Evidence for Spatial Proximity of Two Distinct Receptor Regions in the Substance P (SP)·Neurokinin-1 Receptor (NK-1R) Complex Obtained by Photolabeling the NK-1R with p-Benzoylphenylalanine3-SP*

Andrew A. Bremer, Susan E. Leeman, and Norman D. BoydDagger

From the Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118

Received for publication, January 29, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that 125I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine8-SP (Bpa8SP) covalently attaches to Met181, whereas 125I-Bolton-Hunter reagent-labeled Bpa4SP covalently attaches to Met174, both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue 125I-[D-Tyr0]Bpa3SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of 125I-[D-Tyr0]Bpa3SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide·NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 epsilon -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-3alpha ,6alpha -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 gamma -emission spectrometry. 20% (v/v) beta -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 alpha -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 gamma -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. Nalpha -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 alpha -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 alpha -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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

[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.

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. Nalpha -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.

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).

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

[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.

    ACKNOWLEDGEMENTS

We express our appreciation for the valuable discussions with Dale F. Mierke and Maria Pellegrini during the preparation of this manuscript.

    FOOTNOTES

* This work was supported by Grant NS-31346 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Experimental Therapeutics, L-611, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4387; Fax: 617-638-4329; E-mail: nboyd@bu.edu.

Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M100824200

    ABBREVIATIONS

The abbreviations used are: SP, substance P; NK-1R, neurokinin-1 receptor; rNK-1R, rat NK-1R; Bpa, p-benzoylphenylalanine; BH, Bolton-Hunter reagent; EC, extracellular loop; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; DTT, DL-dithiothreitol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; KRH, Krebs-Ringer HEPES.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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