggering intracellular calcium responses. Both binding and calcium responses are blocked by the specific SP receptor antagonist CP-96345. On photolysis, radioiodinated BPA3-SP, and BPA8-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 electrophoresis, high pressure liquid chromatography, and chemical or enzymatic modification. BPA3-SP and BPA8-SP photo-incorporate 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.