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INTRODUCTION |
The undecapeptide substance P
(SP)1 belongs to the
tachykinin family of bioactive peptides, which are structurally
characterized by the conserved carboxyl-terminal sequence of
-Phe-X-Gly-Leu-Met-NH2, where X is
either a
-branched aliphatic (Val, Ile) or an aromatic (Phe, Tyr)
amino acid residue (1-5). Synthesized and secreted from both neural
and non-neural tissues, SP
(H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) participates in many physiological processes in the cardiovascular, respiratory, gastrointestinal, immune, and nervous systems (3, 6, 7).
Considerable evidence exists that SP plays a role in pain modulation
and neurogenic inflammation (6, 7), and recent studies have associated
this peptide in the pathogenesis of certain affective disorders (8).
The many diverse biological effects of SP are mediated by the
functional interaction of the peptide with its cell-surface G
protein-coupled receptor, the neurokinin-1 receptor (NK-1R).
In our laboratory, we have utilized the biochemical approach of
photoaffinity labeling using SP analogues containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) in different peptide
positions to identify contact sites between specific residues of SP and side chains of receptor residues (9, 10). We have shown that 125I-Bolton-Hunter reagent (BH)-labeled
Bpa8SP covalently labels Met181 (9),
whereas 125I-BH-Bpa4SP covalently labels
Met174 (10) of the rat NK-1R (rNK-1R), information that has
been integral to the three-dimensional modeling of the SP·NK-1R
complex (see the accompanying article (27)).
Maggio and co-workers (11) previously reported that a photoreactive
analogue of SP in which the Bpa residue is substituted at position 3 (125I-[D-Tyr0]Bpa3SP)
covalently labels a residue within the initial 21 amino acid residues
of the N terminus of the NK-1R present in the murine P388D1
cell line. We found this result intriguing in view of our finding that
when Bpa is substituted at position 4 of the peptide, Met174 on the second extracellular loop (EC2) is the site
of covalent attachment (10). We therefore decided that it would be of
interest to study the site of photoincorporation of
125I-[D-Tyr0]Bpa3SP
into rat NK-1Rs expressed in stably transfected Chinese hamster ovary
(CHO) cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Purified SP was purchased from Sigma, and
[D-Tyr0]Bpa3SP was
purchased from Quality Controlled Biochemicals, Inc., following high
performance liquid chromatography (HPLC) purification and mass
spectrometric analysis. 125I-Labeled Bolton-Hunter reagent
and 125I (each with a specific activity of 2200 Ci/mmol)
were obtained from PerkinElmer Life Sciences, and
127I-labeled Bolton-Hunter reagent was synthesized by B. Tomczuk (Eastman Kodak Co.).
Preparation of Iodinated Ligands--
Purified SP was
iodinated by coupling the Lys3 residue
-NH2
group to the monoiodinated 125I-labeled Bolton-Hunter
reagent (N-succinimidyl-3-(4-hydroxyphenyl) propionate) as
described previously (12, 13). Purified
[D-Tyr0]Bpa3SP was iodinated by
coupling the N-terminal D-Tyr0 residue to
either 125I or 127I using the solid-phase
oxidant 1,3,4,6-tetrachloro-3
,6
-diphenylglycouril (IODO-GEN® iodination reagent, Pierce). Briefly,
[D-Tyr0]Bpa3SP was dissolved at
room temperature in 0.1 M sodium borate buffer (pH 8.5) and
incubated with 125I or 127I for 15 min at room
temperature in IODO-GEN® reagent-treated 10 × 75-mm
borosilicate culture tubes. Reversed-phase HPLC on a 0.1% (v/v)
trifluoroacetic acid-equilibrated and derivatized silica gel
C18 column was then performed to separate the iodination reaction products using an acetonitrile/water/trifluoroacetic acid
solvent system. The acetonitrile concentration in the eluant was raised
by a gradient controller/pump system by 0.7%/min, and fractions were
collected every minute at a flow rate of 1.5 ml/min. 127I-[D-Tyr0]Bpa3SP
was identified by measuring the UV absorbance at 262 nm, and 125I-[D-Tyr0]Bpa3SP
was identified by
-emission spectrometry. 20% (v/v)
-mercaptoethanol was added to both the
127I-[D-Tyr0]Bpa3SP
and
125I-[D-Tyr0]Bpa3SP
peptide fractions, and the samples were heated at 90 °C for 2 h
(to reduce the methionine sulfoxide on Met11 of the
peptides to its thioether form). Mass spectrometry was then used to
confirm that the 127I had coupled to the
D-Tyr0 residue of
127I-[D-Tyr0]Bpa3SP.
Both
127I-[D-Tyr0]Bpa3SP
and
125I-[D-Tyr0]Bpa3SP
were shown to coelute in the reversed-phase HPLC system described above.
Cell Culture--
CHO cells stably transfected with the cDNA
encoding the rNK-1R and a Geneticin resistance gene (14) and expressing
500,000 rNK-1Rs/cell were kindly provided by Dr. J. E. Krause
(Neurogen, Brandford, CT). Transfected cells were maintained as
monolayer cultures in
-minimal essential medium (Life Technologies,
Inc.) supplemented with 10% (v/v) Cool CalfTM 2 (Sigma)
and 1 mg/ml Geneticin (G418 sulfate; Life Technologies, Inc.) as
described previously (9, 15). Transfected cells were grown in an
atmosphere of 95% air and 5% CO2 at 37 °C and harvested for experiments using phosphate-buffered saline-based enzyme-free dissociation buffer (Specialty Media).
Equilibrium Displacement Competition
Assay--
rNK-1R-transfected CHO cells were harvested and resuspended
in ice-cold KRH buffer (20 mM HEPES, 1 mM CaCl2, 2.2 mM MgCl2, 5 mM KCl, and 120 mM NaCl (pH 7.4))
supplemented with 6 mg/ml glucose and 0.6 mg/ml bovine serum albumin.
Cells were incubated for 2 h at 4 °C with the radiolabeled
ligand 125I-BH-SP, and binding was measured either alone or
in the presence of increasing concentrations of unlabeled SP or
[D-Tyr0]Bpa3SP. In all
experiments, nonspecific 125I-BH-SP binding was defined as
the binding in the presence of 1 µM unlabeled SP. To
separate bound ligand from free ligand, cells were filtered after
incubation through Whatman GF/C filter paper (soaked >2 h in 0.1%
polyethyleneimine) and washed three times in ice-cold KRH buffer (pH
7.4) with a Brandel Harvester apparatus. Bound radioactivity on the
filters was then quantified by
-emission spectrometry. Competition
assays were performed in triplicate and were repeated at least three times.
Photoaffinity Labeling of Transfected Cells--
Stably
transfected CHO cells were photolabeled with
125I-[D-Tyr0]Bpa3SP
using the procedure described previously (9, 15, 16). Transfected cells
were harvested; pelleted through centrifugation at 100 × g for 10 min; and resuspended in ice-cold KRH buffer (pH
7.4) supplemented with 6 mg/ml glucose, 0.6 mg/ml bovine serum albumin,
3 µg/ml chymostatin, 5 µg/ml leupeptin, and 30 µg/ml bacitracin.
Radiolabeled
125I-[D-Tyr0]Bpa3SP
was added to a final concentration of 1-2 nM, and the
mixtures were incubated in the dark at 4 °C for 2 h with gentle
agitation. Competing peptides or non-peptide antagonists were added at
the concentrations indicated. For preparative scale photolabeling, 4-5 × 109 transfected CHO cells (Cell Culture
Center, Minneapolis, MN) were photolabeled with
125I-[D-Tyr0]Bpa3SP
isotopically diluted with
127I-[D-Tyr0]Bpa3SP
(1:1000). Following incubation, the mixtures were transferred to a
Petri dish, diluted 1:1 with ice-cold KRH buffer (pH 7.4), and
irradiated at 365 nm by exposure to a 100-watt long-wave UV lamp for 15 min at a distance of 6 cm as described previously (9, 15, 16).
Cell Membrane Preparation--
Membranes were prepared by
collecting photolabeled cells through centrifugation and resuspension
in Tris/EDTA buffer (5 mM Tris and 1 mM EDTA
(pH 7.4)) containing 0.1 mM phenylmethylsulfonyl fluoride.
Cell mixtures were sonicated for 10 s twice with a Sonicator Cell
Disrupter to ensure complete homogenization and centrifuged at 500 × g for 10 min to remove nuclear and cellular debris. The remaining supernatants were then sedimented at 38,000 × g for 1 h to collect the membrane pellets. Following
the removal of noncovalently attached radioligand from the membranes by
a hypertonic acid wash (0.2 M acetic acid and 0.5 M NaCl (pH 2.4)), the membranes were washed twice by
resuspension and centrifugation in Tris/EDTA buffer (pH 7.4) and stored
at
20 °C.
Identification of the Photolabeled
Receptor--
125I-[D-Tyr0]Bpa3SP-labeled
membranes were solubilized in sample buffer (0.125 M Tris,
2% SDS, 10% glycerol, and 0.01% bromphenol blue (pH 6.8)), heated at
55 °C for 10 min, and then subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) as described by Laemmli (17). Following
electrophoresis, the gels were dried on filter paper and exposed to
x-ray film (Kodak XAR-5). Prestained molecular mass standards
(14.3-220 kDa; Amersham Pharmacia Biotech) were used to determine the
molecular mass of the radiolabeled complex.
Tryptic Digestion of Photolabeled
Membranes--
125I-[D-Tyr0]Bpa3SP-labeled
membranes were resuspended in 0.1% SDS, 50 mM Tris, and 1 mM CaCl2 (pH 8.0) and digested at room
temperature for 2 h with the amount of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
bovine trypsin specified in the legend to Fig. 3.
N
-p-tosyl-L-lysine
chloromethyl ketone was added to the reaction mixtures in a 1:1000
dilution following incubation to terminate enzymatic activity, and the
samples were then added to sample buffer ± 30 mM
DL-dithiothreitol (DTT). The
125I-[D-Tyr0]Bpa3SP
photoprobe itself is protected from tryptic cleavage under the
conditions used. Tryptic cleavage fragments were then separated and
analyzed using the Tricine gel system of SDS-PAGE (18). Prestained molecular mass standards (2.35-46 kDa; Amersham Pharmacia Biotech) were used to determine the molecular masses of the
radiolabeled tryptic fragments.
Endoglycosidase F Digestion of Photolabeled Tryptic
Fragments--
The radioactive bands of the limit tryptic
fragments were passively eluted from macerated dried Tricine gel slices
in extraction buffer (5 mM Tris and 1 mM EDTA
(pH 8.0)) for 1-4 days at room temperature. The eluted radiolabeled
limit tryptic fragments were then dried by speed vacuum; resuspended in
5 mM Tris, 1 mM EDTA, and 0.5%
n-octyl
-D-glucopyranoside (pH 8.0); and
digested with Flavobacterium meningosepticum endoglycosidase
F (Roche Molecular Biochemicals) overnight at 37 °C. Digestion
products were analyzed with either the Tricine gel system of SDS-PAGE
(18) or the NuPAGE 4-12% BisTris gradient gel system (Novex) using
MES running buffer. For preparative scale experiments, the radioactive
N-terminal limit tryptic fragment was electroeluted from
Tricine/SDS-polyacrylamide gels into extraction buffer as described
above and then adsorbed to wheat germ agglutinin-agarose beads
overnight at 4 °C. The wheat germ agglutinin-agarose beads were then
washed twice in 1% SDS, 5 mM Tris, and 1 mM
EDTA (pH 8.0) and pelleted by centrifugation. The supernatants were
transferred to Centricon-10 microconcentrators (Amicon, Inc.) and
centrifuged at 5000 × g for 1 h. The concentrates were washed in extraction buffer and reconcentrated twice before being
dried by speed vacuum. Dried radiolabeled pellets were then resuspended
in 5 mM Tris, 1 mM EDTA, and 0.5%
n-octyl
-D-glucopyranoside (pH 8.0) and
digested with endoglycosidase F overnight at 37 °C as described above.
Endoproteinase Glu-C (Protease V8) Subcleavage of Photolabeled
Tryptic Fragments--
The radioactive bands of the limit tryptic
fragments were passively eluted from macerated dried Tricine gel slices
as described above and then subjected to subcleavage at a final
concentration of 1 mg/ml endoproteinase Glu-C (protease V8,
Worthington) overnight at 37 °C. Note that in the TE buffer (5 mM Tris and 1 mM EDTA (pH 8.0)) used for these
subcleavage experiments, protease V8 hydrolyzes peptide and ester bonds
specifically at the carboxylic side of both Glu and Asp.
Subcleavage products were analyzed with either the Tricine gel system
of SDS-PAGE (18) or the NuPAGE 4-12% BisTris gradient gel system
using MES running buffer.
Disulfide Bond Reduction in Photolabeled Tryptic
Fragments--
The radioactive bands of the limit tryptic fragments
were passively eluted from macerated dried Tricine gel slices as
described above and then incubated with 30 mM DTT for
1 h at room temperature. The DTT-treated labeled fragments were
analyzed using the Tricine gel system of SDS-PAGE (18).
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RESULTS |
[D-Tyr0]Bpa3SP Specifically
Binds and Photolabels the rNK-1R--
The photoreactive
[D-Tyr0]Bpa3SP ligand
specifically binds to the rNK-1R with high affinity and has an
IC50 of 2.2 ± 0.6 nM, a value that is not
markedly different from that of the parent peptide (0.8 ± 0.3 nM) (Fig. 1). Moreover,
125I-[D-Tyr0]Bpa3SP
specifically photolabels the rNK-1R, as shown by the ability of 1 µM unlabeled SP or RP 67580 (a specific rNK-1R
non-peptide antagonist (19)) to prevent photoincorporation (Fig.
2). The photolabeled rNK-1R migrates as a
diffuse band at ~80 kDa on SDS-polyacrylamide gel due to receptor
glycosylation (16). After treatment with endoglycosidase F (an enzyme
that cleaves asparagine-linked carbohydrates), the photolabeled
receptor is reduced in size to 46 kDa (12, 20), a molecular mass
consistent with that calculated from the rNK-1R cDNA sequence
(21-23).

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Fig. 1.
[D-Tyr0]Bpa3SP displacement
of 125I-BH-SP/rNK-1R binding. The IC50
values for the displacement of 125I-BH-SP binding to the
rNK-1R for SP and [D-Tyr0]Bpa3SP
are 0.83 ± 0.26 and 2.17 ± 0.56 nM,
respectively. The data are shown as a percentage of the specific
control binding determined in the absence of competing ligands and are
representative of three to six similar independent experiments
conducted in triplicate.
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Fig. 2.
Specific photolabeling of the rNK-1R with
125I-[D-Tyr0]Bpa3SP.
Stably transfected CHO cells were photolabeled with
125I-[D-Tyr0]Bpa3SP
as described under "Experimental Procedures." CHO cells expressing
the rNK-1R were equilibrated with 1-2 nM
125I-[D-Tyr0]Bpa3SP
in the dark at 4 °C for 2 h either alone ( ) or in the
presence of 1 µM SP or RP 67580. Membrane preparations
were made and then subjected to SDS-PAGE.
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Tryptic Digestion of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R--
The tryptic digestion patterns of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R generated with increasing amounts of trypsin (0-0.2 mg/ml) and
analyzed in both the absence and presence of 30 mM DTT are shown (Fig. 3, a and
b, respectively). The largest fragment generated by
treatment of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R with 0.2 mg/ml trypsin (in both the presence and absence of DTT)
migrates as a diffuse band at ~40 kDa, suggesting the presence of
carbohydrate side chain residues. The smallest fragment generated by
treatment of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R with 0.2 mg/ml trypsin (in the presence and absence of DTT)
migrates at 5.1 kDa. The intermediate tryptic fragments generated by
treatment of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R with 0.2 mg/ml trypsin in the absence of DTT are extensions of
the smallest fragment and were reduced in size to 6.7 kDa following the
addition of DTT. This confirms the presence of a disulfide bond in
these extended fragments of the rNK-1R, which has previously been shown
to exist between Cys105 and Cys180, linking the
first and second extracellular domains (15).

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Fig. 3.
Tryptic digestion of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R. a and b, tryptic digestion patterns
of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R generated with increasing amounts of trypsin (0-0.2 mg/ml)
under nonreducing conditions (without 30 mM DTT) and
reducing conditions (with 30 mM DTT), respectively. As
described under "Experimental Procedures,"
125I-[D-Tyr0]Bpa3SP-labeled
membranes were resuspended in 0.1% SDS, 50 mM Tris, and 1 mM CaCl2 (pH 8.0) and digested at room
temperature for 2 h with the amount of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
bovine trypsin specified.
N -p-tosyl-L-lysine
chloromethyl ketone was added to the reaction mixtures in a 1:1000
dilution following incubation to terminate enzymatic activity, and the
samples were then added to sample buffer ± 30 mM DTT.
Tryptic cleavage fragments were separated and analyzed using the
Tricine gel system of SDS-PAGE.
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Peptide mapping analysis using the theoretical tryptic fragmentation
restriction map of the rNK-1R suggests that the ~40-kDa tryptic
fragment observed under both nonreducing and reducing conditions
corresponds to the
125I-[D-Tyr0]Bpa3SP
photoligand (calculated molecular mass of 1.8 kDa) covalently attached
to an amino acid within the segment of the rNK-1R extending from
residues 1 to 61 (calculated molecular mass of 6.8 kDa + ~30-kDa
carbohydrate residues). This region of the rNK-1R (designated the
N-terminal tryptic fragment; see Fig. 5) includes the extracellular N
terminus (containing both consensus sequences for N-linked
receptor glycosylation: Asn14 and Asn18), the
first transmembrane region, and the proximal portion of the first
intracellular loop. This ~40-kDa tryptic fragment was not reduced
further in size by digestion with higher concentrations of trypsin,
indicating that it represents a limit tryptic fragment of the
photolabeled rNK-1R.
We can also use the theoretical tryptic fragmentation restriction map
of the rNK-1R to conclude that the 5.1-kDa tryptic fragment, when
analyzed under both nonreducing and reducing conditions, corresponds to
the
125I-[D-Tyr0]Bpa3SP
photoligand (calculated molecular mass of 1.8 kDa) covalently attached
to an amino acid within the segment of the rNK-1R extending from
residues 149 to 177 (calculated molecular mass of 3.3 kDa). This region
of the rNK-1R (designated the core tryptic fragment; see Fig. 5)
includes the fourth transmembrane region and the proximal portion of
EC2. This 5.1-kDa fragment was also not further reduced in size by
digestion with higher concentrations of trypsin, indicating that it
represents a second limit tryptic fragment of the photolabeled rNK-1R.
The intermediate tryptic fragments observed in Fig. 3a were
reduced in size to 6.7 kDa upon addition of DTT, confirming the presence of a disulfide bond. Remarkably, this tryptic digestion pattern closely resembles the tryptic digestion pattern observed with
the 125I-BH-Bpa4SP-labeled rNK-1R (10).
Work in our laboratory has shown that when the rNK-1R is
photolabeled with 125I-BH-Bpa4SP, the
identified 6.7-kDa tryptic fragment corresponds to the 125I-BH-Bpa4SP photoligand (calculated
molecular mass of 1.8 kDa) covalently attached to an amino acid within
the segment of the rNK-1R extending from residues 149 to 190 (calculated molecular mass of 4.9 kDa) (10). This segment of the rNK-1R
contains EC2 residue Cys180, which forms a disulfide bond
with EC1 residue Cys105, linking the first and second
extracellular domains of the receptor (15). Our laboratory subsequently
reported that the direct site of covalent attachment of
125I-BH-Bpa4SP to the rNK-1R is to EC2 residue
Met174 (10). The inability of trypsin to fully cleave the
125I-BH-Bpa4SP-labeled (and in this case, the
125I-[D-Tyr0]Bpa3SP-labeled)
rNK-1R at the rNK-1R Arg177-Val178 cleavage
site might suggest that (i) the photoligand in some manner limits the
enzyme from accessing this particular site, or (ii) the spatial
constraint of the disulfide bond linking the first and second
extracellular domains of the receptor partially protects this receptor
region from the active site of the enzyme by positioning it near the
plasma membrane.
Unlike previous results observed with the peptide mapping
of both 125I-BH-Bpa4SP-labeled (10) and
125I-BH-Bpa8SP-labeled (9) rNK-1Rs, peptide
mapping of the
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R revealed two limit tryptic fragments, suggesting that
125I-[D-Tyr0]Bpa3SP
covalently attaches to two distinct residues far apart from one another
within the rNK-1R primary sequence: one site of covalent attachment
being to a receptor amino acid between residues 1 and 61 on the
extracellular N terminus and the other site of covalent attachment
being to a receptor amino acid between residues 149 and 177 on EC2.
Subcleavage Mapping of the
125I-[D-Tyr0]Bpa3SP/rNK-1R
Contact Points--
As expected, treatment of the 5.1-kDa core tryptic
fragment with additional reducing agents did not alter the size of the fragment due to the lack of the cysteine-cysteine disulfide bond (Fig.
4a, lane 3).
However, digestion of the 5.1-kDa core tryptic fragment with protease
V8 resulted in the generation of a 2.4-kDa fragment (Fig.
4a, lane 5). This protease V8-generated fragment corresponds to the
125I-[D-Tyr0]Bpa3SP
photoligand (calculated molecular mass of 1.8 kDa) covalently attached
to an amino acid within the segment of the rNK-1R extending from
residues 173 to 177 (calculated molecular mass of 0.6 kDa) (see Fig.
5). Therefore, these results show that one site of covalent attachment
of
125I-[D-Tyr0]Bpa3SP
to the rNK-1R is to an amino acid located on the proximal portion of
EC2 between residues 173 and 177.

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Fig. 4.
Subcleavage mapping of the
125I-[D-Tyr0]Bpa3SP/rNK-1R
contact points. a, subcleavage mapping of the core
tryptic fragment. The radioactive band of the core tryptic fragment was
passively eluted from Tricine/SDS-polyacrylamide gel slices as
described under "Experimental Procedures." Fragments were then
either incubated with 30 mM DTT or subjected to subcleavage
at a final concentration of 1 mg/ml endoproteinase Glu-C (protease V8)
in TE buffer (which permits protease V8 hydrolysis at the carboxylic
side of both Glu and Asp). Fragments were separated and analyzed using
the Tricine gel system of SDS-PAGE. Lanes 1 and
4, core tryptic fragment (5.1 kDa); lanes 2 and
6,
125I-[D-Tyr0]Bpa3SP
photoligand (1.8 kDa); lane 3, core tryptic fragment + DTT
(5.1 kDa); lane 5, core tryptic fragment + protease V8 (2.4 kDa). b, subcleavage mapping of the N-terminal tryptic
fragment. The radioactive band of the N-terminal tryptic fragment was
passively eluted from Tricine/SDS-polyacrylamide gel slices as
described under "Experimental Procedures." Fragments were then
digested with F. meningosepticum endoglycosidase F and
subcleaved at a final concentration of 1 mg/ml endoproteinase Glu-C
(protease V8) in TE buffer. The order of enzyme addition did not affect
the results. Fragments were separated and analyzed using the Tricine
gel system of SDS-PAGE. Lanes 7 and 10,
N-terminal tryptic fragment (~40 kDa); lane 8,
N-terminal tryptic fragment + endoglycosidase F (8.6 kDa);
lane 9, N-terminal tryptic fragment + endoglycosidase F + protease V8 (3.0 kDa); lane 11, N-terminal tryptic fragment + protease V8 (~33 kDa); lane 12, N-terminal tryptic
fragment + protease V8 + endoglycosidase F (3.0 kDa).
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Treatment of the ~40-kDa N-terminal tryptic fragment with
endoglycosidase F resulted in the generation of an 8.6-kDa fragment (Fig. 4b, lane 8). This result shows that the
N-terminal tryptic fragment contains ~30 kDa of carbohydrate residues
and must therefore also contain the two asparagine residues on the
extracellular N terminus capable of being glycosylated
(Asn14 and Asn18). The deglycosylated 8.6-kDa
fragment itself corresponds to the 125I-[D-Tyr0]Bpa3SP
photoligand (calculated molecular mass of 1.8 kDa) covalently attached
to an amino acid within the segment of the rNK-1R extending from
residues 1 to 61 (calculated molecular mass of 6.8 kDa). Subsequent
digestion of the 8.6-kDa fragment with protease V8 resulted in the
generation of a 3.0-kDa fragment (Fig. 4b, lane 9). This protease V8-generated fragment corresponds to the
125I-[D-Tyr0]Bpa3SP
photoligand (calculated molecular mass of 1.8 kDa) covalently attached
to an amino acid within the segment of the rNK-1R extending from
residues 11 to 21 (calculated molecular mass of 1.2 kDa) (Fig.
5). Therefore, these results show that a
secondary site of covalent attachment of
125I-[D-Tyr0]Bpa3SP
to the rNK-1R is to an amino acid located on the extracellular N
terminus between residues 11 and 21; interestingly, this segment of the
rNK-1R contains both consensus sequences for N-linked
glycosylation, the importance of which is still undetermined.
Importantly, the same 3.0-kDa protease V8-generated
125I-[D-Tyr0]Bpa3SP-labeled
rNK-1R fragment was generated when the N-terminal tryptic fragment was
analyzed in the converse manner, i.e. when the glycosylated N-terminal tryptic fragment was first subjected to protease V8 digestion and then to treatment with endoglycosidase F (Fig.
4b, lanes 11 and 12).

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Fig. 5.
Summary of rNK-1R regions containing sites of
125I-[D-Tyr0]Bpa3SP
photoincorporation. The N-terminal tryptic fragment and the core
tryptic fragment are shown in gray. Sites of cleavage by
endoproteinase Glu-C (protease V8) are shown by the solid
bars. Underlined receptor residues
(Leu11-Glu21 and
Thr173-Arg177) are the regions of the rNK-1R
containing a site of
125I-[D-Tyr0]Bpa3SP
covalent attachment. Note that the extended form of the core tryptic
fragment extends from Val149 to Arg190 and
contains Cys180, which links this fragment to EC1
(E1). aa, amino acids; C1 and
C2, first and second intracellular loops,
respectively.
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 |
DISCUSSION |
[D-Tyr0]Bpa3SP is, in our
studies to date, the only photoreactive analogue of SP that covalently
labels residues on two distinct domains of the rNK-1R. The sites of
[D-Tyr0]Bpa3SP covalent
attachment to the rNK-1R are located at a considerable distance from
each other in the primary amino acid sequence of the receptor molecule:
one is located within the proximal portion of EC2, and the other is
located within the extracellular N terminus. An interpretation of the
results presented in this report is that two distinct ligand·receptor
complexes exist at equilibrium: one complex in which an amino acid of
the receptor between residues 173 and 177 on EC2 is in close spatial
proximity to the Bpa3 residue of
125I-[D-Tyr0]Bpa3SP
and a second complex in which an amino acid of the receptor between
residues 11 and 21 on the extracellular N terminus is in close spatial
proximity to the Bpa3 residue of
125I-[D-Tyr0]Bpa3SP.
However, since the results of our competition binding studies comparing
the binding properties of Bpa3SP with those of SP for the
rNK-1R do not suggest the existence of multiple peptide·rNK-1R
complexes, a more likely interpretation of the photolabeling data
showing the ability of
125I-[D-Tyr0]Bpa3SP
to photolabel the aforementioned receptor segments is that the
glycosylated segment of the extracellular N-terminal domain of the
rNK-1R is in close spatial proximity to the experimentally determined
proximal region of EC2 in the high affinity SP·NK-1R equilibrium complex.
Previously, the same photoligand
([D-Tyr0]Bpa3SP) has been used to
map the peptide-binding domains of the NK-1R present in the macrophage/monocyte cell line P388D1 (11). The conclusion
of this study was that the site of
125I-[D-Tyr0]Bpa3SP
covalent attachment to the NK-1R was to an amino acid of the NK-1R
within the extracellular N-terminal segment extending from residues 1 to 21. In this report, we have confirmed these previous results
documenting
125I-[D-Tyr0]Bpa3SP
photolabeling of the N terminus of the NK-1R and have restricted the
site of covalent attachment to an amino acid between residues 11 and
21, a segment of the N terminus that contains the two consensus sequences for N-linked glycosylation.
In the previous study using the
125I-[D-Tyr0]Bpa3SP
photoligand (11), however, no photolabeling of EC2 was reported. The
difference in the results obtained is probably due to the differences
in the methods used for analysis of the photolabeled receptor
fragments. We analyzed the
125I-[D-Tyr0]Bpa3SP-photolabeled
NK-1R tryptic fragments and subcleavage products by an SDS-PAGE system
modified for small peptide analysis (17, 18) and autoradiography. In
contrast, Li et al. (11) used HPLC analysis following
receptor trypsinization. In our experience, hydrophobic tryptic
fragments, such as those derived from EC2, are resistant to elution by
standard HPLC procedures.
Thus, our data add new information that contributes experimentally
determined spatial constraints that are important for the three-dimensional modeling of the SP·NK-1R complex (see accompanying article (27)). Whereas large glycoprotein hormones (e.g.
thyrotropin and follicle-stimulating hormone) bind to the extended
extracellular N-terminal region of their G protein-coupled receptors
(24, 25), small neurotransmitters (e.g. acetylcholine and
norepinephrine) bind to multiple transmembrane-spanning sequences of
their G protein-coupled receptors (25, 26). The mechanisms by which
small flexible peptides such as SP interact with their membrane-bound G
protein-coupled receptors is still unknown. The insights we are
obtaining on the multiple contacts established between SP and the NK-1R
should prove useful for the study of the structure-function
relationships of other peptides of similar size with their G
protein-coupled receptors.