Further definition of the substance P (SP)/neurokinin-1 receptor complex -: MET-174 is the site of photoinsertion of p-benzoylhenylalanine SP

被引:18
作者
Li, HZ
Macdonald, DM
Hronowski, XP
Costello, CE
Leeman, SE
Boyd, ND
机构
[1] Boston Univ, Sch Med, Dept Pharmacol, Boston, MA 02118 USA
[2] Boston Univ, Sch Med, Dept Biophys, Boston, MA 02118 USA
关键词
D O I
10.1074/jbc.M007397200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Bpa4-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.
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页码:10589 / 10593
页数:5
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