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(Received for publication, September 9, 1994) From the
The tachykinin substance P (SP) is a peptide transmitter of
primary afferents. Its actions on both central and peripheral targets
are mediated by a G-protein-coupled receptor of known primary
structure. To identify contact sites between the undecapeptide SP and
its receptor, we prepared radiolabeled photoreactive analogs of SP
(H-RPKPQQFFGLM-NH A large majority of the known receptors belong to the
G-protein-coupled receptor superfamily (Baldwin, 1994). These receptors
are characterized by the presence of seven hydrophobic regions of
primary structure thought to represent transmembrane domains. The
receptors lie in the bilayer such that the amino-terminal region of the
protein is extracellular and the carboxyl-terminal region is
cytoplasmic. The agonists which bind to and activate G-protein-coupled
receptors vary widely in size, from glycoprotein hormones (>30 kDa)
to single photons. The larger agonists (>10 kDa; e.g. thyroid stimulating hormone and follicle stimulating hormone) bind
to the amino-terminal region of their G-protein-coupled receptors,
while the smaller agonists (<0.2 kDa; e.g. norepinephrine,
serotonin, and photons) bind within the plane of the bilayer between
the seven transmembrane domains (Bockaert, 1991). Essentially all
characterized receptors for bioactive peptides (0.5-5 kDa) are
also members of the G-protein-coupled receptor superfamily, but which
regions of their receptors interact with these agonists of intermediate
size has not yet been defined. The undecapeptide substance P (SP) ( Chimeric and point-mutated SP receptors have been
constructed to probe receptor structure-function in an attempt to
identify binding domains for peptide agonists and nonpeptide
antagonists as well as domains associated with agonist-stimulated
second messenger responses (e.g. Cascieri et al., 1994; Fong et al., 1992a, 1992b, 1993, 1994a 1994b;
Gether et al., 1993a, 1993b, 1993c, 1994; Huang et al., 1994a, 1994b; Jensen et al., 1994; Sachais et al., 1993; Yokota et al., 1992; Zoffmann et al., 1993). These studies have indicated that both the extracellular
and transmembrane domains of the SP receptor are important for the
binding of agonist, and several specific residues conserved in all
species examined have been identified as important for peptide binding.
Analysis of SP analogs further suggested the COOH-terminal carboxyamide
of SP may interact with residues in the second transmembrane domain
(Huang et al., 1994b). However, the identification of a
particular residue as necessary for agonist binding does not
necessarily imply direct interaction of that side chain with agonist,
as loss of function may instead result from changes in protein folding.
Since the binding of SP (1350 Da) must involve a larger number of
receptor/ligand contacts than small nonpeptide agonists (e.g. norepinephrine, 170 Da), it has not been possible to define the
interaction of SP and its receptor by mutagenesis alone. Photoaffinity labeling has been proven to be a useful tool in
identifying structural domains of receptors involved in ligand binding (e.g. Dohlman et al., 1991). This technique offers a
unique approach by directly identifying the contact regions of a
receptor and its ligands. As an essential complement to mutagenesis
approaches, we have applied photoaffinity labeling to identify agonist
peptide binding domains of the SP receptor. p-Benzoyl-L-phenylalanine (BPA), a photoreactive
amino acid, has been used to replace amino acids in peptides for
receptor photoaffinity labeling (Dorman and Prestwich, 1994).
Photoactivated (triplet biradical) BPA reacts preferentially with C-H
bonds but has low reactivity toward water; furthermore, the chromophore
can be activated in the visible, avoiding protein-damaging UV
wavelengths. In previous work by others (Boyd et al., 1991a,
1991b, 1994; Kage et al., 1993), an SP derivative containing
BPA at position 8 and acylated with 3-(3-iodo-4-hydroxyphenyl)
propionic acid at the side chain of Lys P388D
The
isolated L-BPA
Fluorescence measurements were made using a Nikon microscope
optically linked to a PTI Deltascan instrument (Photon Technologies)
that produces dual excitation at 340 and 380 nm. Emitted light was
collected after passing though a 510-nm band pass filter. A 40X Nikon
fluor objective was used and the field was limited to about 15-20
cells for data collection.
After photolysis, the cells were washed twice (5
ml) with phosphate-buffered saline and transferred to a microcentrifuge
tube to collect cell pellets by centrifugation at 16,000
Figure 1:
Primary
structures of SP, BPA
Figure 2:
SP, BPA
Figure 3:
Autoradiography of P388D
Figure 4:
Autoradiography of Endo F deglycosylated
photolabeled SP receptor following SDS-PAGE (12% gel).
Acetone-precipitated
Figure 5:
Autoradiography of V8 protease digest of
photolabeled SP receptor following SDS-PAGE. Acetone precipitated
[
Peptide fragments from V8 digestion of
[
Figure 6:
Analysis of the BPA
BPA V8 digests of SPR
photolabeled with [ V8 protease-digested
fragments of [
Figure 7:
Analysis of the BPA
Replacement of amino acid residues at the third
(Lys Upon near UV irradiation (>310 nm), This intact cell-photolabeling technique demonstrates that the
SPR expressed in this natural (i.e. nontransfected) cell line
is highly glycosylated. Reports of photolabeling of SPR prepared from
various other sources suggests a heterogeneity of molecular size. Dam et al.(1987), using a photoreactive SP analog in which
Phe Limited V8 digestion of
[ Consistent with the size prediction, HPLC elution behavior confirms
that SPR 173-183 (1.3 kDa) is the photolabeled receptor fragment.
SPR 173-183 is a part of the relatively polar second
extracellular domain of the SPR (between transmembrane domain (TM) IV
and TMV), with a hydrophobic index (Engelman et al., 1986) of
-1.8. The addition of such a fragment to BPA Limited V8 digestion of
[ Photoaffinity
labeling identifies receptor domains in close proximity to the bound
photoligand. Another approach to receptor-ligand interactions,
site-directed mutagenesis, identifies domains necessary for function,
but which are not necessarily proximal to the site of that function.
Chimeras of the substance P receptor with other tachykinin receptors (e.g. substance K receptor) demonstrated the agonist ligand
specificity of the tachykinin receptors is mainly determined by the
region around TMII TMIV and also partly by the extracellular
NH Combining the present results
with those of previous mutagenesis studies, a model of the agonist
peptide-binding site of the SPR can be constructed (Fig. 8). In
this model, the COOH-terminal hydrophobic sequence -GLM-NH
Figure 8:
Schematic model of the peptide agonist
binding site of the murine SP receptor. Black circles represent the contact regions of SPR with SP analogs (BPA
Studies of other G-protein-coupled receptors have
demonstrated that those which bind larger (>10 kDa) agonists have
agonist-binding sites within their NH
Volume 270,
Number 3,
Issue of January 20, 1995 pp. 1213-1220
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cells with Photolabile Agonists (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) by replacing amino acids in the peptide
with p-benzoyl-L-phenylalanine (BPA). SP,
BPA
-SP, and BPA
-SP bind with high affinity (K
)has been identified as a neurotransmitter associated with
pain modulation and neurogenic inflammation (Pernow, 1983; Otsuka and
Yoshioka, 1993). SP belongs to the tachykinin peptide family which is
characterized by a conserved COOH-terminal sequence
-FXGLM-NH, where X is an aromatic or
aliphatic amino acid (Maggio, 1988). The SP receptor (also known as the
neurokinin-1 or NK-1 receptor) has been cloned from several species
including human, mouse, rat, and guinea pig and displays a very high
degree of primary sequence homology across species (Gerard et al., 1993). The SP receptor (SPR) is a member of the G-protein-coupled
receptor superfamily, as are receptors for other peptides in the
tachykinin family. has been
synthesized to study the SP receptor. Photolysis of this ligand with
membrane-bound SP receptors from rat submaxillary gland led to about
70% incorporation of bound label into two polypeptides (46 and 53 kDa);
enzymatic studies suggested that the smaller protein resulted from
proteolysis of the larger (Kage et al., 1993). cells, a nontransfected murine macrophage/monocyte cell line
(Dawe and Potter, 1957), express a high density of functional SP
receptors (Persico et al., 1988; Li et al., 1994) but
no detectable levels of other tachykinin receptors. ()The SP
receptors of this cell line are coupled to Ca mobilization (Li et al., 1994). In the present study we
have used two site-specific, high affinity photolabile analogs of SP
(incorporating BPA in the third (BPA
) or eighth
(BPA) position) to label the SP receptor of P388D cells and map the peptide-binding domains of the receptor for
each ligand.
Preparation of
Fluoren-9-ylmethoxycarbonyl-4-benzoylphenylalanine
(Fmoc-BPA)
Racemic DL-BPA was synthesized and resolved
into L- and D-BPA as described by Kauer et
al.(1986). The resolved amino acid (or alternatively the racemic
mixture) was treated directly with Fmoc-chloroformate (Aldrich) or
Fmoc-hydroxysuccinimide (Sigma) to provide the protected amino acid
(Fmoc-BPA) for solid-phase synthesis.Peptide Synthesis
Peptide synthesis of the SP
analogs, BPA-SP and BPA-SP, was performed by
our departmental Biopolymers Facility or by Quality Control
Biochemicals (Hopkinton, MA) using a standard Fmoc solid-phase
synthetic strategy (Maggio et al., 1992). The crude synthetic
peptide was then purified by reverse-phase high performance liquid
chromatography (HPLC) using a C
column (Vydac 4.6 
250 mm, 5 µm, 300 Å) on a Waters Liquid Chromatographic
System equipped with a variable wavelength UV detector. The column was
eluted with a linear water-acetonitrile gradient (26-56%
acetonitrile, 1%/3 min; 1.0 ml/min) containing 10 mM trifluoroacetic acid. The racemic peptide had two major UV active
(254 nm) components of equal intensity. The L-BPA peptide was
identified by elution position; that is, L-BPA-SP
(synthesized from Fmoc-L-BPA) showed only one UV active peak
which was coincident with the earlier eluting HPLC peak of DL-BPA-SP (synthesized from Fmoc-DL-BPA).
Thus, the earlier HPLC peak corresponds to L-BPA-SP and the later peak to D-BPA-SP. In addition, Marfey's reagent
(1-fluoro-2,4-dinitrophenyl-5-L-alanineamide) formed an adduct
(Marfey, 1984) with isolated L-BPA that precedes that formed
from D-BPA. When the L-BPA-SP and D-BPA-SP fractions were tested for receptor
binding and biological activity, the earlier eluting peptide, i.e.L-BPA-SP, was much more active than the later
eluting D-BPA analog. By analogy to the greater
activity of L-Phe-SP relative to D-Phe-SP (Fournier et al., 1982), this
result further confirms that the first of the paired HPLC peaks is the L-BPA-isomer. The L-BPA diastereomer has also been
shown to precede the D-BPA diastereomer of other peptides in
reverse-phase HPLC elution (Shoelson et al., 1993).-SP was analyzed for purity and
correct structure by amino acid analysis, laser desorption mass
spectroscopy, and sequence. The peptide was sequentially Y R
P
K
P
Q
Q
F
(BPA) G
L
M
with m/z 1616.2
((M+H)
). Neither BPA or its phenylthiohydantoin
derivative elute from the analyzer column under standard conditions
(Kauer et al., 1986). Nevertheless, the high UV extinction
coefficient of BPA at 254 nm,
= 21
10,
verifies that BPA is incorporated (Kauer et al., 1986),
confirming the results of mass spectrometry. Tyrosine was partially
destroyed under the hydrolysis conditions employed.Preparation of Radioligands
The radioligand
[I]BPA
-SP
([I]iodotyrosyl
-L-BPA-SP)
was formed using general peptide iodination techniques previously
described (Too and Maggio, 1991). Typically 10 nmol of dry peptide was
dissolved in 50 µl of 0.5 M sodium phosphate buffer, pH
7.5, and vortexed with 1 mCi of NaI (10 µl, Amersham
Corp.). Chloramine-T (10 µg in 10 µl of water) was added to
activate iodine incorporation, and Na
SO
(100 µg in 10 µl of water) was added after 1 min of
vortexing to quench the reaction. The mixture was diluted and acidified
with 0.6 ml of 60 mM trifluoroacetic acid, and 25 µl of 2%
bovine serum albumin was added to limit nonspecific adsorption. To
separate the peptide from the unincorporated I, the
mixture was then applied to an activated C
Sep-Pak
cartridge (Waters) and the iodide and peptide eluted with a series of
0.5-ml portions of 10 mM trifluoroacetic acid solutions of
increasing alcohol (ethanol/methanol, 1:1) content, 10, 10, 20, 40, 60,
80, 90, 95, and 100%. Unincorporated iodide elutes immediately, while
the peptide is retained until the alcohol concentration reaches about
60%. The peptide fractions (containing both oxidized and reduced
methionine), eluting with 60-90% alcohol, were pooled and reduced
in volume under a nitrogen stream. After the addition of 20% (v/v)
-mercaptoethanol, the sample was heated at 90 °C for 2 h to
reduce methionine sulfoxide to its thioether. Further purification was
achieved by reversed-phase HPLC on a Vydac C column as
above. The eluate was collected in fractions during gradient elution
and the fractions counted for radioactivity. The reduced (Met) and
monoiodinated (Tyr) peptide eluted in a wellresolved peak (34.6%
acetonitrile), predictably later than the original compound (33.2%) or
the oxidized products, but prior to the diiodinated reduced peptide.
The reduced monoiodinated tracer (specific activity 2000
C
/mmol; 1C = 37 GBq) was protected from
oxidation by 0.5% -mercaptoethanol (v/v) added immediately after
purification and stored at -20 °C until use. Radioiodinated
BPA-SP was prepared similarly.Cell Culture
The murine cell line P388D (Dawe and Potter, 1957; Persico et al., 1988) was a gift
of Dr. J. Jackie (Harvard Medical School) and has been maintained in
our laboratory in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (FCS).Calcium Measurements
P388D cells were
cultured on 12-mm diameter round glass coverslips that had been
pretreated with laminin. These were used within 24-48 h after
plating. Dye loading was achieved by exposing the cells to fura-2
acetoxymethyl ester at a concentration of 8 µM for 30 min
at room temperature. The cells were then washed with 2% bovine serum
albumin and kept on ice until used. Experiments were performed using a
standard saline buffer with the following components: NaCl 120
mM, KCl 4.2 mM, CaCl 2.5 mM,
MgSO
1.0 mM, NaHPO 1.0
mM, glucose 12 mM, and HEPES 10 mM, pH 7.40. Ligand Binding of P388D
P388DCells cells (5 10 cells/well) were inoculated on FCS precoated 24-well plates and
cultured overnight. The confluent cells (1
10
cells/well) were washed twice (0.5 ml/well) with ice-cold buffer
(Dulbecco's modified Eagle's medium + 20 mM HEPES, pH 7.2) and incubated with 0.5 ml of buffer on ice for at
least 10 min. Then radioactive ligand, in the presence or absence of
unlabeled displacers, was added to a final concentration of 150 pM (5
10 cpm/ml) and incubated for 2 h.
Nonspecific binding is defined as binding in the presence of 10
µM unlabeled SP. After incubation, the cells were washed
twice with 0.5 ml/well phosphate-buffered saline (104 mM NaCl,
2.7 mM KCl, 10 mM NaHPO, 1.8
mM KHPO, pH 7.2), then solubilized by
0.5 ml of lysis buffer (1% Nonidet P-40, 0.2% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0) for 20 min and transferred for
gamma counting. Unlabeled SP and CP-96345 were stored as 10 mM stock solutions in dimethyl sulfoxide. Dimethyl sulfoxide at less
than 3% (v/v) had no detectable effects on the binding assay.Photoaffinity Labeling of P388D
Cells (5 Cells 10) were inoculated on
FCS-pretreated dishes (60 mm) and grown for at least 12 h before
labeling. The cultured cells (1
10
) were
washed twice (5 ml) with ice-cold buffer (Dulbecco's modified
Eagle's medium + 20 mM HEPES, pH 7.2) and incubated
with 5 ml of buffer on ice for at least 10 min. The photolabile
radioligand, in the presence or absence of unlabeled displacers, was
added to a final concentration of 2 nM (6
10 cpm/ml) and incubated 2 h. The dishes were then
irradiated on ice for 15 min using a focused HBO 100-watt mercury short
arc lamp through an optical filter to eliminate light below 310 nm. A
second filter removed infrared wavelengths to minimize sample heating
during photolysis. g for 10 min. The pelleted cells were resuspended in 0.3 ml of 5
mM Tris-HCl, pH 8.0, and hypotonically lysed for 30 min at
room temperature. Then the samples were homogenized and centrifuged at
500 g for 15 min to remove debris. The resulting
supernatants were sedimented at 16,000 g for 30 min
and the membrane pellets stored at -20 °C until analysis. The
presence or absence of a mixture of protease inhibitors (bacitracin,
chymostatin, and leupeptin) did not affect the results of binding or
photolysis experiments.Partial Purification of the Labeled
Complex
SDS-polyacrylamide gel electrophoresis (PAGE) was
performed as described by Laemmli(1970) using 1.5-mm 8% gels. The
labeled cell membranes were solubilized in 1 SDS sample buffer
(10% glycerol, 5% 2-mercaptoethanol, 3% SDS, 0.025% bromphenyl blue,
125 mM Tris-HCl, pH 6.8) for 30 min at room temperature. After
electrophoresis, the gel was directly dried on a filter paper and
exposed to x-ray film (Kodak XAR-5) with an intensifying screen
(DuPont). The labeled bands were isolated from the preparative gel
using a passive elution protocol similar to that described by Blanton
and Cohen(1994). After autoradiography, radioactive bands of the
labeled complex were excised from dried gels and rehydrated with
extraction buffer (0.1% SDS, 100 mM NHHCO, pH 7.8). The gel slices were
macerated and eluted for 1-4 days with extraction buffer. The
eluted protein was filtered (Whatman No. 1) and concentrated using
Centriprep-10 or Centricon-10 (Amicon). Finally, the labeled complex
was precipitated by cold acetone (85-90%, v/v) overnight at
-20 °C. The precipitate was dried and stored at -20
°C until use. For all electrophoretic gels, the ratio of
bis-acrylamide to acrylamide was 3%. For gels above 12% acrylamide, 1%
glycerol was added to the running buffer to prevent cracking of gels
during drying.Endoglycosidase F Digestion of the Partially Purified
Complex
The acetone precipitate was resuspended in 10 mM EDTA, 0.1% SDS, 0.5% N-octylglucoside, 100 mM NHHCO, pH 7.8, and then digested with Flavobacterium meningosepticum endoglycosidase F (Endo F)
(Boehringer Mannheim) for 2 days at room temperature.Protease Digestion of the Partial Purified
Complex
The acetone precipitate was resuspended in 0.1% SDS, 100
mM NHHCO, pH 7.8, and then digested
with Staphylococcus aureus V8 protease (V8) (Boehringer
Mannheim) for 2-4 days at room temperature or L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated
bovine trypsin (Sigma) for 1-4 days at room temperature. Both
BPA-SP and BPA-SP completely resist cleavage by
these proteases under these conditions (not shown) as both ligands lack
glutamic acids residues, and basic residues are protected by adjacent
prolines (Fig. 1).
-SP, and BPA-SP. Addition
of Tyr at the NH terminus facilitates radioiodination.
Lys and Phe are respectively replaced by BPA to
give BPA-SP and
BPA-SP.
HPLC of Enzymatic Digests
The V8-digested samples
were loaded into a Vydac C column (2.1 150 mm, 5
µm, 300 Å) and eluted with increasing solvent B (0.09%
trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol) in solvent A
(0.1% trifluoroacetic acid in water) at a flow rate of 0.25 ml/min. The
elution of I-labeled SPR fragments was monitored by gamma
counting of the fractions. More than 90% of injected radioactivity was
recovered in the eluate for all HPLC experiments.
5,5`-Dithio-bis(2-nitrobenzoic acid) (DTNB)
Modification
The HPLC fractions were dried and resuspended in
100 mM NHHCO, pH 7.8. Dithiothreitol
was added to a final concentration of 25 mM and the mixture
incubated for 30 min at 50 °C, then DTNB (final concentration 75
mM) was added and allowed to react with the peptide for at
least 30 min. The mixture was then diluted with 20% solvent B and
loaded onto reverse-phase-HPLC as described.
Specificity of BPA
Both BPA-SP and
BPASP-SP and BPA-SP (Fig. 1) are full and potent agonists relative to SP for the
calcium responses of P388D cells. This action is completely
blocked by CP-96345, a specific nonpeptide antagonist of the SP
receptor (Fig. 2). Both photoreactive ligands bound to SPR (Table 1) with the same affinity (IC values
3
nM) as SP and the binding of each was similarly inhibited by
CP-96345 (IC
values
35 nM).
-SP, and
BPA-SP induce calcium responses in P388D cells.
[Ca]
was
measured with fura-2 using 12-18 cells in an optical field.
Agonists (10 nM) were perfused over the cells for 30 s.
CP-96345 (1 µM) was preincubated with the cells for 30 s
before adding the mixture of agonist and CP-96345. Experiments were
carried out at room temperature (22
°C).
Photoaffinity Labeling of SP Receptor
After
photoinsertion of radioiodinated BPA-SP or
BPA-SP bound to P388D cells, two radioactive
bands were observed on SDS-PAGE. A major broad band of 75 kDa accounted
for about 95% of the radioactivity, while a minor band of 205 kDa
accounted for about 5% (Fig. 3). Labeling of both bands was
completely inhibited by SP (Fig. 3) or CP-96345 (not shown). For
BPA-SP, 46 ± 3% of bound ligand is recovered in the
broad 75 kDa band; for BPA-SP, the major band represents
about 6% of the total bound radioactivity. For both ligands,
deglycosylation with Endo F shifted the broad major band to a sharp
band of 42 kDa (Fig. 4), indistinguishable from the molecular
mass of the murine SPR calculated from its cDNA sequence (Sundelin et al., 1992). The major band labeled by both photoprobes was
partially purified by preparative SDS-PAGE and used for further
studies. Incubation of cells with the photoprobes in the dark resulted
in no detectable incorporation into protein.
cell
membranes photoaffinity labeled with
[I]BPA
-SP and
[I]BPA
-SP following SDS-PAGE (8%
gel). Cell culture, photolysis, cell membrane preparation, and
solubilization were as described under ``Experimental
Procedures.'' After electrophoresis, the gel was stained with 0.1%
Coomassie Blue in MeOH/AcOH/HO (4:1:5) and destained in the
same solvent. The same amount of protein was found in each lane (not
shown). Lanes 1 and 2,
[I]BPA
-SP-labeled membranes; lanes 3 and 4,
[I]BPA
-SP-labeled membranes. The
labeling was carried out in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 10
µM unlabeled SP. Molecular weights are indicated at the right.
I-labeled SPR (5000-8000 cpm)
from preparative SDS-PAGE was dissolved in 0.1% SDS, 10 mM EDTA, 0.5% N-octylglycoside, 100 mM NH
HCO, pH 7.8, and digested by Endo F (0.8
unit) for 2 days at room temperature. The gel was directly dried
without fixation or staining prior to autoradiography. Lanes 1 and 2,
[I]BPA
-SP-labeled SPR; lanes 3 and 4,
[I]BPA
-SP-labeled SPR. The samples
were treated with (lanes 1 and 3) or without (lanes 2 and 4) Endo F. Molecular weights indicated
are at the right.
V8 Digestion of the Labeled SP Receptor
V8
protease (Glu-C) cleaves proteins specifically at the COOH-terminal
side of glutamate residues under the conditions employed. V8 digestion
of partially purified
[I]BPA
-SP-labeled SPR showed five
detected bands (33, 25, 19, 9, and 3.2 kDa). The larger proteolytic
fragments were converted into the smaller ones at higher concentration
of V8 protease, with the smallest fragment, designated
BPA-SPR-3.2k, being the limit digest (Fig. 5A). Double digestion of
[I]BPA
-SP-labeled SPR with Endo F
and V8 protease revealed the same five-band pattern seen with V8
digestion alone.
I]labeled SPR (5000 8000 cpm) from preparative
SDS-PAGE was dissolved in 0.1% SDS, 100 mM NH
HCO, pH 7.8, and digested by the
indicated amount of V8 protease for 4 days at room temperature. The gel
was directly dried for autoradiography. A,
[I]BPA
-SP-labeled SPR (17.5% gel);
molecular weights are indicated at the left. B,
[I]BPA
-SP-labeled SPR (18% gel);
molecular weights are indicated at the right.
I]BPA
-SPlabeled SPR were isolated
by HPLC (Fig. 6A). One major peak (33% solvent B),
accounting for most of the eluted radioactivity, corresponded to
BPA
-SPR-3.2k (Fig. 6B).
BPA-SPR-3.2k and BPA-SP tracer eluted in a
similar position on reverse-phase HPLC but were cleanly resolved by
SDS-PAGE (Fig. 6B). A second peak of radioactivity
eluting from the HPLC column, accounting for most of the recovered
radioiodine not in BPA-SPR-3.2k, corresponded to
BPA-SPR-9k. Further digestion of BPA-SPR-9k
with V8 protease converted this fragment to BPA-SPR-3.2k
(not shown).
-SPR-3.2k
fragment. [I]BPA
-SP-labeled SPR was
digested by V8 protease as described in Fig. 5. The digests were
separated by reverse-phase HPLC as described under ``Experimental
Procedures'' (microbore C column, organic phase 60%
acetonitrile, 40% isopropanol). The solvent gradient (flow rate 0.25
ml/min) was as follows: 20-70% solvent B in 62.5 min;
70-100% solvent B in 15 min. Fractions (0.5 ml each) were counted
for I (A). The digests and HPLC fractions were
dried and analyzed by SDS-PAGE on an 18% gel (B). Lane
1, minor HPLC peak, fraction 44-48; lane 2, major
HPLC peak, fractions 27-29; lane 3, V8 digest prior to
HPLC fractionation; lane 4,
[
I]BPA
-SP; molecular weights are
indicated at the right. BPA-SPR-3.2k fractions were dried
and treated with or without DTNB as described, then separated by HPLC
using a solvent gradient of 20 50% solvent B in 37.5 min (C). Arrow indicates the elution position of
[I]BPA
-SP.
-SPR-3.2k, the limit digest, was reacted
with DTNB, a specific sulfhydryl modification reagent which converts
free peptidyl -SH groups to mixed disulfides of 2-nitro-5-thiobenzoic
acid. Treatment of BPA-SPR-3.2k with DTNB shifted the HPLC
elution position of the peptide to later elution by 3.2% solvent B (Fig. 6C). A parallel sample incubated identically but
without DTNB showed no change in elution position.I]BPA
-SP
displayed a different pattern of proteolytic fragments than digests of
SPR photolabeled with [I]BPA
-SP (Fig. 5). A major fragment of 40 kDa (BPA-SPR-40k)
was detected under conditions which reduced SPR photolabeled with
[I]BPA
-SP to peptides of less than
10 kDa. Very high concentrations of V8 produced, in addition to the
major band at 40 kDa, minor bands at about 3 and 10 kDa. Endo F
digestion of BPA-SPR-40k produced a 3.5-kDa fragment,
BPA-SPR-3.5k. The same fragment was produced by double
digestion with Endo F and V8 protease of SPR photolabeled with
[I]BPA
-SP.I]BPA
-SP-labeled SPR
were separated by HPLC. A major peak containing >70% of eluted
radioactivity passed through without being retarded by the
reverse-phase column. These fractions were dried, digested with Endo F,
and reanalyzed by HPLC. The Endo F-treated sample
(BPA-SPR-3.5k) was retained by the HPLC column and eluted
at 30% solvent B (Fig. 7). BPA-SPR-3.5k and
BPA-SP were separable by HPLC. A control sample
(identically treated in the absence of Endo F) still passed through the
HPLC column without retention.
-SPR-40K
fragment. [I]BPA
-SP-labeled SPR was
digested by V8 protease as described in Fig. 5. The digests were
separated by HPLC as described in Fig. 6A. Fractions
eluting at the void volume (unretained by the reverse-phase column)
were dried and treated with or without Endo F. The samples then
reanalyzed by HPLC (A) as described in Fig. 6C and by SDS-PAGE (B) on an 18% gel. Lane 1, HPLC
fractions 43-48 after Endo F; lane 2, HPLC fractions
9-19 without Endo F; molecular weights are indicated at the right. Arrow (A) indicates the elution
position of
[I]BPA
-SP.
Trypsin Digestion of the Labeled SP
Receptor
Double digestion of
[I]BPA
-SP-labeled SPR with Endo F
and trypsin revealed the same pattern of radioactive fragments as seen
with trypsin digestion alone. However, double digestion (with Endo F
and trypsin) of [I]BPA
-SP-labeled
SPR produced a different pattern of fragments than that seen with
trypsin treatment alone. As seen with Endo F and V8, digestion of the
major high molecular mass (>45 kDa) tryptic fragment of
[I]BPA
-SP-labeled SPR with Endo F
converted it to a much smaller fragment.
) or eighth (Phe) positions of SP by BPA
and addition of Tyr at the NH-terminal (Tyr)
gave analogs (Fig. 1) which triggered calcium responses of
P388D cells (Fig. 2) with the same potency as the
parent peptide. The calcium response was inhibited by CP-96345, a
specific SPR antagonist. Binding of I-labeled
BPA
-SP and BPA-SP to P388D cells
was blocked by cold SP and CP-96345 at nM concentrations (Table 1). The two photolabile ligands thus are high affinity
full agonists of the SPR of P388D cells. Previous studies
showed that Phe of SP could be structurally modified
without a marked decease in activity on affinity on several bioassays
(Lee et al., 1983; Maggio, 1988; Viger et al., 1983).
Boyd et al. (1991a, 1991b) have demonstrated that replacement
of Phe of SP by BPA is well tolerated in binding affinity
and biological activity. The results of the present study illustrate
that substitution of Lys of SP with BPA also maintains
biological activity and binding affinity at the SPR of murine
P388D cells. To facilitate radioiodination, we added a
tyrosine at the NH terminus of the peptide; the Tyr peptides also retain full biological activity and binding
affinity (Sachais et al., 1993; Cascieri et al.,
1994).I-labeled BPA
-SP and BPA-SP are
photoincorporated into a major broad radiolabeled band of 75 kDa
in P388D
cells (Fig. 3). The broad range of
molecular mass reflects heterogeneous glycosylation, as Endo F
treatment dramatically converted the broad 75 kDa band to a sharp one
of 42 kDa (Fig. 4). This size is consistent with the value
deduced from the cDNA sequence for the mouse SPR (Sundelin et al., 1992). The sensitivity of photolabeling with these ligands to SP
and CP-96345 (Table 1, Fig. 3) further indicated that
BPA
-SP and BPA-SP were cross-linked with the
SPR. was replaced by p-azidophenylalanine,
demonstrated specific photolabeling of a single polypeptide, 46
kDa, in a rat brain membrane preparation. Boyd et al.(1994)
reported that the molecular mass of SPR in rat tissues labeled with
I-3-(3-iodophenyl-4-hydroxylphenyl)propionyl-
-Lys
-BPA-SP
varied from 53 and 46 kDa for submaxillary or parotid gland to 72 kDa
for large intestine and 90 kDa for striatum or olfactory bulb.
Deglycosylation of each of these photolabeled receptors from different
tissues yielded a discrete radiolabeled band of 46 kDa, while in
salivary gland an additional band at
36 kDa was also observed.
I]BPA
-SP-labeled SPR implies that
the smallest labeled complex, BPA-SPR-3.2k, represents the
interaction site of the ligand and receptor, as all other fragments are
converted to the 3.2-kDa fragment at high concentrations of protease.
Since BPA-SP has a molecular mass of 1.7 kDa, a SPR V8
fragment peptide with molecular mass of
1.5 kDa is involved in the
BPA
-SPR-3.2k complex. DTNB reaction indicates that this
peptide contains a cysteine residue. The V8 digestion map of the mouse
SPR deduced from its cDNA sequence shows only four cysteine-containing
peptides, with values of 10.7 kDa (SPR 79-172, 2 Cys), 1.3 kDa
(SPR 173-183, 1 Cys), 3.8 kDa (SPR 194-227), 1 Cys), and
11.1 kDa (SPR 239-312, 6 Cys). The two larger fragments (M
> 10 kDa) are excluded based on their
molecular masses. Of the remaining two, the smaller (1.3 kDa) is
clearly a much better candidate than the larger (3.8 kDa) for the
1.5-kDa fragment deduced from the V8 digestion studies.
-SP
(hydrophobic index -15.1) would have little effect on HPLC
elution position, as is observed experimentally (Fig. 6). Both
the tracer alone, and its photoadduct with the receptor fragment, elute
as sharp peaks at the same (33% solvent B) position in the solvent
gradient. In sharp contrast, SPR 194-227 (
3.8 kDa) is
comprised mainly of the very hydrophobic TMV region of the receptor and
has a hydrophobic index of 35. Hydrophobic peptides elute from
reverse-phase HPLC columns as relatively broad peaks at very high
solvent concentrations. For example, a photolabeled transmembrane
domain (
-M4, residues 401-428, hydrophobic index =
36.9) from the Torpedo nicotinic acetylcholine receptor, with
very similar molecular weight and amino acid composition to SPR
194-227, elutes in a very broad peak at about 74% solvent B in
the same HPLC system (Blanton and Cohen, 1994). Such behavior is
inconsistent with the reverse-phase-HPLC elution of
BPA-SPR-3.2k (Fig. 6). Thus, molecular weight, HPLC
elution behavior, and presence of cysteine, taken together, establish
that the SPR region photolabeled by BPA-SP is SPR
173-183 of the second extracellular loop, whose primary sequence
is TMPSRVVCMIE. Using a different SP tracer containing BPA at position
8, Boyd et al.(1993) also found labeling of the second
extracellular loop of rat SPR in transfected hamster cells, a finding
consistent with the present results. Because the radiolabel in these
probes is located at a site distinct from the photoreactive amino acid,
radiochemical sequencing cannot be used to define the specific amino
acid of the SPR labeled by BPA.I]BPA
-SP-labeled SPR suggested
that the glycosylated peptide complex, BPA-SPR-40k,
represents the interaction site of the ligand and receptor because Endo
F plus V8 mixed digestion shifted the 40-kDa complex to a much smaller
fragment, BPA-SPR-3.5k. This is further confirmed by HPLC
analysis. BPA-SPR-40k passed though the reverse-phase
column in the void volume, behavior common to very polar biopolymers
such as carbohydrates. Deglycosylation of BPA-SPR-40k
converted the complex to a smaller peptide (3.5 kDa) which was
retained by the reverse-phase column and eluted by the solvent gradient
at about 30% solvent B. There are two potential sites
(N-X-S/T) for N-linked glycosylation in the SP
receptor. Both are located in the NH
-terminal extracellular
tail of the receptor, based both on primary sequence (Sundelin et
al., 1992) and experimental results (Boyd et al., 1991b,
1994). The V8 digestion map of the murine SPR indicated that
NH-terminal peptide (SPR 1-21, 2.3 kDa) contains
two N-linked glycosylation sites, while all other fragments
have none. The hydrophobicity index of this (deglycosylated) peptide is
-27.3, which predicts that the deglycosylated receptor fragment
cross-linked with BPA
-SP would elute earlier than the free
photoprobe. Consistent with this prediction, the complex does elute
earlier in the solvent gradient than
[I]BPA
-SP. Thus, molecular size of
the complex, the presence of carbohydrate, and HPLC elution behavior,
taken together, establish that the NH-terminal
extracellular tail of the receptor (SPR 1-21, whose primary
sequence is MDNVLPVDSDLFPNTSTNTSE) is the insertion site of
[I]BPA
-SP.-terminal domain of the receptors (Yoshifumi et al., 1992). Fong et al. (1992b) found that extracellular
domains of SPR (also known as NK-1R), including a segment of
NH-terminal tail and the first extracellular loop were
essential for high affinity binding of agonist peptides. Furthermore,
they identified several residues in the NH-terminal domain
(Asn, Gln
, and Phe
), first
extracellular (also known as E2) loop (Asn
,
His
), and part of second extracellular (also known as E3)
loop (Ser
-Glu
) which are required for high
affinity binding of peptides (Fong et al., 1992a). Other
mutagenesis studies demonstrated that residues in TMII
(Asn
, Asn
, Tyr
) and TMVII
(Tyr
) are also required for high affinity binding of
peptide agonists (Huang et al., 1994b). Analysis of SP analogs
further suggested the COOH-terminal carboxyamide of SP may interact
with Asn
in the second transmembrane domain (Huang et
al., 1994b). Taken together, these data demonstrate that both the
extracellular and transmembrane domains of SPR are important for the
peptide binding. The present studies identify the interaction sites of
the third (BPA
) and eighth (BPA) positions of
SP as (i.e. BPA and BPA contact and
photolabel) the NH-terminal extracellular tail (SPR
1-21) and the second extracellular loop (SPR 173-183) of
the receptor, respectively. The results of the present photolabeling
experiments and those of previous mutagenesis experiments are distinct,
in that the different regions of the SPR are identified, but not
inconsistent. The photolabeling results do not match the predictions of
a graphics-computer-generated model (Trumpp-Kallmeyer et al.,
1994) of SP bound to its receptor. of SP inserts into a hydrophobic ligand binding pocket between
the transmembrane domains and between the extracellular surface and
center of the bilayer. This binding pocket is formed by TMII and TMVII
with contributions from other transmembrane domains. The carboxyamide
penetrates to the level of and interacts with Asn (Huang et al., 1994b). Other than this COOH-terminal tail, the
remainder of the SP molecule interacts with amino acids on the
extracellular face on the receptor. Specifically, position 8 of SP
interacts with the second extracellular loop (SPR 173-183) and
position 3 of SP with the NH
-terminal extracellular tail
(SPR 1-21). These regions of the SPR are highly conserved across
species; 10 of 11 amino acids of SPR 173-183 and 18 of 21 amino
acids of SPR 1-21 are invariant across the four mammalian species
whose SPR cDNA sequences have been reported (Gerard et al.,
1993). The binding site for specific, high affinity nonpeptide
antagonists of the SPR is at a distinct location (Cascieri et al., 1994; Fong et al., 1992a, 1992b, 1993, 1994a, 1994b;
Gether et al., 1993a, 1993b, 1993c, 1994; Huang et al., 1994a; Jensen et al., 1994; Sachais et al., 1993; Yokota et al., 1992; Zoffmann et al., 1993).
and BPA). The third and the eighth positions of SP,
respectively, interact with the NH-terminal extracellular
tail (SPR 1-21, MDNVLPVDSDLFPNTSTNTSE) and second extracellular
loop (SPR 173-183, TMPSRVVCMIE) of the SP receptor. Shaded
circles indicate residues essential for high affinity binding of
SP as identified by site-directed mutagenesis. A, view in the
plane of the bilayer; B, view from the extracellular side,
normal to the plane of the bilayer. See text for further
explanation.
-terminal
extracellular domains. In contrast, receptors of this superfamily which
bind smaller (<0.5 kDa) nonpeptide agonists have agonist-binding
sites deep within the bilayer between the transmembrane domains
(Bockaert, 1991; Dohlman et al., 1991). The smallest
neuropeptide, thyrotropin-releasing hormone (360 Da) apparently also
binds within this same region (Perlman et al., 1994). Recently
Gerszten et al.(1994) found that the specificity of thombin
receptors for peptide agonists was determined by the extracellular face
of the receptor. Substance P, a peptide agonist of intermediate size,
apparently interacts with both the extracellular region and
transmembrane region of its receptor. Thus, the regions of interaction
between SP and its receptor include elements of both the large and
small agonist-receptor systems. Other bioactive peptides among the
dozens in this intermediate size range may similarly interact with both
the extracellular and transmembrane domains of their own
G-protein-coupled receptors.
)-SP,
YBPA-SP; BPA-SP,
YBPA-SP; CP-96345,
(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-aza-bicyclo[2.2.2]octan-3-amine;
DTNB, 5,5`-dithio-bis(2-nitrobenzoic acid); Endo F, F.
meningosepticum endoglycosidase F; FCS, fetal calf serum; Fmoc,
fluoren-9-ylmethoxycarbonyl; HPLC, high performance liquid
chromatography; NK-1R, neurokinin-1 receptor (same as SPR); PAGE,
polyacrylamide gel electrophoresis; SPR, substance P receptor (same as
NK-1R); TM, transmembrane; V8, S. aureus V8 protease; cpm,
counts/minute.)
We thank J. B. Cohen, G. R. Strichartz, M. Blanton, C.
E. Dahl, D. E. Wingrove, I. Hubaçek-Veselska, and
H.-P. Too for advice and discussion. We thank Pfizer for a gift of
CP-96345.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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