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INTRODUCTION |
Substance P (SP)1 is a
peptide neurotransmitter that has a high affinity (10
10
M) interaction with the NK-1 receptor (also Substance P
receptor) (1). Identification of side-chain interactions between SP and the NK-1 receptor is important to understand the molecular basis for
high affinity peptide binding and receptor activation. In the absence
of high resolution structural data, molecular biological and
biochemical approaches, particularly when combined, have provided useful information that allows the localization of specific
interactions between SP and the NK-1 receptor. We have developed a
methodology using the direct approach of photoaffinity labeling for the
determination of contact sites between a peptide ligand and its
receptor (1). In previous work, an analogue of SP in which a
photoreactive amino acid
p-benzoyl-L-phenylalanine (Bpa) was incorporated
into position 8 of the peptide Bpa8-SP was used to
covalently label the NK-1 receptor (1, 3-5). Importantly, the
introduction of the Bpa residue into this position does not alter the
ability of the SP analogue to bind the NK-1 receptor with high affinity
or to induce a functional response. Furthermore, Bpa8-SP
was shown to be a specific and an efficient photoprobe of the NK-1
receptor expressed in Chinese hamster ovary (CHO) cells. The site of
Bpa8-SP covalent attachment was identified by peptide
mapping strategies and matrix-assisted laser desorption/ionization time
of flight (MALDI-TOF) mass spectrometry to be Met-181 of the E2 loop of the receptor (2, 3)).
In the present study, to define further the peptide binding domain of
the NK-1 receptor as well as to orient the SP peptide within the
binding pocket, a second photoprobe, Bpa4-SP, in which
proline 4 is replaced by the photoreactive Bpa residue (Bpa4-SP), has been used to covalently label the NK-1
receptor. The introduction of the Bpa residue into this position does
not alter the ability of this analogue to bind to the NK-1 receptor
with high affinity or to function as an NK-1 receptor agonist. The attachment site of radioiodinated 125I-Bpa4-SP
was identified by peptide mapping and MALDI-TOF mass spectrometry to be
Met-174.
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EXPERIMENTAL PROCEDURES |
Materials--
p-Benzoyl-L-phenylalanine4-substance
P (Bpa4-SP) was synthesized and radioiodinated with a
specific activity of 2200 Ci/mmol using the Bolton-Hunter reagent as
described previously (1, 2).
Cell Transfection and Culture--
CHO cells were stably
transfected with the rat SP receptor cDNA as described previously
(6). The transfected CHO cells expressing 1 × 105 SP
binding sites/cell were maintained in
-minimum Eagle's
medium (Sigma) supplemented with 10% fetal bovine serum and 0.8 mg/ml G418 (Life Technologies, Inc.). For preparative experiments,
cells were obtained in quantity by culture in suspension using spinner flasks to a cell density of 2-3 × 106 cells/ml.
Receptor Binding Assay--
For the saturation binding
experiments, transfected CHO cells were incubated at 4 °C for 2 h with increasing concentrations of
125I-Bpa4-SP (0.1-5 nM) in HEPES
buffer (20 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.2 mM MgCl2, 1 mM CaCl2, pH 7.4), supplemented with 6 mg/ml glucose and 0.6 mg/ml bovine serum albumin. Binding experiments using
photoaffinity ligands were performed in the dark. Nonspecific binding
was determined by the addition of excess unlabeled SP (1 µM). Binding was terminated by the addition of 5 ml of
ice-cold HEPES buffer and rapid filtering through a glass fiber filter (Whatman GF/C) that had been soaked for at least 2 h in
polyethyleneimine (0.1%). The filters were then assayed for
radioactivity by
spectrometry. Binding affinity
(Kd) was calculated from the Scatchard transformation of the binding data.
Intracellular Calcium Measurement--
Cells were plated on
9 × 22-mm glass coverslips in a tissue culture dish and
maintained in
-minimum Eagle's medium containing 10% fetal
bovine serum and 0.8 mg/ml G418. When the cells reached 70-90% confluence, the coverslips were washed with a HEPES-buffered saline solution (10 mM HEPES, 140 mM
NaCl, 5 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 10 mM glucose, pH 7.4).
The cells were loaded with the fluorescent calcium indicator, 4 µM Fura-2/AM (Molecular Probes), with 0.01% pluronic
acid (9:1, v/v) in 2 ml of HEPES-buffered saline and incubated for 25 min at room temperature. The coverslip was then washed with
HEPES-buffered saline and placed into a fluorescence spectrophotometer
in a cuvette containing HEPES-buffered saline. Upon the addition of
peptide ligands, fluorescent emission was measured at 510 nm with
excitation wavelengths of 340 and 380 nm (Fura-2 bound and unbound to
calcium, respectively). [Ca2+]i was
determined by the ratio of the fluorescence at the two excitation
wavelengths as described previously (7).
Photoaffinity Labeling of Transfected CHO Cells--
Cells were
resuspended at 107 cells/ml in HEPES buffer and incubated
with 125I-labeled Bpa4-SP (0.1 nM)
for 2 h at 4 °C in the dark. The cells were then irradiated at
a distance of 6 cm from a 100-watt long-wave (365 nm) UV lamp
(Blak-ray, San Gabriel, CA) for 15 min on ice. For large scale
photolabeling, cells were obtained in large quantity (5-liter
suspension culture) and incubated with
125I-Bpa4-SP isotopically diluted with
127I-labeled Bpa4-SP (1:1000).
After photolysis, the cells were obtained by centrifugation and washed
twice with HEPES buffer. The cell pellets were resuspended in 5 mM Tris-HCl containing 1 mM EDTA (pH 7.0),
homogenized briefly, and centrifuged at 1000 × g for
15 min to remove cellular debris. The photolabeled membrane pellet was
obtained by centrifugation at 40,000 × g for 30 min
and stored at
20 °C. To test the specificity of the photoaffinity
ligand, labeling was also carried out in the presence of excess
unlabeled SP (1 µM).
Enzymatic and Chemical Cleavage of Photolabeled NK-1
Receptors--
Membrane preparations containing the photolabeled
receptor were partially digested for 18 h with
L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin in 50 mM Tris-HCl and 1 mM CaCl2 (pH 8.0) at room temperature. The
amount of trypsin used per mg of membrane protein is specified in the
figure legends. The digestion was stopped by the addition of SDS-PAGE
sample buffer (10% glycerol, 1% SDS, 0.05% bromphenol blue, 125 mM Tris-HCl, pH 6.8). The solubilized peptide fragments
were separated on an SDS-PAGE gel using the Tricine gel system of
Schagger and von Jagow (8). Following electrophoresis, the gel
was dried and exposed to x-ray film (Kodak XAR-5) with an intensifying
screen (DuPont). The molecular masses of the radiolabeled fragments
were determined using molecular mass markers from Amersham
Pharmacia Biotech (2.35-46 kDa).
Following autoradiography, the radioactive bands were excised from
dried gels and macerated into pieces. The partially digested receptor
fragments were then eluted passively in either extraction buffer (0.1%
SDS, 100 mM NH4HCO3, pH 7.8) for
subcleavage by Staphylococcus aureus V8 protease or 0.1 N HCl for CNBr (cyanogen bromide) subcleavage. The eluted
tryptic fragments were also incubated with 100 mM
dithiothreitol (DTT) for 1 h at room temperature and then
reanalyzed by SDS-PAGE.
For cleavage of the intact receptor with CNBr, photolabeled membranes
were first absorbed to C18-derivatized silica beads (Alltech) at 1 mg
of C18 beads/mg of membrane protein. The silica beads were washed twice
with 0.1% trifluoroacetic acid in water, followed by 70%
acetonitrile in 0.1% trifluoroacetic acid/water to remove the unbound
ligand. The membrane-attached silica beads were then incubated
overnight with 50 mg/ml CNBr in 0.1 N HCl in the dark at
room temperature. CNBr-generated peptide fragments were eluted from the
silica beads with SDS-PAGE sample buffer and were subject to
electrophoresis and autoradiography.
To evaluate the effects of CNBr on the ligand, which also contains a
methionine residue, 127I-Bpa4-SP was
treated with 50 mg/ml CNBr in 0.1 N HCl at room temperature overnight. The reaction mixture was further separated by C18 HPLC and
analyzed by mass spectrometry.
Partial Purification of the Labeled Fragments by
SDS-PAGE--
For preparative experiments, a small receptor fragment
(2 kDa) generated from CNBr cleavage was partially purified on
preparative SDS-PAGE Tricine gel (3 mm). The small labeled receptor
fragment was eluted from the gel in 0.1% trifluoroacetic acid for
further purification by HPLC.
To determine the yield of photolabeled receptor fragments, the
autoradiographs were aligned with the dried gels, the radiolabeled polypeptides were cut out, and the amount of radioactivity was measured.
Purification of the CNBr Cleavage Fragment by HPLC--
The
eluted receptor fragment (2 kDa) in 0.1% trifluoroacetic acid was
further purified by reverse-phase HPLC using an Alltech C18 column
(4.6 × 250 mm) and a Vydac C4 column (4.6 × 250 mm). The
columns were eluted with increasing Solvent B (60% acetonitrile/0.08% trifluoroacetic acid/39% water) against Solvent A (0.08%
trifluoroacetic acid in water) at a constant flow rate of 1.5 ml/min at
45 °C. The concentration of Solvent B was raised from 20 to
60% at a rate of 1%/min. Fractions were collected every minute, and
the 125I radioactivity of eluted fractions was monitored by
spectrometry. The UV absorbance of each fraction was also recorded
at a wavelength of 210 nm.
Receptor Fragment Peptide Analysis by Mass Spectrometry--
The
molecular mass of the purified 2-kDa receptor fragment was determined
at the Mass Spectrometry Resource at Boston University School of
Medicine using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in less than picomole quantities. The
Finnigan Vision 2000 instrument (Thermo BioAnalysis, Franklin, MA) is equipped with an LSI nitrogen laser (337 nm, 3-ns pulse duration). The matrix, 2,5-dihydroxybenzoic acid, was dissolved in
1:4 water/acetonitrile (10 mg/ml).
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RESULTS |
Characterization of Iodinated Bpa4-SP--
The
addition of a 127I-labeled Bolton-Hunter conjugate
of Bpa4-SP to transfected CHO cells expressing the NK-1
receptor induced a Ca2+ response comparable with that
induced by the same concentration of the parent peptide SP (Fig.
1). The response evoked by both peptides
was completely inhibited in the presence of 1 µM
RP-67,580, a specific nonpeptide antagonist of the rat NK-1
receptor.

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Fig. 1.
SP and
127I-Bpa4-SP induced calcium
mobilization in CHO cells expressing the rat NK-1 receptor.
[Ca2+]i was measured using Fura-2/AM and
expressed as the fluorescent ratio at 340 and 380 nm as described under
"Experimental Procedures." The change in
[Ca2+]i was monitored after the addition of 1 nM agonist in the absence or presence of the antagonist
RP-67,580 (1 µM).
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The binding affinity of radioiodinated
125I-Bpa4-SP to intact CHO cells expressing the
NK-1 receptor was determined by saturation binding experiments. The
observed equilibrium binding was characterized by Kd = 1.4 ± 0.2 nM, a value about 5-fold higher than that
previously reported for the corresponding radioiodinated conjugate of
the parent peptide (3). In contrast, the number of NK-1 receptors/cell
(Bmax) was the same for both derivatives.
Photoaffinity Labeling of the Rat NK-1 Receptor and Fragmentation
Analysis--
NK-1 receptors expressed on intact CHO cells were
photolabeled with 125I-Bpa4-SP, and the
resulting ligand-receptor complex was analyzed by SDS-PAGE and
autoradiography. Photolabeled receptors were observed as a broad band
centered at 80 kDa (Fig. 2, lane
1). The diffuseness of the band is attributable to heterogeneous
glycosylation of NK-1 receptors expressed in CHO cells (5).
Quantitative analysis of the radioactivity incorporated into the NK-1
receptor indicated that photolabeling was highly efficient; ~40% of
the specifically bound photoligand became covalently linked to the
receptor upon UV exposure.

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Fig. 2.
Limited tryptic digestion and CNBr cleavage
of the 125I-Bpa4-SP labeled SP receptor.
Rat NK-1 receptors expressed in CHO cells were photoaffinity-labeled
with 125I-Bpa4-SP (lane 1), and a
membrane preparation derived from these cells was subjected to limited
cleavage by trypsin (0.5 mg/ml) (lane 2) or by CNBr (1 mg/ml) (lane 3) under conditions described under
"Experimental Procedures." The cleavage fragments were analyzed by
Tricine SDS-PAGE under nonreducing conditions followed by
autoradiography. Lane 4 is the photoaffinity probe,
125I-Bpa4-SP.
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Membrane preparations of photolabeled receptors were treated under
nonreducing conditions with trypsin, and the resulting fragments were
analyzed by SDS-PAGE. Three major photolabeled fragments of 22, 14, and
5 kDa were observed (Fig. 2, lane 2). These bands were
eluted from the gel. Further digestion by trypsin of the eluted
fragments converted the 22- and 14-kDa fragments to the 5-kDa fragment
(data not shown), thus establishing that the 5-kDa fragment is the
limit peptide produced by trypsin digestion.
Previously we have shown that the NK-1 receptor contains a disulfide
bond linking Cys-105 on the E1 loop to Cys-180 on the E2 loop and that
this bond plays an important role in the high affinity binding of SP
(3). The analysis of receptor fragments generated in the absence or
presence of DTT is valuable for the identification of receptor
fragments held together by a disulfide bond between Cys-105 and
Cys-180. Therefore, each of the photolabeled bands obtained from
tryptic digestion under nonreducing conditions was further treated with
DTT and reanalyzed by SDS-PAGE. Upon reduction by DTT, the mobility of
the 22-kDa band increased to 14 kDa, whereas the 14-kDa band increased
to 7 kDa (Fig. 3, lanes 1-4).
In contrast, the mobility of the 5-kDa fragment was not changed by
treatment with DTT, indicating that this photolabeled receptor
fragment, unlike the other two larger fragments, lacks the disulfide
bond (Fig. 3, lanes 5 and 6). Based on the
experimentally determined molecular mass of the limit fragment
and extended forms, together with their susceptibility to reductive
cleavage by DTT, we can conclude that digestion of the photolabeled
receptor with trypsin generates an ~5-kDa fragment corresponding to
residues Leu-142 to Arg-177, which has a calculated molecular mass of
5.8 kDa when the mass of the covalently attached probe, 1.8 kDa, is added.

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Fig. 3.
Reduction of the disulfide bond within the
photolabeled tryptic fragments. Isolated fragments from limited
tryptic digestion were treated with 100 mM DTT, reanalyzed
by Tricine SDS-PAGE, and visualized by autoradiography. Lanes
1 and 2, 22-kDa fragment; lanes 3 and
4, 14-kDa fragment; lanes 5 and 6,
5-kDa fragment.
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This 5-kDa fragment contains a single cleavage site for S. aureus V8 protease at residue Glu-172. To define more
closely the residue that serves as the site of photoincorporation, the
limit tryptic fragment of Mr 5000 was
treated with S. aureus V8 protease. After cleavage of the
5-kDa fragment with S. aureus V8 protease, a small fragment
of 2.4 kDa in agreement with the calculated mass (2.3 kDa) of the
photoligand covalently attached to a receptor fragment extending from
residues 173 to 177 (TMPSR) was obtained (Fig.
4, lane 2). The
Mr 5000 limit tryptic fragment was also treated
with CNBr, a reagent specific for cleavage after Met, generating a
fragment of Mr ~2000 (Fig. 4, lane
3), a value that is close to that of the photoligand itself.

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Fig. 4.
Subcleavage of the tryptic 5-kDa fragment by
S. aureus V8 protease and CNBr. An
isolated tryptic fragment (5 kDa) (lane 1) was further
treated with either S. aureus V8 protease (0.5 mg/ml)
(lane 2) or CNBr (1 mg/ml) (lane 3). The
subcleaved fragments were resolved by Tricine SDS-PAGE and visualized
by autoradiography. Lane 4 is the photoaffinity probe,
125I-Bpa4-SP, with a molecular mass of 1.8 kDa.
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Identification of the Residue on the NK-1 Receptor That Is
Covalently Labeled by Bpa4SP--
To obtain a sufficient
amount of the 2-kDa fragment for accurate determination of molecular
mass MALDI-TOF mass spectrometric analysis, transfected CHO cells
(109) were photolabeled with
125I-Bpa4-SP isotopically diluted with
127I-Bpa4-SP to a specific activity of 2 Ci/mmol and then subjected to solid-phase CNBr cleavage (2).
Approximately 100 pmol of the photolabeled receptor was absorbed onto
C18-derivatized silica beads. Following treatment with CNBr, the
generated fragments were resolved using Tricine-based SDS-PAGE. The
2-kDa radiolabeled fragment was eluted and further purified by HPLC
using both C18 and C4 columns (Fig. 5,
A and B). The final yield of the purified 2-kDa
fragment was 8 pmol, representing 8% of the starting photolabeled receptor, an amount more than sufficient to permit MALDI-TOF mass spectrometric analysis.

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Fig. 5.
Purification of the CNBr-generated 2-kDa
receptor fragment by HPLC. The partially purified 2-kDa receptor
fragment from CNBr cleavage was isolated from the SDS-PAGE gel and was
further purified by reverse-phase HPLC using a C18 column
(A), followed by a C4 column (B). Radioactivity
was monitored by -emission spectrometry.
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MALDI-TOF mass spectrometric analysis of the CNBr-generated fragment
yielded a (M+H)+ with an m/z value of
1801.3 ± 1.8 (Fig. 6). This mass
spectrum not only confirms the purity of the isolated fragment but also defines the chemical structure of the fragment. The observed molecular mass (1801.3 ± 1.8) could only be generated from a
photolabeled receptor by the covalent attachment of the probe
127I-Bpa4-SP (molecular weight = 1776.9) to the methyl group of a methionine residue, followed
by CNBr cleavage of the bond between the
-carbon and sulfur on the
methionine side chain to form a thiocyanate (
CH2SCN)
derivative. In addition, the C-terminal methionine amide of the
covalently attached probe is converted by CNBr to a homoserine lactone.
The observed (M+H)+ of m/z
1801.3 ± 1.8 of the final cleavage product is in good agreement
with the calculated (M+H)+ of m/z
1802.9.

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Fig. 6.
The MALDI-TOF mass spectrum of the isolated
CNBr-generated 2-kDa fragment. [M+H]+ is the
protonated molecular ion at 1801.3 ± 2. Additional low abundance
peaks are derived from the parent ion as indicated.
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Because there is only one methionine residue (Met-174) in the
photolabeled receptor fragment (residues 173-177), we can
conclude that the site of photoincorporation of
Bpa4-SP is Met-174 (Fig.
7).

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Fig. 7.
Schematic representation of the NK-1 receptor
tryptic fragment (residues 142-177). The disulfide bond between
Cys-105 and Cys-180 is indicated. Cleavage by S. aureus V8
protease converts the tryptic fragment to a fragment of
Mr ~2400. CNBr cleavage identifies Met-174 as
the site of covalent attachment.
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DISCUSSION |
Our previous results have shown that Bpa8-SP, which
contains the photoreactive Bpa residue in the eighth position of the SP peptide within the conserved C-terminal region that defines the tachykinin peptide family, covalently attaches to a single residue, Met-181, on the NK-1 receptor (2). The present study characterizes the
site of a covalent attachment of a second photoreactive analogue of SP
with the photoreactive residue located in the nonconserved N terminus
of the peptide, specifically position 4 (Bpa4-SP).
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. The results
presented in this report show that Bpa4-SP covalently
attaches to a single residue Met-174, implying that position 4 of SP is
in close spatial proximity to Met-174 when bound to the NK-1 receptor.
Interestingly, in a separate study, Girault et al. (9)
identified Met-174 as the attachment site on the human NK-1 receptor for the SP analogue Bpa8-Pro9-SP, the same
residue that is labeled in the present study by Bpa4-SP.
Although the basis for the discrepancy in these studies is unknown, the
introduction of a proline residue at the 9th position of SP could
potentially alter the confirmation of the peptide in such a manner that
the Bpa residue of Bpa8-Pro9-SP is positioned
in close proximity to Met-174 rather than Met-181. Additional
explanations could include (i) species differences (human
versus rat) or (ii) experimental differences
(e.g. labeling of membrane preparations versus
labeling of intact cells).
The results obtained here with radioiodinated Bpa4-SP, when
combined with our previous findings (2, 3) with radioiodinated Bpa8-SP, establish the importance of the initial
sequence of the E2 loop extending beyond transmembrane 4 of the NK-1
receptor (in peptide binding). Based on these findings, it is likely
that the bound SP peptide is oriented parallel to this region of the
receptor with its 4th position adjacent to Met-174 and its 8th position adjacent to Met-181. Because of its location in the E2 loop just after
it emerges from the lipid bilayer, Met-174 is positioned close to the
membrane. Furthermore, although Met-181 is located in the middle of the
E2 loop, it is adjacent to Cys-180, which participates in a disulfide
bond with Cys-105 (3), a residue that is close to the membrane
interface. Thus, Met-181 is also positioned near the membrane interface
of the peptide. Interestingly, we have recently obtained evidence (10)
that these two methionines on the E2 loop are also spatially close to
each other.