©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping Peptide-binding Domains of the Substance P (NK-1) Receptor from P388D Cells with Photolabile Agonists (*)

(Received for publication, September 9, 1994)

Yue-Ming Li Margarita Marnerakis Evelyn R. Stimson John E. Maggio (§)

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2)) by replacing amino acids in the peptide with p-benzoyl-L-phenylalanine (BPA). SP, BPA^3-SP, and BPA^8-SP bind with high affinity (K


INTRODUCTION

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) (^1)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(2), 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.

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^3 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).

P388D(1) 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. (^2)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^3) or eighth (BPA^8) position) to label the SP receptor of P388D(1) cells and map the peptide-binding domains of the receptor for each ligand.


EXPERIMENTAL PROCEDURES

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^3-SP and BPA^8-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(18) column (Vydac 4.6 times 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^8-SP (synthesized from Fmoc-L-BPA) showed only one UV active peak which was coincident with the earlier eluting HPLC peak of DL-BPA^8-SP (synthesized from Fmoc-DL-BPA). Thus, the earlier HPLC peak corresponds to L-BPA^8-SP and the later peak to D-BPA^8-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^8-SP and D-BPA^8-SP fractions were tested for receptor binding and biological activity, the earlier eluting peptide, i.e.L-BPA^8-SP, was much more active than the later eluting D-BPA^8 analog. By analogy to the greater activity of L-Phe^8-SP relative to D-Phe^8-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).

The isolated L-BPA^8-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 times 10^3, 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^8-SP ([I]iodotyrosyl^0-L-BPA^8-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(2)S(2)O(5) (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(18) 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) beta-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(18) 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(i)/mmol; 1C(i) = 37 GBq) was protected from oxidation by 0.5% beta-mercaptoethanol (v/v) added immediately after purification and stored at -20 °C until use. Radioiodinated BPA^3-SP was prepared similarly.

Cell Culture

The murine cell line P388D(1) (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(1) 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) 2.5 mM, MgSO(4) 1.0 mM, Na(2)HPO(4) 1.0 mM, glucose 12 mM, and HEPES 10 mM, pH 7.40.

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.

Ligand Binding of P388D(1)Cells

P388D(1) cells (5 times 10^5 cells/well) were inoculated on FCS precoated 24-well plates and cultured overnight. The confluent cells (1 times 10^6 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 times 10^5 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 Na(2)HPO(4), 1.8 mM KH(2)PO(4), 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(1) Cells

Cells (5 times 10^6) were inoculated on FCS-pretreated dishes (60 mm) and grown for at least 12 h before labeling. The cultured cells (1 times 10^7) 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 times 10^6 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.

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 times 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 times g for 15 min to remove debris. The resulting supernatants were sedimented at 16,000 times 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 times 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 NH(4)HCO(3), 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 NH(4)HCO(3), 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 NH(4)HCO(3), 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^3-SP and BPA^8-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).


Figure 1: Primary structures of SP, BPA^3-SP, and BPA^8-SP. Addition of Tyr at the NH(2) terminus facilitates radioiodination. Lys^3 and Phe^8 are respectively replaced by BPA to give BPA^3-SP and BPA^8-SP.



HPLC of Enzymatic Digests

The V8-digested samples were loaded into a Vydac C(8) column (2.1 times 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 NH(4)HCO(3), 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.


RESULTS

Specificity of BPA^3-SP and BPA^8SP

Both BPA^3-SP and BPA^8-SP (Fig. 1) are full and potent agonists relative to SP for the calcium responses of P388D(1) 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).


Figure 2: SP, BPA^3-SP, and BPA^8-SP induce calcium responses in P388D(1) 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^3-SP or BPA^8-SP bound to P388D(1) 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^8-SP, 46 ± 3% of bound ligand is recovered in the broad 75 kDa band; for BPA^3-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.


Figure 3: Autoradiography of P388D(1) cell membranes photoaffinity labeled with [I]BPA^3-SP and [I]BPA^8-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/H(2)O (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^8-SP-labeled membranes; lanes 3 and 4, [I]BPA^3-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.




Figure 4: Autoradiography of Endo F deglycosylated photolabeled SP receptor following SDS-PAGE (12% gel). Acetone-precipitated 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(4)HCO(3), 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^8-SP-labeled SPR; lanes 3 and 4, [I]BPA^3-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^8-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^8-SPR-3.2k, being the limit digest (Fig. 5A). Double digestion of [I]BPA^8-SP-labeled SPR with Endo F and V8 protease revealed the same five-band pattern seen with V8 digestion alone.


Figure 5: Autoradiography of V8 protease digest of photolabeled SP receptor following SDS-PAGE. Acetone precipitated [I]labeled SPR (5000 8000 cpm) from preparative SDS-PAGE was dissolved in 0.1% SDS, 100 mM NH(4)HCO(3), 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^8-SP-labeled SPR (17.5% gel); molecular weights are indicated at the left. B, [I]BPA^3-SP-labeled SPR (18% gel); molecular weights are indicated at the right.



Peptide fragments from V8 digestion of [I]BPA^8-SPlabeled SPR were isolated by HPLC (Fig. 6A). One major peak (33% solvent B), accounting for most of the eluted radioactivity, corresponded to BPA^8-SPR-3.2k (Fig. 6B). BPA^8-SPR-3.2k and BPA^8-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^8-SPR-3.2k, corresponded to BPA^8-SPR-9k. Further digestion of BPA^8-SPR-9k with V8 protease converted this fragment to BPA^8-SPR-3.2k (not shown).


Figure 6: Analysis of the BPA^8-SPR-3.2k fragment. [I]BPA^8-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(8) 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^8-SP; molecular weights are indicated at the right. BPA^8-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^8-SP.



BPA^8-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^8-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.

V8 digests of SPR photolabeled with [I]BPA^3-SP displayed a different pattern of proteolytic fragments than digests of SPR photolabeled with [I]BPA^8-SP (Fig. 5). A major fragment of 40 kDa (BPA^3-SPR-40k) was detected under conditions which reduced SPR photolabeled with [I]BPA^8-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^3-SPR-40k produced a 3.5-kDa fragment, BPA^3-SPR-3.5k. The same fragment was produced by double digestion with Endo F and V8 protease of SPR photolabeled with [I]BPA^3-SP.

V8 protease-digested fragments of [I]BPA^3-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^3-SPR-3.5k) was retained by the HPLC column and eluted at 30% solvent B (Fig. 7). BPA^3-SPR-3.5k and BPA^3-SP were separable by HPLC. A control sample (identically treated in the absence of Endo F) still passed through the HPLC column without retention.


Figure 7: Analysis of the BPA^3-SPR-40K fragment. [I]BPA^3-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^3-SP.



Trypsin Digestion of the Labeled SP Receptor

Double digestion of [I]BPA^8-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^3-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^3-SP-labeled SPR with Endo F converted it to a much smaller fragment.


DISCUSSION

Replacement of amino acid residues at the third (Lys^3) or eighth (Phe^8) positions of SP by BPA and addition of Tyr at the NH(2)-terminal (Tyr^0) gave analogs (Fig. 1) which triggered calcium responses of P388D(1) 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^8-SP and BPA^3-SP to P388D(1) 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(1) cells. Previous studies showed that Phe^8 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^8 of SP by BPA is well tolerated in binding affinity and biological activity. The results of the present study illustrate that substitution of Lys^3 of SP with BPA also maintains biological activity and binding affinity at the SPR of murine P388D(1) cells. To facilitate radioiodination, we added a tyrosine at the NH(2) terminus of the peptide; the Tyr^0 peptides also retain full biological activity and binding affinity (Sachais et al., 1993; Cascieri et al., 1994).

Upon near UV irradiation (>310 nm), I-labeled BPA^8-SP and BPA^3-SP are photoincorporated into a major broad radiolabeled band of 75 kDa in P388D(1) 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^8-SP and BPA^3-SP were cross-linked with the SPR.

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^8 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^3-BPA^8-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.

Limited V8 digestion of [I]BPA^8-SP-labeled SPR implies that the smallest labeled complex, BPA^8-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^8-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^8-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(r) > 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.

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^8-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 (alpha-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^8-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^8-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.

Limited V8 digestion of [I]BPA^3-SP-labeled SPR suggested that the glycosylated peptide complex, BPA^3-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^3-SPR-3.5k. This is further confirmed by HPLC analysis. BPA^3-SPR-40k passed though the reverse-phase column in the void volume, behavior common to very polar biopolymers such as carbohydrates. Deglycosylation of BPA^3-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(2)-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(2)-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^3-SP would elute earlier than the free photoprobe. Consistent with this prediction, the complex does elute earlier in the solvent gradient than [I]BPA^3-SP. Thus, molecular size of the complex, the presence of carbohydrate, and HPLC elution behavior, taken together, establish that the NH(2)-terminal extracellular tail of the receptor (SPR 1-21, whose primary sequence is MDNVLPVDSDLFPNTSTNTSE) is the insertion site of [I]BPA^3-SP.

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(2)-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(2)-terminal tail and the first extracellular loop were essential for high affinity binding of agonist peptides. Furthermore, they identified several residues in the NH(2)-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^3) and eighth (BPA^8) positions of SP as (i.e. BPA^3 and BPA^8 contact and photolabel) the NH(2)-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.

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(2) 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(2)-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).


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^3 and BPA^8). The third and the eighth positions of SP, respectively, interact with the NH(2)-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.



Studies of other G-protein-coupled receptors have demonstrated that those which bind larger (>10 kDa) agonists have agonist-binding sites within their NH(2)-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.


FOOTNOTES

*
This work was supported by Public Health Service Grant GM-15904 (to J. E. M.) from the National Institutes of Health. Portions of this work were published in abstract form ((1994) Soc. Neurosci. Abst.20, 905). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Tel.: 617-432-0757; Fax: 617-432-3833.

(^1)
The abbreviations used are: SP, substance P; BPA, p-benzoyl-L-phenylalanine; BPA^3-SP, Y^0BPA^3-SP; BPA^8-SP, Y^0BPA^8-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.

(^2)
H.-P. Too and J. E. Maggio, unpublished results.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Baldwin, J. M. (1994) Curr. Opinion Cell Biol. 6, 180-190 [Medline] [Order article via Infotrieve]
  2. Blanton, M. P., and Cohen, J. B. (1994) Biochemistry 33, 2859-2872 [Medline] [Order article via Infotrieve]
  3. Bockaert, J. (1991) Curr. Opinion Neurosci. 1, 32-42
  4. Boyd, N. D., Macdonald, S. G., Kage, R., Luber-Narod, J., and Leeman, S. E. (1991a) Ann. New York Acad. Sci. 632, 79-93 [Medline] [Order article via Infotrieve]
  5. Boyd, N. D., White, C. F., Cerpa, R., Kaiser, E. T., and Leeman, S. E. (1991b) Biochemistry 30, 336-342 [Medline] [Order article via Infotrieve]
  6. Boyd, N. D., Kage, R., Dumas, J. J., Krause, J. E., and Leeman, S. E. (1993) J. Neurochem. Suppl. 61, S63
  7. Boyd, N. D., Kage, R. K., and Leeman, S. E. (1994) The Tachykinin Receptors (Buck, S. H., ed) pp. 219-236, Humana Press, Totowa, NJ _
  8. Cascieri, M. A., Macleod A. M., Underwood, D., Shiao, L. L., Ber, E., Sadowski, S., Yu, H., Merchant, K. J., Swain, C. J., Strader, C. D., and Fong, T. M. (1994) J. Biol. Chem. 269, 6587-6591 [Abstract/Free Full Text]
  9. Dam, T.-V., Escher, E., and Quirion, R. (1987) Biochem. Biophys. Res. Commun. 149, 297-303 [Medline] [Order article via Infotrieve]
  10. Dawe, C. J., and Potter, M. (1957) Am. J. Pathol. 33, 603
  11. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  12. Dorman, G., and Prestwich, G. D. (1994) Biochemistry 33, 5661-5673 [Medline] [Order article via Infotrieve]
  13. Engelman, D. M., Steitz, T. A., and Goldman, A. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321-353 [CrossRef][Medline] [Order article via Infotrieve]
  14. Fong, T. M., Huang, R. R. C., and Strader, C. D. (1992a) J. Biol. Chem. 267, 25664-25672 [Abstract/Free Full Text]
  15. Fong, T. M., Yu, H., Huang, R. R. C., and Strader, C. D. (1992b) Biochemistry 31, 11806-11811 [Medline] [Order article via Infotrieve]
  16. Fong, T. M., Cascieri, M. A., Yu, H., Bansal, A., Swain, C., and Strader, C. D. (1993) Nature 362, 350-353 [CrossRef][Medline] [Order article via Infotrieve]
  17. Fong, T. M., Yu, H., Cascieri, M. A., Underwood, D., Swain, C. J., and Strader, C. D. (1994a) J. Biol. Chem. 269, 2728-2732 [Abstract/Free Full Text]
  18. Fong, T. M., Yu, H., Cascieri, M. A., Underwood, D., Swain, C. J., and Strader, C. D. (1994b) J. Biol. Chem. 269, 14957-14961 [Abstract/Free Full Text]
  19. Fournier, A., Couture, R., Regoli, D., Gendreau, M., and St.-Pierre, S. (1982) J. Med. Chem. 25, 64-68 [Medline] [Order article via Infotrieve]
  20. Gerard, N. P., Bao, L., He, X. P., and Gerard, C. (1993) Regul. Peptides 43, 21-35 [CrossRef][Medline] [Order article via Infotrieve]
  21. Gerszten, R. E., Chen, J., Ishii, M., Ishii, K., Wang, L., Nanevicz, T., Turck, C. W., Vu, T-K. H., and Coughlin, S. R. (1994) Nature 368, 648-651 [CrossRef][Medline] [Order article via Infotrieve]
  22. Gether, U., Johansen, T. E., and Schwartz, T. W. (1993a) J. Biol. Chem. 268, 7893-7898 [Abstract/Free Full Text]
  23. Gether, U., Johansen, T. E., Snider, R. M., Lowe, J. A.,III, Nakanishi, S., and Schwartz, T. W. (1993b) Nature 362, 345-347 [CrossRef][Medline] [Order article via Infotrieve]
  24. Gether, U., Yokota, Y., Edmonds-Alt, X., Breliere, J. C., Lowe, J. A., III, Snider, R. M., Nakanishi, S., and Schwartz, T. W. (1993c) Proc. Natl. Acad. Sci. U. S. A. 90, 6194-6198 [Abstract]
  25. Gether, U., Edmonds-Alt, X., Breliere, J. C., Fujii, T., Hagiwara, D., Pradier, L., Garret, C., Johansen, T. E., and Schwartz, T. W. (1994) Mol. Pharmacol. 45, 500-508 [Abstract]
  26. Huang, R. R. C., Yu, H., Strader, C. D., and Fong, T. M. (1994a) Mol. Pharmacol. 45, 690-695 [Abstract]
  27. Huang, R. R. C., Yu, H., Strader, C. D., and Fong, T. M. (1994b) Biochemistry 33, 3007-3013 [Medline] [Order article via Infotrieve]
  28. Jensen, C. J., Gerard, N. P., Schwartz, T. W., and Gether, U. (1994) Mol. Pharmacol. 45, 294-299 [Abstract]
  29. Kage, R. K., Leeman, S. E., and Boyd, N. D. (1993) J. Neurochem. 60, 347-351 [Medline] [Order article via Infotrieve]
  30. Kauer, J. C., Erickson-Viitanen, S., Wolfe, H. R., Jr., and DeGrado, W. F. (1986) J. Biol. Chem. 261, 10695-10700 [Abstract/Free Full Text]
  31. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  32. Lee, C.-M., Javitch, J. A., and Snyder, S. H. (1983) Mol. Pharmacol. 23, 563-569 [Abstract]
  33. Li, Y.-M., Wingrove, D. H., Too, H.-P., Marnerakis, M., Stimson, E. R., Strichartz, G. R., and Maggio, J. E. (1995) Anesthesiology, in press
  34. Maggio, J. E. (1988) Annu. Rev. Neurosci. 11, 13-28 [CrossRef][Medline] [Order article via Infotrieve]
  35. Maggio, J. E., Stimson, E. R., Ghilardi, J. R., Allen, C. J., Dahl, C. E., Whitcomb, D. C., Vigna, S. R., Vinters, H. R., Labenski, M. E., and Mantyh, P. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5462-5466 [Abstract]
  36. Marfey, P. (1984) Carlsberg Res. Commun. 49, 591-596
  37. Otsuka, M., and Yoshioka, K. (1993) Physiol. Rev. 73, 229-308 [Free Full Text]
  38. Perlman, J. H., Thaw, C. N., Laakkonen, L., Bowers, C. Y., Osman, R., and Gershengorn, M. C. (1994) J. Biol. Chem. 269, 1610-1613 [Abstract/Free Full Text]
  39. Pernow, B. (1983) Pharmacol. Rev. 35, 85-141 [Medline] [Order article via Infotrieve]
  40. Persico, F. J., Kimball, E. S., and Vaught, J. L. (1988) J. Immunol. 141, 3564-3569 [Abstract/Free Full Text]
  41. Sachais, B. S., Snider, R. M., Lowe, J. A., III, and Krause, J. E. (1993) J. Biol. Chem. 268, 2319-2323 [Abstract/Free Full Text]
  42. Shoelson, S. E., Lee, J., Lynch, C. S., Backer, J. M., and Pilch, P. F. (1993) J. Biol. Chem. 268, 4085-4091 [Abstract/Free Full Text]
  43. Sundelin, J. B., Provvedini, D. M., Wahlestedt, C. R., Laurell, H., Pohl, J. S., and Peterson, P. A. (1992) Eur. J. Biochem. 203, 625-631 [Abstract]
  44. Too, H.-P., and Maggio, J. E. (1991) Methods. Neurosci. 6, 232-247
  45. Trumpp-Kallmeyer, S., Hoflack, J., and Hibert, M. (1994) The Tachykinin Receptors (Buck, S. H., ed) pp. 237-255, Humana Press, Totowa, NJ
  46. Viger, A., Beaujouan, J. C., Torrens, Y., and Glowinski, J. (1983) J. Neurochem. 40, 1030-1038 [Medline] [Order article via Infotrieve]
  47. Yokota, Y., Akazawa, C., Ohkubo, H., and Nakanishi, S. (1992) EMBO J. 11, 3585-3591 [Abstract]
  48. Zoffmann, S., Gether, U., and Schwartz, T. W. (1993) FEBS Lett. 336, 506-510 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.