Ym. Li et al., MAPPING PEPTIDE-BINDING DOMAINS OF THE SUBSTANCE-P (NK-1) RECEPTOR FROM P388D(1) CELLS WITH PHOTOLABILE AGONISTS, The Journal of biological chemistry, 270(3), 1995, pp. 1213-1220
The tachykinin substance P (SP) is a peptide trans mitter of primary a
fferents, Its actions on both central and peripheral targets are media
ted by a G-protein-coupled receptor of known primary structure. To ide
ntify 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 intra
cellular calcium responses. Both binding and calcium responses are blo
cked by the specific SP receptor antagonist CP-96345. On photolysis, r
adioiodinated BPA(3)-SP and BPA(8)-SP covalently label a heterogeneous
ly glycosylated protein of about 75 kDa; labeling is abolished by exce
ss 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 i
nto 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 e
xtracellular loop (residues 173-183) of the SP receptor. A model for t
he agonist peptide binding sites of the SP receptor is proposed based
on photoaffinity labeling and mutagenesis studies.