Cellular and Molecular Neurobiology, Vol. 12, No. 6, 1992

Antibodies to the Rat Substance P Receptor: Production and Characterization M. S. Gilbert, ~ N. W. Bunnett, 2'3 and D. G. Payan 1'4-6 Received November 10, 1991; accepted April 10, 1992 KEY WORDS: substance P; substance P receptor (SPR); protein A - S P R fusion protein; anti-SPR Fab antiserum.

SUMMARY 1. A protein A - r a t substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (~,Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca 2÷ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not l Department of Microbiology-Immunology, University of California, San Francisco, San Francisco, California 94143. 2 Department of Physiology, University of California, San Francisco, San Francisco, California 94143. 3 Department of Surgery, University of California, San Francisco, San Francisco, California 94143. 4 Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94143. 5 Department of Medicine, University of California, San Francisco, San Francisco, California 94143. 6 To whom correspondence should be addressed at University of California, San Francisco, Third and Paranassus Avenue, San Francisco, California 94143-0724. 529 0272-4340/92/1200-0529506.50/0© 1992PlenumPublishingCorporation

530

Gilbert, Bunnett, and Payan

induce intracellular C a 2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions. INTRODUCTION

The tachykinin family of peptides consists of several neuropeptides, all with a common carboxy-terminal sequence: - P h e - X - G l y - L e u - M e t - N H 2 (Erspamer, 1981). Substance P (SP), 7 the first member of the tachykinin family, was discovered in extracts from equine brain and intestine and shown to lower blood pressure and stimulate smooth muscle contraction (von Euler and Gaddum, 1931). The mammalian tachykinin family has now been expanded to include substance K (also known as NKA), neuromedin K (also known as NKB), neuropeptide K (NPK), neuropeptide gamma (NP~,), and neurokinin A (MacDonald et al., 1989). SP has been the most extensively studied of all the tachykinins. It is associated with several areas of the central nervous system (CNS) and peripheral nervous system (PNS), including the spinal chord, brain-stem axons, cell bodies, and nerve terminals (Pernow, 1983; Payan et al., 1987). It is presumed to function as a neurotransmitter (Nicoll et al., 1980; Pernow, 1983), having the ability to depolarize nerve membranes, resulting in the excitation of postsynaptic neurons (Pernow, 1983). The many different actions of the tachykinin family of neuropeptides are mediated by three distinct classes of receptors. The NK-1, NK-2, and NK-3 receptors are defined by their rank order of potency for patricular tachykinins based on results obtained from specific bioassays and ligand binding experiments (Helke et al., 1990). The NK-1 receptor, which preferentially binds SP over the other members of the tachykinin family and is henceforth referred to as the substance P receptor (SPR), is located throughout the CNS and the PNS, where it is expressed by both neurons and glial cells (Nakanishi, 1987). In nonneuronal tissue, it is located in smooth muscle cells, endothelial cells, fibroblasts (Helke et al., 1990), and specific cells of the immune system such as lymphoblasts and lymphocytes (Gilbert and Payan, 1991). Recently a cDNA for the NK-1 receptor from rat brain has been cloned and expressed in frog oocytes and the mammalian COS cell line (Yokata et al., 1989; Hershey and Krause, 1989). This receptor consists of 407 amino acids, with a calculated molecular mass of 46 kDa. There are seven putative transmembrane spanning domains with a high degree of sequence homology with the family of G protein-coupled receptors such as the adrenergic receptor (Dohlman et al., 1982), serotonin receptor (Julius, 1988), and rhodopsin receptor (Natahn and Hogness, 1983). 7Abbreviations used: bp, base pairs; BSA, bovine serum albumin; CNS, central nervous system; DTT, dithiothreitol; FACS, fluorescence-activated cell sorter; HBSS, Hank's buffered saline solution; MCS, multiple cloning site; NPK, neuropeptide K; ~'Pr, heat-inducible promoter; PBS, phosphate-buffered saline; PNS, peripheral nervous system; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; SP, substance P; rSPR, rat substance P receptor; TE, Tris-HCI/EDTA; TST, Tris-HCl/NaC1/Tween-20.

Antibodies to SubstanceP Receptor

531

The binding of SP to the SPR results in the hydrolysis of inositol phospholipids (Payan et al., 1987), suggesting signal transduction by the phosphatidyl inositol-calcium second-messenger system involving G proteinmediated interactions with the activated receptor (Buck and Burcher, 1986). Although current evidence indicates that SPRs act via the association with G proteins (Mitsuhashi et al., 1992; Nakajima et al., 1992), a unique G protein or proteins has yet to be identified, as in the case of the fl-adrenergic receptor (Lefkowitz et al., 1983). Thus, the exact mode of signal transduction of the activated tachykinin receptor remains unknown. Antisera to specific receptors such as the SPR are important as they can be used to enhance our understanding of such receptor desensitization mechanisms and other cellular processes involved in signal transduction. Here we report the production of an antiserum that recognizes surface expressed SPR. Over the last 5 years several attempts have been made to produce antiserum that specifically recognizes the SPR by using synthetic peptides based on the known sequence of the receptor or cell lines known to express the SPR, as immunogens. However, recent advances in recombinant DNA technology provide a means of producing recombinant full-length rat SPR in bacteria such as Escherichia coli. One such system is the protein A expression system, which allows inserted D N A to be expressed in the form of a protein A fusion protein (Ulen et al., 1984). The advantages of using this expression system is that it allows controlled expression of the protein of interest, driven by the heat-inducible promoter (ZPr), and the fusion protein can be isolated by a single-step purification method that produces the fusion protein in a form which can be used directly as an immunogen. In this study the rat SPR cDNA was used to produce a full-length SPR fusion protein which was used to raise polyclonal antiserum to SPR in rabbits. The data presented in this report confirm that the antisera produced can be used to detect cell surface SPR by FACS analysis and immunohistochemistry. Furthermore, the antibodies can also be used to immunoprecipitate the SPR and modulate SP binding.

METHODS Construction, Expression, and Purification of the Substance P Receptor Fusion Protein The original cDNA clone containing rat SPR (prTKR2) was kindly supplied by Dr. S. Nakanishi (Kyoto University, Japan). The manipulations performed on the plasmid prTKR2 and the expression vector of choice pRIT2T (Pharmacia, Piscataway, N J) are outlined in Fig. 1. The recombinant vector (pRIT2TSPR) was transformed into the supplied host bacterial strain E. coli N4830-1 using the method described by the supplier. Clones were selected on ampicillin-containing agar plates grown at 28°C and the putative clones were analyzed by specific restriction digests to confirm the presence of the cDNA insert. The 3' and 5' junctions were also sequenced to

532

Gilbert, Bunnett, and Payan

Pstl MCS xI \

Bgl II Pst! ~

.Saul I

off/

~ , , ~ ~

Pstl

Smal EcoRI Ixz/I

X~[~/~PR

orl

1. Saul digest/ Klenow fill in 2. Pstl digest 3. 1584bp fragment gel purified Saul SPR cDNA I

1. Smal/Pstl digest 2. Largefragment gel purified Pstl

Sinai

Ligation

/ ~

ori~

k

Bgl II

"Bglll

'

pRIT2TSPR ~ " 7/~PR

5.9Kb

/

" BgIII

Fig. 1. The constriction of the rat substance P receptor fusion protein expressing plasmid pRIT2TSPR. Schematic diagram showing the method of subeloning used to insert the rat substance P receptor cDNA into the expression vector PRIT2T. Vectors and substance P receptor cDNA are not shown to scale. The positions of the ampicillin resistance gene (Ampr), the origin of replication (ori), the multiple cloning site (MCS), and the lambda promoter (~.Pr) are all shown. The correct orientation and size of the insert were verified by restriction digest analysis using the restriction enzymes BglII, BglII/PstI, and EcoRI/PstI. A 1% (w/v) agarose gel of the specific restriction digests of the recombinant pRIT2T/rat SPR cDNA ptasmid was run and showed the expected fragment sizes (not shown).

Antibodies to SubstanceP Receptor

533

verify that the rSPRcDNA was in the correct frame and orientation. Plasmid DNA samples from positive clones were obtained using the miniprep procedure (Sambrook et al., 1989). All restriction enzymes and buffers were supplied by Gibco BRL (Gaithersburg, MD). All enzyme reactions were performed according to the suppliers instructions. Positive clones were grown in LB (Luria-Bertani broth; 10 g bacto-tryptone, 5 g bacto-yeast extract, and 10 g NaCl/liter) at 28°C. At an OD6o0 of 0.8, an equal volume of LB (prewarmed to 54°C) was added and the temperature raised to 42°C, leading to direct expression of the fusion protein by the APt (Ulen et al., 1984). Cultures were incubated in LB for 90 min at 42°C. Cells were pelleted (5000 g, 5 min), washed once with TE (10 mM Tris-HC1, pH 7.4, 1 mM EDTA, pH 8), freeze-thawed, and sonicated (3 × 30sec, 20W). The homogenate was centrifuged at 20,000g for 30 min and the supernatant diluted with 5 vol of TST (50 mM Tris-HC1, pH 7.6, 150 mM NaCI, 0.05% Tween-20). The supernatant was applied to a preequilibrated IgG Sepharose-6 fast-flow column (Pharmacia, Piscataway). Samples were passed over the column twice. The column was washed with 10 bed vol of TST and 2 vol of 5 mM ammonium acetate, pH 5.0. The bound protein was eluted in 5 ml 0.5 M acetic acid, pH 3.4. Fractions were immediately neutralized with Tris base, lyophilized, and resuspended in PBS (136mM NaCI, 2.7mM KC1, 10mM Na2HPO4, 1.4mM KH2PO4, pH 7.4). Protease inhibitors were added to all the buffers used in the purification step as follows: leupeptin (inhibition of acid proteases; 1 mM), chymostatin (inhibition of serine proteases; 1 mM), and phenylmethylsulfonyl fluoride (inhibition of serine proteases; 1 mM). Analysis of the SPR Fusion Protein

Purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Laemmli, 1970). Samples were solubilized in SDS-sample buffer: 100 mM Tris-HCl, pH 8.8, 4% (w/v) SDS, 5 mM EDTA, 15% (w/v) sucrose, 10 mM dithiothreitol (DT-I'), 0.001% (w/v) bromophenol blue and separated by SDS-PAGE (7.5-10% polyacrylamide). Gels were stained with Coomassie brilliant blue [0.1% (w/v) Coomassie, 10% (v/v) acetic acid, and 5% (v/v) methanol] and destained with 10% (v/v) acetic acid, 5% (v/v) methanol. Production of Polyclonal Antiserum to SPR Fusion Protein

Four New Zealand white rabbits were initially immunized with 250/tg of purified SPR fusion protein in Freund's complete adjuvant (Harlow and Lane, 1988). Animals were boosted at montly intervals with 250/lg SPR fusion protein in Freund's incomplete adjuvant. Bleeds (50 ml) were obtained 10-12 days after immunization/boost. After 4 months the rabbits were rested for 2 months prior to a final boost. Total IgG was first purified using a protein A column (Pierce, Rockford, IL), and the purified IgG was cleaved with papain (Coulter and Harris, 1983) to obtain Fab fragments. The papain-digested IgG was passed over the protein A column to remove all of the Fc fragments and the anti-Fab fragments

534

Gilbert, Bunnett, and Payan

directed against the protein A portion of the fusion protein. The Fab fragments were passed over a protein A - S P R affinity column (50/~g protein A-SPR fusion protein coupled to 5 ml of cyanogen bromide-activated Sepharose) to obtain Fab fragments against the SPR portion of the fusion protein. The eluted anti-SPR Fab was lyophilized and resuspended in PBS/0.1% BSA/0.02% sodium azide at the desired concentration and stored at -70°C.

Analysis of Anti-SPR Immunoaflinity-Purified Fab Antibodies Western Blot Analysis For Western blot analysis, proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). Membranes were incubated at room temperature for 2 hr with primary antibody (0.5/tg/ml). Specific proteins were detected using a goat anti-rabbit (H + L-specific) horse radish peroxidase-conjugated antibody (Pel Freez, Rogers, AR) at a final dilution of 1/15,000. Membranes were incubated with antibody at room temperature for 2 hr. The bound antibody was detected by chemiluminescence according to the supplier (Amersham, Amersham, UK). Immunoprecipitation The ability of the anti-SPR Fab antibody to immunoprecipitate the SPR was examined using a rat pancreatic cell line (AR42J) which constitutively expresses functional SPR (Womack et al., 1985) and a rat kidney cell line (KNRK) transfected with rat SPRcDNA (KNRKSPR) to express functional SPR (Mitsuhashi et al., 1992). The untransfected, wild-type KNRK cells were used as controls. Cells were grown on 60-era dishes at 37°C/5%CO2 to approximately 80-90% confluence. Cells were rinsed with PBS and incubated in either methionine-free RPMI-1640 (AR42J) or methionine-free DME-H16 (KNRK/KNRKSPR) for l hr. 35S-Methionine (Trans-label; ICN Costa Mesa, CA) was added to the medium at a final concentration of 200 t~Ci/ml and the cells were incubated for 4 hr at 37°C/5% CO2. Cells (still adherent) were washed once with Tris-saline (50 mM Tris-C1, pH 7.5, 150 mM NaC1) and then incubated on ice for 30 rain with 500/~1 of lysis buffer [RIPA; 50 mM Tris-Cl, pH 8.0, 150 mM NaC1, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholic acid, 0.1% sodium lauryl sulfate]. The lysate was removed from the dish and collected. Cell debris was removed by centrifugation at 10,000g for 15 min. The supernatant was precleared by incubation with protein A-agarose beads for 30 rain at 4°C. Beads were removed by centrifugation prior to incubation with specific anti-SPR Fab antiserum, In all cases, the samples were incubated with the primary antibody (5/~g/ml) for at least 12 hr at 4°C. The Fab/SPR complexes were cross-linked for 6 hr at 4°C with goat anti-rabbit Fab (10/~g/ml) prior to precipitation with protein Ar-agarose beads. The pellets were washed 3× with lysis buffer (RIPA) and, finally, resuspended in 25-30/~1 SDS-sample buffer (see Analysis of SPR Fusion Protein above). Samples were placed in a boiling water bath for 5 min and centrifuged at 10,000g to remove any insoluble material. After protein bands were separated by SDS-PAGE, the gels were dried down onto 3 MM paper and exposed to X-ray film for the required amount of time (1-4 days).

Antibodies to SubstanceP Receptor

535

Cell Surface Expression of SPR Analyzed in a Fluorescence-Activated Cell Sorter (FA CS)

Cells were detached from flasks and resuspended in Hank's buffered saline solution (HBSS) containing 0.1% BSA. Desensitization of cell surface SPR was accomplished by preincubating cells with SP (Peninsula Laboratories, Belmont, CA) at a final concentration of 10-8M for l h r at 37°C/5% CO2 prior to detachment. Cells (1 x 104) were resuspended in a final volume of 200/~1 buffer containing either anti-SPR Fab antiserum (1/~g/ml) or preimmune Fab serum (1/~g/ml). Samples were then incubated at 4°C for 1 hr, pelleted (3000g for 5 rain), washed once with HBSS/0.1% BAS, and finally, resuspended in 200/~1 of buffer containing the secondary antibody (fluorescein-conjugated goat anti-rabbit Fab; Pel Freez) at a dilution of 11100. Samples were incubated at 4°C for I hr. Prior to analysis, propidium iodide was added at a final concentration of 1 #g/ml in order to assess cell viability. Immunohistochemistry

Cells were smeared onto polylysine-coated slides, dried for 30 min at room temperature, and washed (3 x 10min) with PBS/1% normal goat serum/0.3% Triton X-100. Cells were incubated with either preimmune serum, anti-SPR Fab serum, or "blocked" antiserum at 4°C overnight. Blocked antiserum was produced by incubating anti-SPR Fab antiserum (1/tg/ml) with 20#g/ml gel-purified SPR fusion protein at 4°C overnight. After incubation slides were washed in buffer as above (3 x 10 min), the secondary antibody (fluoresceinconjugated goat anti-rabbit Fab; Pel Freez) was added diluted in buffer at a final concentration of 1150. Slides were incubated for 4hr at room temperature, washed 3 x 10 min in PBS, and coverslipped. Functional Studies Measurement of Calcium Response to SP. AR42J cells were incubated in HBSS containing 0.1% (w/v) BSA (BSA-HBSS) and loaded with the acetoxymethyl ester of Fura-2 (2.5 #M) for 60 min at 37°C/15% COz (Mitsuhashi and Payan, 1988). Cells were washed with BSA-HBSS, gently removed from the tissue culture flask using cell dissociation buffer as described by the supplier (GIBCO BRL), and finally, resuspended in BSA-HBSS at a cell density of 5 x 105 cells/ml. The effect of anti-SPR Fab antiserum on the mobilization of intracellular calcium [Ca2+]i was investigated by initially incubating Fura-2-1oaded AR42J cells with either BSA-HBSS buffer alone or BSA-HBSS buffer plus anti-SPR Fab antiserum (0.1-10/~g]ml) for various amounts of time at 37°C. At the desired time, SP was added to the cells at a final concentration of 10 -8 M. The intracellular Fura-2 signals (F) were measured at 340-nm excitation and 510-nm emission using a fluorimeter. The values of [Ca2+]i were calculated by exposing cell suspensions to 0.1% Triton X-100 (Fmax), followed by the addition of 10 mM EGTA, pH 10 (Fmi,). [Ca2+]i values were then derived from the following formula (Mitsuhashi and Payan, 1988):

[CaZ+]i(nM) = ka(F - Fmin)/(Fm.x - F) where kd = 224 nM.

Gilbert, Bunnett, and Payan

536

Binding Studies. The ability of the anti-SPR Fab antiserum to inhibit binding of 125I-SP (Amersham; sp act, 2200 Ci/mmol) was carried out according to Mitsuhashi et al. (1992). Nonspecific binding was determined in the presence of 10 -6 M cold SP (Peninsula Labs). Inhibition of binding was tested by the addition of anti-SPR Fab antiserum or preimmune Fab antiserum at various concentrations for 30 min at room temperature prior to the addition of 125I-SP. Samples were incubed on ice for 2 hr prior to counting.

RESULTS Cloning and Expression of the SPR The recombinant plasmid pRIT2TSPR (Fig. 1) was transformed into the host E. coli strain N4830-1, with the clones being selected for ampicillin resistance. Verification of the presence of the recombinant plasmid, in the correct orientation and frame, was confirmed by specific restriction endonuclease digests and sequencing of the junctions between the inserted cDNA and the protein A sequences (results not shown). Expression of the protein A-SPR fusion protein was under the control of the lambda promoter (APt). Under normal growth conditions (cultures incubated at 28°C), the E. coli N4830-1 strain constitutively produces the ~,CI857 repressor protein, which inhibits expression from the ~'Pr- When the temperature was raised to 42°C the repressor protein was inactivated, allowing expression to occur. The soluble fusion protein was released from the host bacterium by a series of freezing and thawing steps followed by sonication (see Methods). Throughout the whole lysis and purification process, several protease inhibitors were added to the a

KDa

1

2

3

4

200 -109 -71--

~--- 8 2 K D a

44--

29-b

KDa 109 - -

71--

44--

27 F

.,.--Fusion protein

Fig. 2. Expression of substance P receptor fusion protein. (a) Coomassie-stained 7.5% (w/v) SDSPAGE of bacterial proteins showing (1) protein molecular weight markers (kDa), (2) uninduced total cell lysate of bacterial strain containing recombinant plasmid, (3) induced total cell lysate of bacterial strain containing recombinant plasmid, and (4) column-purified protein A - S P R fusion protein (82 kDa). One hundred micrograms of protein was run in lanes 2 and 3, and 25/~g of protein was run in lane 4. (b) Western blot analysis of bacterial proteins using anti-SPR Fab antiserum at a final dilution of 1/2000. (1) Uninduced total cell lysate from bacterial strain containing recombinant plasmid; (2) induced total cell lysate from bacterial strain containing recombinant plasmid, (3) purified protein A - S P R fusion protein. Protein molecular weight markers are in kiloDaltons. Arrow indicates the position of the protein A - S P R fusion protein. One hundred micrograms of protein was run in lanes 1 and 2, and 25/~g of protein was run in lane 3.

Antibodies to SubstanceP Receptor

537

sample to minimize degradation of the fusion protein. The fusion protein was purified using an IgG-Sepharose fast-flow column which binds those proteins that contain the protein A sequence (IgG binding domains). These proteins were eluted using an acidic buffer and analyzed initially by SDS-PAGE (Fig. 2a). The position of the purified protein A-SPR fusion protein is indicated by the arrow. This novel protein migrates with an apparent molecular weight of 82kDa, comparable to the expected size of the fusion protein (SPR, 46 kDa, + truncated protein A, 34.9 kDa). The level of expression observed was typically less than 0.5% of the total cellular protein. Several other protein bands were also present. The major one, with an apparent molecular weight of 72 kDa, appears to be a contaminating E. coli protein that binds to the IgG column (as shown in the Western blot results; see Fig. 2b). Smaller bands ( m

W

-100 200

2&o

400 TIME

400

(sec)

b

40 antiSPR Fab • Preirnmune Fab

80-

CO~

~0 -

,o_

q

0

T

10

F

q

20

30

40

50

60

Fab ~glml) Fig. 5. Functional studies. (a) Representative responses of Fura-2-1oaded AR42J cells resuspended in BSA-HBSS with respect to [Ca2+]i signal. (A) Control response of AR42J cells incubated with SP ( 1 0 - a M ) at the indicated time (arrow). (B) Response of AR42J cells preincubated with anti-SPR Fab serum (10#g/ml) for 10rain prior to the addition of SP (10 - s M) at the time indicated. A set of typical results reproduced three times. (b) Graph showing the ability of anti-SPR Fab antiserum to inhibit ~zSI-SP binding. Values obtained in the presence of Fab are calculated as percentage inhibition of total binding.

542

Gilbert, Bunnett, and Payan DISCUSSION

The presence of SP and the SPR in several regions throughout the body has been extensively reported in the literature (Pernow, 1983; Gilbert and Payan, 1991). Understanding the mechanisms by which SP/SPR interactions lead to their diverse actions requires an understanding of the cellular processes that are initiated as a result of ligand binding to the receptor. Several "second-messenger" pathways have been reported, such as phosphatidylinositol turnover, protein kinase C activation, and increase in [cAMP] (Cohen, 1988; Freissmuth et al., 1989; Nakanishi et al., 1990; Nakajima et aL, 1992). Activation of these pathways, by ligand-receptor interactions, results in intracellular phosphorylation of specific proteins which regulate the activities of specific enzymes associated with physiological cellular responses. To study the mechanisms of ligand/receptor activation, a means of detecting and isolating the receptor of choice is required. One way of achieving such receptor recognition is to obtain specific polyclonal or monoclonal antiserum to the whole receptor molecule or portions of the receptor. Previously, synthetic peptides to suspected antigenic regions, such as extracellular domains, have been used. In some cases, such as the dopamine D2 receptor, this method has proven successful, with the antiserum being used to localize the receptor in rat brain (McVittie et al., 1991). In our laboratory we were bale to produce polyclonal antisera to the substance K receptor using synthetic peptides (unpublished results). The antiserum to these peptides was shown to have a slight crossreactivity with the SPR, which is not surprising considering the degree of homology between the two receptors in these regions (Parnet et al., 1991). However, this serum was of limited usefulness with respect to specificity toward the SPR and was unable to detect cell surface SPR or immunoprecipitate the receptor. A similar attempt to use synthetic peptides to the SPR to raise antiserum proved unsuccessful (unpublished results). The decision to use a SPR fusion protein utilizing the bacterial protein A system as the initial immunogen was based on the published reports showing that such fusion proteins render the eukaryotic portion present more immunogenic than when the native protein is used (Harris, 1983; Marston, 1985). Also, expression of eukaryotic proteins in the form of chimeric fusion proteins has previously resulted in high levels of expression of the inserted protein (>50% total cellular protein). This observed increase in expression results from an increased stability of the fusion protein (Marston, 1985). However, in our particular case, the level of expression observed was very low, with the resultant recovery of soluble fusion protein being less than 0.5% of the total cellular protein. Attempts were made to increase the level of expression by increasing the time of heat inducibility of 42°C from 90 to 120 min and to start the induction of fusion protein at a higher cell density. However, neither of these modifications in the protocol resulted in any increase in the level of expression. It is possible that, even with the presence of the protein A moiety, the stability of the chimeric SPR fusion protein is low. This observation appears to be confirmed by the presence of degradation products present in the column-purified sample and the induced cell lysate as seen in Figs

Antibodies to SubstanceP Receptor

543

2a and b. It appears that the observed degradation occurs prior to lysis of the bacteria, as the presence of a cocktail of protease inhibitors in the lysis buffer (see Methods) fails to prevent degradation. In this report, we utilized the purified SPR-protein A fusion protein to raise polyclonal antiserum to SPR in rabbits. Initially, using IgG-purified serum, the results obtained were inconclusive, as the background cross-reactivity encountered was high (results not shown). To overcome this problem it was necessary to produce Fab fragments from the serum which were eventually affinity purified using a SPR-protein A column (see Methods.) After this step, the results obtained in various experiments, as outlined above, showed that the anti-SPR Fab antiserum could recognize both cell surface-expressed (FACS analysis, immunohistology) and solubilized (immunoprecipitation) forms of the SPR. The immunoprecipitation of both AR42J and KNRKSPR cells shows the SPR at approximately 46 kDa; this suggests that the SPR is not glycosylated at either of the two possible sites in these particular cells (Yokata et al., 1989). The results obtained in the functional studies, which show that the anti-SPR Fab serum has the ability to inhibit partially SP-induced [Ca2+]i mobilization and 125I-SP binding, suggest that the antiserum may be recognizing an epitope or epitopes at or near the suspected ligand binding site on the SPR. Alternatively, the presence of the anti-SPR Fab antiserum may produce a conformational change in the external domains of the receptor which results in decreased efficiency of ligand binding. This observation is under further investigation. The corss-reactivity of the anti-SPR Fab serum with other members of the tachykinin receptor family was not investigated at this time, as suitable cells lines expressing these receptors were not available. However, it is feasible that due to the high degree of homology between the tachykinin receptors, cross-reactivity is a possibility (Parnet et al., 1991). This has yet to be investigated. As we have reported here, we have successfully produced antiserum (Fab) to the rat SPR. This serum has been shown to be able to detect cell surfaceexpressed SPR in two cell lines and also to immunoprecipitate the solubilized form of the receptor. In the future it is planned to use this antiserum to help elucidate the many cellular processes/responses that occur as a result of SP binding to SPR. In particular, the mode of rapid desensitization of the receptor, which is unique to this particular member of the tachykinin family, will be investigated further using this anti-SPR Fab antiserum.

ACKNOWLEDGMENTS

We would like to thank Paul Dazin for his technical assistance and expertise in the FACS analysis. This work was supported by NIH Grant NS21710 (D.G.P), NIH Grand DK39957 (N.W.B.), and the Howard Hughes Medical Institute (M.S.G., D.G.P.).

544

Gilbert, Bunnett, and Payan

REFERENCES Buck, S. H., and Burcher, E. (1986). The tachykinins: A family of peptides with a brood of "receptors." Trends Physiol. Sci. 7:65-68. Cohan, P. (1988). Protein phosphorylation and hormone action. Proc. R. Soc. Lond. B234:115-144. Coulter, A., and Harris, R. (1983). Simplified preparation of rabbit Fab fragments. J. Immunol. Meth. 59:199-203. Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1982). The multiple membrane spanning topography of the fl-adrenergic receptor. J. Biol Chem 262:1428214288. Erspamer, V. (1981). The tachykinin peptide family. Trends Neurosci. 4:267-269. Freissmuth, M., Casey, P. J., and Gilman, A. G. (1989). G proteins control diverse pathways of transmembrane signalling. FASEB J. 3:2125-2131. Gilbert, M. S., and Payan, D. G. (1991). Interactions between the nervous and the immune systems. Front. Neuroendocrinol. 12:299-322. Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 53-139. Harris, T. J. R. (1983). Expression of eukaryotic genes in E. coli. In Genetic Engineering, Vol. 4 (R. Williamson, Ed.), Academic Press, London. Helke, C. J., Krause, J. E., Mantyh, P. W., Couture, R., and Bannon, M. J. (1990). Diversity in mammalian tachykinin peptidergic neurons: Multiple peptides, receptors, and regulatory mechanisms. FASEB J. 4:1606-1615. Hershey, A. D., and Krause, J. E. (1989). Molecular characterization of a functional cDNA encoding the rat substance P receptor. Science 247:958-962. Julius, D., MacDermott, A. B., Axel, R., and Jessel, T. M. (1988). Molecular characterization of a functional cDNA encoding the serotonin 1C receptor. Science 241:558-564. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277:680-:683. Lefkowitz, R. J., Stadel, A., and Caron, M. G. (1983). Adenylate cyclase-coupled fl-adrenergic receptors: Structure and mechanism of activation and desensitization. Annu. Rev. Biochem. 52:159-186. Marston, F. A. O. (1985). The Purification of Eukaryotic Peptides Expressed in Escherichia coli. In DNA Cloning: A Practical Approach, Vol. 111 (D. Glover, Ed).), IRL Press, Oxford. Mitsuhashi, M., and Payan, D. G. (1988). Characterization of functional histamine H1 receptor on a cultured smooth muscle cell line. J. Cell. Physiol. 134:367-375. Mitsuhashi, M., Ohashi, Y., Shichijo, S., Christian, C., Sudduth-Klinger, J., Harrowe, G., and Payan, D. G. (1992). Multiple intracellular signaling pathway of the neuropeptide substance P receptor. J. Neurosci. Res. MacDonald, M. R., Takeda, J., Rice, C. M., and Krause, J. E. (1989). Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, oL, fl and ~,-preprotachykinin. J. Biol. Chem. 264:15578-15592. McVittie, L. D., Ariano, M. A., and Sibley, D. R. (1991). Characterization of anti-peptide antibodies for the localization of D2 dopamine receptors in rat striatum. Proc. Natl. Acad. Sci. USA 88:1441-1445. Nakajima, T., Tsuchida, K., Negishi, M., Ito, S., and Nakanishi, S. (1992). Direct linkage of three techykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic AMP cascades in transfected chinese hamster ovary cells. J. Biol. Chem. 267:2437-2442. Nakanishi, S. (1987). Substance P precursor and kinogen: Their structures, gene organizations, and regulation. Physiol. Rev. 67:1117-1140. Nakanishi, S., Ohkubo, H., Kakizuka, A., Yokota, Y., Shigemoto, R., Sasai, Y., and Takumi, T. (1990). Molecular characterization of mammalian tachykinin receptors and a possible epithelial potasium channel. Res. Prog. Horm. Res. 46:59-84. Nathans, J., and Hogness, D. S. (1983). Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 34:807-814. Nicoll, R. A., Schenker, C., and Leeman, S. E. (1980). Substance P as a transmitter candidate. Annu. Rev. Neurosci. 3:1529-1534. Parnet, P., Mitsuhashi, M., Turk, C. W., Kerdelhue, B., and Payan, D. (3. (1991). Tachykinin receptor cross talk. Immunological cross-reactivity between the external domains of the substance K and substance P receptors. Brain Behav. lmmun. 5:73-83. Payan, D. G., McGillis, J. P., Renold, F. K., Mitsuhashi, M., and Goetzl, E. J. (1987). Neuropeptide modulation of leukocyte function. In Neuroimmune Interactions: Proceedings o f the Second

Antibodies to Substance P Receptor

545

International Workshop on Neuroimmodulation (B. Jankovic, B. Markovic, and N. Spector,

Eds.), New York Academy of Science, New York, pp. 183-191. Pernow, B. (1983). Substance P. Pharmacol. Rev. 35:85-140. Sambrook, J., Fritseb, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1.40-1.52. Ulen, M. (1984). Complete sequence of Staphylococcal gene encoding protein A. A gene evolved through multiple duplication. J. Biol. Chem. 259:1695-1702. von Euler, U. S., and Gaddum, J. H. (1931). An unidentified depressor substance in certain tissue extracts. J Physiol. 72:577-583. Womack, M. D., Hanley, M. R., and Jessell, T. M. (1985). Functional substance P receptors on a rat pancreatic acinar cell line. J. Neurosci. 5:3370-3378. Yokata, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R., Kakizuka, Y., Obkudo, H., and Nakanishi, S. (1989). Molecular characterization of a functional cDNA for rat substance P receptor. J. Biol. Chem. 264:17649-17652.