CγH2 of Met174 Side Chain Is the Site of Covalent Attachment of a Substance P Analog Photoactivable in Position 5

Emmanuelle Sachon,Gérard Bolbach,Gérard Chassaing,Solange Lavielle,Sandrine Sagan

doi:10.1074/jbc.m207242200

Abstract

Analogs of substance P (H-RPKPQQFFGLM-NH(2)) incorporating a photoreactive para-benzoyl-l-phenylalanine (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence have been synthesized and pharmacologically characterized previously as full NK-1 receptor agonists. In this study we show that all analogs, [BAPA(0), (p-Bzl)Phe(x), Met(O(2))(11)]SP also display high yields (40-70%) of NK-1 receptor photolabeling. To identify the site of photoinsertion in the receptor, covalent ligand/receptor complexes were digested with enzymes or chemically cleaved with cyanogen bromide and purified with streptavidin-coated magnetic beads before matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Only the analog photoreactive at position 5 gave irreversible, reproducible, and unequivocal covalent linkage. Sequential digestions of the covalent complex, substance P analog photoreactive at position 5/NK-1 receptor, with trypsin, endo-GluC and carboxypeptidase Y, led to the identification of the tripeptide (173)TMP(175) in the second extracellular loop of the hNK-1 receptor as the site of photoinsertion. Reaction of cyanogen bromide on the pentapeptide TMPSR did not yield the expected cleavage on the carboxylic side of methionine. The high precision of mass spectrometry analysis on the mass measured led us to determine that C(gamma)H(2) of Met(174) was the site of covalent linkage of the photoreactive substance P analog. Such an insertion (photolinked ligand) on its C(gamma)H(2) renders methionine refractory to CNBr cleavage.

Highlights

The substance P (H-RPKPQQFFGLM-NH2) NK-1 receptor is a member of the class I family within the superfamily of Gprotein-coupled receptors
Using a photoreactive analog at position 8 of SP, Kage et al reported the site of covalent attachment as Met[181] in the rat (5), whereas we identified Met[174] in the second extracellular loop of the human (6, 7) or the rat NK-1 receptor (7)
During the course of this study, Bremer et al have reported that photoinsertion of a photoreactive analog of SP at position 3 is located both within the segments 173–177 of the second extracellular loop and 11–21 of the N terminus of the rat NK-1 receptor (8)

Summary

EXPERIMENTAL PROCEDURES

Materials—The photoreactive peptide analogs of SP have been synthesized and characterized previously (11). Determination of the Yield of Photoaffinity Labeling—Membranes (10 ␮g of proteins) were incubated for 10 min at room temperature with the photoreactive analogs at a concentration equal to their affinity (Ki) previously determined in competition experiments with [113H][Pro9]SP, i.e. 5 nM for [BAPA0, (p-Bzl)Phe[4], Met(O2)11]SP, 20 nM for [BAPA0, (p-Bzl)Phe[5], Met(O2)11]SP, 0.5 nM for [BAPA0, (p-Bzl)Phe[6], Met(O2)11]SP, 13 nM for [BAPA0, (p-Bzl)Phe[9], Met(O2)11]SP, and 18 nM for [BAPA0, (p-Bzl)Phe[10], Met(O2)11]SP in 100 ␮l of 50 mM Tris-Cl buffer pH 7.4 containing 1 mM EDTA, 10 mM MgCl2, 10 mM MnCl2, 100 ␮M phenylmethylsulfonyl fluoride, and 400 ␮g/ml bovine serum albumin. After endo-GluC cleavage, samples were again purified with the magnetic beads and subjected either to further digestion with CBPY or MALDI-TOF MS analysis as described (6, 7). Peptide receptor domains corresponding to the mass peaks obtained were identified by using the Protein Analysis WorkSheet (PAWS) freeware edition (Proteomics, www.proteomics.com) and applied to the hNK-1 receptor using the different cleavages

RESULTS

MHϩ measured

MHϩ expected

DISCUSSION