Mapping peptide-binding domains of the substance P (NK-1) receptor from P388D1 cells with photolabile agonists.
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.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Y M
YM
Li
M
M
Marnerakis
E R
ER
Stimson
J E
JE
Maggio
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D007941:Leukemia P388; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D018040:Receptors, Neurokinin-1; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin