Abstract
The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.
Highlights
The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid pbenzoyl-L-phenylalanine (Bpa) in position 8 of the Cterminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor
Photoaffinity Labeling of the Rat NK-1 Receptor and Fragmentation Analysis—NK-1 receptors expressed on intact CHO cells were photolabeled with 125I-Benzoyl-L-phenylalanine4-substance P (Bpa4-SP), and the resulting ligand-receptor complex was analyzed by SDS-PAGE and autoradiography
The substitution of the 4th position of SP (Pro-4) by Bpa is well tolerated by the receptor, as evidenced by its high affinity binding to the NK-1 receptor and its ability to stimulate an increase in intracellular calcium in the in vitro assay
Summary
The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid pbenzoyl-L-phenylalanine (Bpa) in position 8 of the Cterminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa4-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide.
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