WO1992019723A1

WO1992019723A1 - Method of isolating ligands which bind neutrophil receptors

Info

Publication number: WO1992019723A1
Application number: PCT/US1992/003432
Authority: WO
Inventors: Javier Navarro
Original assignee: The Trustees Of Boston University
Priority date: 1991-04-29
Filing date: 1992-04-27
Publication date: 1992-11-12

Abstract

Disclosed are methods of determining whether a ligand is capable of binding to a neutrophil receptor. The methods involve (a) providing a neutrophil-derived DNA clone which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene or with a DNA fragment which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene; (b) introducing the DNA clone into a cell which presents substantially no neutrophil receptor on its surface so that the recombinant protein encoded by the DNA clone is presented on the surface of the cell; (c) contacting the cell, or a membrane fragment thereof, with the ligand to allow formation of an affinity complex between the cell (or membrane) and the ligand; and (d) detecting any such complex formation.

Description

METHOD OF ISOLATING LIGANDS WHICH BIND NEUTROPHIL RECEPTORS

Background of the Invention This invention relates to the isolation of ligands, particularly ligands which specifically bind to neutrophil cell surface receptors.

Neutrophils (or poly orphonuclear leukocytes, PMNs) are members of the granulocyte class of white blood cells. Under normal circumstances, neutrophils circulate in the blood without infiltrating the endothelial cells lining the blood vessels or entering the tissue. Invasion of. the body by infectious bacteria or foreign agents, however, results in a mobilization of the circulating neutrophils to the afflicted site in order to limit the spread of infection. The initial trigger for this mobilization is provided by certain biochemical factors which may be produced either by the infectious agent or by the host itself in response to the infectious agent. These factors serve to both stimulate and attract the neutrophils to sites of infection or trauma. In response to stimulation by such factors, the neutrophils undergo a number of biochemical and morphological changes to achieve an activated state. Once in the activated state neutrophils become tightly adherent to endothelial cells lining the capillaries and venules of the microvasculature through which they eventually pass (extravasate) , moving up the gradient of stimulatory factor(s) into the tissue by a process termed chemotaxis. During their migration toward the source of the stimulatory factors, the activated neutrophils begin to release cytotoxic molecules, such as oxygen radicals and hydrolytic enzymes, as a means of destroying the foreign cells or agents. Although this provides an important early protective response against infectious agents, it can also be highly damaging to bystander cells as well as the foreign target cells because these cytotoxic molecules are non-specific in their action. An understanding of neutrophil function depends upon an understanding of its responses to host cell signals, e.g., those signals which direct neutrophil migration to a site of injury, trigger neutrophil activation, and promote neutrophil recognition of an infectious agent. These signals are likely mediated by a battery of factors, each of which binds to a neutrophil cell surface receptor and triggers a particular cascade of biological events leading to neutrophil function. An identification of the receptors on the surface of a neutrophil cell would facilitate the identification of the neutrophil-regulatory factors (i.e. ligands) . Isolation of such ligands would guide the development of therapeutics designed to prevent and/or treat neutrophil- ediated disorders, e.g., the inflammatory disorders, psoriasis, rheumatoid arthritis, and reperfusion injury (such as that occurring following periods of ischemia, e.g., due to myocardial infarction or shock).

Summary of the Invention In general, the invention features a method of determining whether a ligand is capable of binding to a neutrophil receptor. The method involves (a) providing a neutrophil-derived DNA clone which hybridizes with the second transmembrane domain-encoding sequence of a GTP binding protein-coupled (i.e., G-protein-coupled) receptor gene or with a DNA fragment which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene; (b) introducing the DNA clone into a cell which presents substantially no neutrophil receptor on its surface so that the recombinant protein encoded by the DNA clone is presented on the surface of the cell; (c) contacting the cell or a membrane fragment thereof with the ligand to allow formation of an affinity complex between the cell or membrane and the ligand; and (d) detecting any such complex formation.

In a related aspect, the invention features a method of determining whether a ligand is capable of binding to a neutrophil receptor, involving (a) providing a neutrophil-derived DNA clone which hybridizes with the second transmembrane domain-encoding sequence of a G- protein-coupled receptor gene or with a DNA fragment which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene; (b) introducing the DNA clone into a cell which expresses substantially no neutrophil receptor so that the recombinant protein encoded by the DNA clone is expressed; (c) isolating the recombinant protein; (d) immobilizing the recombinant protein on a solid " substrate; (e) contacting the immobilized recombinant protein with-the ligand to allow formation of an affinity complex between the immobilized recombinant protein and the ligand; and (f) detecting any such complex formation.

In various preferred embodiments, the second transmembrane domain-encoding sequence of a G-protein- coupled receptor gene includes the sequence:

3 • TTG ATG AAG GAC GAC TCG GAC CGG ACI CGI CTG GAI TAG TAC 5^r (SEQ ID NO:l); the DNA fragment includes the sequence of Fig. 1 (SEQ ID NO:2); the cell is a eukaryotic cell, preferably, a mammalian cell, for example, a COS cell; the solid substrate is a column; the neutrophil receptor is derived from a human; and the neutrophil receptor is derived from a rabbit.

By "neutrophil receptor" is meant all or a ligand- binding portion of a receptor polypeptide which is present on the surface of a neutrophil cell or its precursor at some point in its in vivo existence; by "polypeptide" is meant any chain of amino acids, regardless of length or post-translational modification (e.g. , glycosylation) . 5 By "ligand" is meant a molecule which has a specific affinity for a neutrophil receptor. Preferably, the affinity arises by virtue of the ligand possessing a three-dimensional structure complementary to that of the receptor. The ligand may be proteinaceous, e.g., a 0 soluble protein, e.g., a growth factor, a lymphokine, or a hormone; or it may be a molecule on the surface of another interacting cell, e.g., a cell adhesion molecule, e.g., a carbohydrate involved in cell adhesion, a cadherin, a cell adhesion molecule (e.g., cell-CAM) , a laminin, a fibronectin, an integrin, a lectin, or a toxin. The ligand may also be present or an organism foreign to the host, e.g., a pathogen (e.g., a bacteria, virus, fungus, or protozoan) and may be antigenic. Finally, the ligand may be a molecule other than that molecule which naturally interacts with the receptor.

For example, the ligand may be a molecule which acts as a v receptor agonist (i.e., mimics or enhances the activity of the natural ligand) or as a receptor antagonist (i.e., binds the receptor but does not trigger the normal series of biological events associated with the ligand:receptor interaction) ; such ligands may be of a proteinaceous or non-proteinaceous composition.

By "hybridizes" is meant interacts under moderately stringent conditions, e.g., those hybridization conditions described herein.

By "neutrophil-derived DNA clone" is meant a genomic or cDNA molecule which encodes a neutrophil receptor (as defined above) , but which is free of the genes that, in the naturally-occurring genome of the organism from which the DNA is derived, flank the gene encoding the receptor.

By "second transmembrane domain sequence of a G-protein-coupled receptor" is meant a nucleic acid sequence identical or substantially identical to (i.e., greater than 80% identical to) the conserved nucleic acid sequence of the second membrane-spanning region of a G-protein-coupled receptor. By a "G-protein-coupled receptor" is meant a cell surface receptor which receives extracellular signals and transmits such signals to the cytoplasm through a G protein (or GTP-binding protein) ; such G proteins are membrane-localized, bind GTP, and function through their regulation of adenyl cyclase. Examples of G-protein-coupled receptors are provided in Findlay et al. (TIPS 11:492, 1990) and a list containing the amino acid sequences of the second transmembrane domain of a subset of G-protein-coupled receptors is provided in Fig. 2.

By "affinity complex" is meant a pair of molecules which have a specific affinity for each other. The affinity complexes herein include a neutrophil receptor (e.g., on the surface of a cell or included in a membrane) and a ligand specific for the receptor.

By "derived from" is meant naturally encoded by the genome .of that organism and, as used herein, present on the surface of a subset of that organism's cells (i.e., neutrophils or their precursors).

Isolation of neutrophil cell surface receptors facilitates the identification of ligands which regulate neutrophil function. Such an identification is not easily accomplished without the isolated receptor for the following reasons: (1) such receptors are generally present in very small quantities and are therefore difficult to isolate by traditional protein purification techniques; (2) many of the ligand:receptor interactions occurring on the surface of a neutrophil cell lead to degranulation (including the release of degradative enzymes) and the resultant destruction of the receptor- bearing cell; and (3) identification of a particular ligand interacting with any given receptor cannot be carried out using intact neutrophil cells because of the presence on the surface of such cells of related receptors. Isolation of the receptor genes (e.g. as described herein) allows the expression of the receptor protein in a cell type remote from neutrophils (e.g., J558 or SP2 myeloma cells or COS cells) , effectively uncoupling the receptor from its normal cytotoxic signaling pathway and dissociating the receptor from the other related receptors on the neutrophil cell surface. Production of the receptor in quantity by recombinant techniques facilitates in vitro screening methods for the identification of ligands which control neutrophil function. Examples of such in vitro screening methods are described herein; these methods are rapid and simple to perform. Ligands so identified are useful for the development of therapeutics to treat neutrophil-mediated disorders, in particular, inflammatory disorders such as psoriasis, rheumatoid arthritis, and reperfusion injury, e.g., occurring following periods of ischemia (such as in myocardial infarction or shock) .

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.

Description of the Preferred Embodiments The drawing will first briefly be described. Drawing

FIG. 1 is the nucleic acid sequence of the hybridization probe which may be used to isolate human neutrophil receptor cDNAs (SEQ ID NO:2) . FIG. 2 is the amino acid sequence of the second transmembrane domain of a number of G-protein-coupled receptor proteins (SEQ ID NOS:3-18). Neutrophil Receptor Clones Neutrophil-derived receptor clones of the invention are identified as those which hybridize with a sequence encoding the second transmembrane domain of a G- protein-coupled receptor. Design of such a hybridization probe has been facilitated by the identification of a large number of such cell surface receptors, examples of which are provided, e.g., in Findlay et al. (TIPS 11:492, 1990) . The second transmembrane domain is identified as the second hydrophobic stretch of amino acids (i.e., second from. the amino terminus) ; hydrophobicity is determined using, e.g., the Chou-Fasman method (Chou and Fasman, Ann . Rev. Biochem . 12:251, 1978). The second transmembrane domain is highly conserved among the superfamily of G-protein-coupled receptors. The amino acid sequence of a subset of such transmembrane domains (and the receptors from which the sequences were derived) are provided in Fig. 2. Using the sequences of Fig. 2 (and, if desired, others available in the art; e.g., the sequences of the receptors described in Findlay et al., supra) _r one can design a hybridization probe (e.g., an oligonucleotide probe) which includes a nucleic acid sequence corresponding to the amino acid sequence of the most highly conserved region of the transmembrane domain. Methods for the design and synthesis of such a probe is described in Ausubel et al. (supra) . Once hybridization probes are synthesized, they are used to screen a neutrophil genomic or, preferably, cDNA library. These libraries may be derived from the neutrophils of any organism using methods well known to those skilled in the art of molecular biology (see, e.g., Ausubel et al., supra) or, alternatively, the libraries may be obtained, e.g., from Clontech (Palo Alto, CA) . Hybridization screening of the neutrophil library for neutrophil receptor clones is carried out, e.g., as described in Ausubel et al., supra; or as described herein.

There now follows a description of the isolation of a number of cDNAs encoding rabbit and human neutrophil receptors. There also follows a description of assays which may be used to identify the ligands which bind these and other (similarly isolated) neutrophil receptors. These examples are provided for the purpose of illustrating the invention and should not be construed as limiting. Any neutrophil receptor isolated by the methods described herein and expressed as a recombinant protein may be used in combination with any in vitro assay (e.g., those described herein) to identify neutrophil-regulatory ligands. Rabbit and Human Neutrophil Receptor Clones

Rabbit neutrophil receptor clones according to the invention are isolated as follows.

Rabbit peritoneal neutrophils are isolated from rabbits by the method of Becker and Showell ( Immunitatsforsch . Exp. Klin . Immunol . 143:466, 1972) and used as a source of poly(A)⁺ RNA. The RNA is prepared, transcribed into cDNA, and cDNA fragments inserted into the EcoRI site of λgtll (all by the methods of Maniatis et al., Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1982) to produce a rabbit neutrophil cDNA library. Recombinant plaques are screened for those which hybridize to an antisense oligonucleotide of sequence:

3^« TTG ATG AAG GAC GAC TCG GAC CGG ACI CGI CTG GAI TAG TAC 5' (SEQ ID N0:1).

This probe is designed based on the sequence derived from the second transmembrane domain of G- protein-coupled receptors (see, e.g., Hartig et al., TIBS 10:64, 1989).

This probe is 5'-end-labeled with [³²P]ATP (Du Pont-New England Nuclear, Boston, MA) and T4 kinase (New England Biolabs, Beverly, MA) by the methods of Maniatis et al., supra. The hybridization conditions are as follows: 6X SSPE, 1% SDS, 0.1% sodium pyrophosphate, IX Denhardt's, 100 μg/ml poly(A) , and 40 μg/ml denatured calf thymus DNA at 42°C for 12h. Filters are washed with 2X SSC, 0.1% SDS at 50°C. Plaques are re-purified and screened three times by hybridization with the transmembrane domain probe.

Using this technique several rabbit clones which hybridized to the oligonucleotide probe were isolated; ten of these clones were termed RL-1, RL-2, RL-3, RL-4, RL-5, RL-6, RL-7, RL-8, RL-9, and RL-10.

Human neutrophil receptor clones according to the invention are isolated as follows.

• A human peripheral blood leukocyte λgtll cDNA library (5' s,tretch) obtained from Clontech (Palo Alto, CA) is screened with a 652 base pair EcoRI-BamEI fragment (including nucleotides -27 to 625; SEQ ID NO:2) of a rabbit neutrophil cDNA termed F3R. The sequence of this probe is shown in Fig. 1. The F3R-derived probe is labeled with [³²P]dCTP by random priming as described above. Filters are hybridized with a solution containing 50% formamide, 200 μg/ml denatured calf thymus DNA, 5X SSPE, 1% SDS, 5X Denhardt's solution, and 0.1% sodium pyrophosphate, and incubated at 42°C for 16 hours. Filters are then washed with 2X SSC and 0.1% SDS at 45°C. Plaques are re-purified and screened three times by hybridization with the 652 bp probe (SEQ ID NO:2) .

Using this technique, several human clones which hybridized to the rabbit neutrophil receptor probe were isolated; eight of these clones were termed HL-1, HL-2, HL-3, HL-4, HL-5, HL-6, HL-7 , and HL-8.

Human neutrophil receptor clones may also be isolated using the probe described above for the rabbit receptor clones. Preferably, a human peripheral blood leukocyte λgtll cDNA library (5¹ stretch) obtained from Clontech is screened with an oligonucleotide based on the second transmembrane domain of G-protein-couple receptors, e.g., of sequence: 3' TTG ATG AAG GAC GAC TCG GAC CGG ACI CGI CTG GAI

TAG TAC 5' (SEQ ID NO:l).

This probe is 5'-end-labeled with [³²P]ATP (Du Pont-New England Nuclear, Boston, MA) and T4 kinase (New England Biolabs, Beverly, MA) by the methods of Maniatis et al., supra. The hybridization conditions are as follows: 6X SSPE, 1% SDS, 0.1% sodium pyrophosphate, IX Denhardt's, 100 μg/ml poly(A) , and 40 μg/ml denatured calf thymus DNA at 42°C for 12h. Filters are washed with 2X SSC, 0.1% SDS at 50°C. Plaques are re-purified and screened three times by hybridization with the transmembrane domain probe, --Expression of Neutrophil Receptors

The neutrophil receptors according to the invention may be produced by transformation of a suitable host cell with a receptor-encoding cDNA (e.g., described above) in a suitable expression vehicle.

Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant receptor protein. The precise host cell used is not critical to the invention, however the following host cells are preferred: SP2 cells, Chinese Hamster Ovary (CHO) cells, COS-7 cells, and fibroblast cells, such as mouse 3T3 cells. Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, MD; Accession Nos. CRL 1581, CCL 61, CRL 1651, and CCL 163, respectively) . The method of transfection and the choice of expression vehicle will depend on the host system selected. Mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) ; expression vehicles may be chosen from those provided, e.g., in Cloning Vectors : A Laboratory Manual (P.H. Pouwels et al. , 1985, Supp. 1987). One preferred expression system is the mouse 3T3 fibroblast host cell transfected with a pMAMneo expression vector (Clontech, Palo Alto, CA) . pMAMneo provides: an RSV-LTR enhancer linked to a dexamethasone- inducible MMTV-LTR promoter, an SV40 origin of replication (which allows replication in mammalian systems) , a selectable neomycin gene, and SV40 splicing and polyadenylation sites. DNA encoding the neutrophil - receptor (as described above) would be inserted into the pMAMneo vector in an orientation designed to allow expression. The recombinant receptor protein would be isolated as described below. Other preferable host cells which may be used in conjunction with the pMAMneo expression vehicle include COS cells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61, respectively) . Another particularly preferred expression system is the COS host cell (ATCC Accession No. CRL 1650) transiently transfected (e.g., as described above) with the pSVL vector (Pharmacia, Piscataway, NJ) into which a receptor-encoding cDNA has been inserted in an orientation which permits expression of the receptor protein.

The neutrophil receptor protein may also be produced by a stably-transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra) ; methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra) . In one example, cDNA encoding the receptor is cloned into an expression vector which includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the receptor-encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 μM ethotrexate in the cell culture medium (as described in Ausubel et al. , supra) . This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra) ; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. DHFR- containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV(A) (described in Ausubel et al., supra) . Any of the host cells described above or, preferably, a DHFR- deficient CHO cell line (e.g., CHO DHFR^" cells, ATCC Accession No. CRL 9096) are among the host cells preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification. One particularly preferred stable expression system is the myeloma cell line, J558 (ATCC Accession No. T1B6) or SP2 (ATCC Accession No. CRL 1581) stably transfected with pSV2-gpt (ATCC Accession No. 37145) . pSV2-gpt provides: an SV40 early promoter and a selectable qpt marker (i.e., E. coli xanthine-guanine phosphoribosyl transferase) . Such a stable expression system may be constructed as described in Ausubel et al. (supra) .

Once the recombinant receptor protein is expressed, it is isolated, e.g., by preparing a β- galactosidase-receptor fusion protein and using affinity chromatography as described in Ausubel et al. (supra) . Lysis and fractionation of receptor-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra) . Once isolated, the β-galactosidase receptor protein is used to produce a polyclonal antiserum by the method of Ausubel et al, supra; such a polyclonal antiserum includes antibodies specific for the neutrophil receptor protein. These antibodies facilitate the isolation and purification of the recombinant neutrophil receptor protein (e.g. , by affinity chromatography, see above) , e.g., from a supernatant derived from recombinant cells stably expressing the protein. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980) . Identification of Ligands which Bind Neutrophil Receptors As discussed above, one aspect of the invention features a screening assay for the identification of compounds which specifically bind to the neutrophil receptors described herein. Such an assay may be carried out using a recombinant receptor protein expressed on the surface of a transfected cell.

In one example, the neutrophil receptor component is produced by a cell that naturally presents substantially no receptor, e.g., by engineering such a cell to contain nucleic acid encoding the receptor component in an appropriate expression system. Suitable cells are, e.g. , those discussed above with respect to the production of recombinant receptor, most preferably myeloma J558 or SP2 cells or COS cells. Preferably, the membranes of recombinant cells are prepared by sucrose gradient centrifugation (e.g., by the method of Colman, Transcription and Translation , IRL Press, Oxford, 1986) and a labelled candidate ligand (e.g., radiolabelled using 125I or 3H) is added. The membrane preparation is subjected to vacuum filtration through Whatman GF/C filters (by the method of Williamson, Biochemistry 22:5371, 1988) and ligand interaction with the receptor is assayed as label in association with the Whatman filter.

Specific interaction of a ligand with a recombinant receptor may also be assayed by fixing the cell expressing the neutrophil receptor component to a solid substrate (e.g., a microtiter well, a test tube, or a column) by means well known to those in the art (see, e.g., Ausubel et al. , supra) , and presenting labelled candidate ligand to the cell. The assay format may be any of a number of suitable formats for detecting specific binding, such as a radioim unoassay format. For example, cells transiently or stably transfected with a neutrophil receptor expression vector are immobilized on a solid substrate (e.g. , the well of a microtiter plate) and reacted with a candidate ligand which is detectably labelled, e.g., with a radiolabel, e.g., 125I or an enzyme which can be assayed, e.g., alkaline phosphatase or horseradish peroxidase. Binding is assayed by the detection label in association with the receptor component (and, therefore, in association with the solid substrate) . A molecule which specifically binds to the neutrophil receptor-expressing cells (i.e., with substantially greater affinity or in a substantially greater amount than that bound to an identical host cell not expressing the recombinant receptor) is considered to be a ligand for that particular neutrophil receptor. The ligand may be purified (or substantially purified) and tested by the above assay, or the ligand may be one component of a mixture of labelled ligands (e.g. , an extract obtained from cells metabolically labelled with ³⁵S-methionine; Ausubel et al., supra) . In a mixed ligand assay, the recombinant receptor-expressing cells are contacted with the ligand mixture, and a specific ligand:receptor interaction detected in a two- step process. Cells are first washed (e.g., with a low salt wash) to remove non-specifically bound ligands. The ligand of interest (i.e., the ligand which is specifically bound to the receptor following washing) is then released from the receptor (e.g., by a high salt wash) . The ligand is purified from the supernatant by standard biochemical techniques (using the label as a means of detection) and characterized, e.g., by amino acid sequencing and PCR cloning (as described below) . In an alternative in vitro binding assay, a " candidate ligand which is purified or substantially purified is adhered to the solid substrate (e.g, a microtiter plate; using methods similar to those for adhering antigens for an ELISA assay; see, e.g. , Ausubel et al., supra) and the ability of labelled neutrophil receptor-expressing cells (e.g., labelled with ³H- thymidine; see Ausubel et al., supra) can be used to detect specific receptor binding to the immobilized candidate ligand.

In one particular example of this assay, a vector expressing a neutrophil receptor is transfected into COS- 7 cells (ATCC Accession No. CRL 1651) by the DEAE dextran-chloroquine method (Ausubel et al., supra) . The candidate ligand does not bind to untransfected host cells or cells bearing the parent vector alone. 10 cm. tissue culture dishes are seeded with neutrophil receptor-expressing COS-7 cells (750,000 cells, dish) 12-18h post- ransfectio . Forty-eight hours later, triplicate dishes are incubated with 0.5nM radioiodinated candidate ligand (-200 Ci/mmol) . Binding of the ligand to the receptor-expressing cells is assayed as association of the detectable label with the tissue culture dish following washing (i.e., an association which is greater than the non-specific interaction, if any, which is observed with the control cells) .

The recombinant receptors may also be used to identify ligands by affinity chromatography. Recombinant receptor is purified by standard techniques, from cells engineered to express the receptor (e.g., those described above) ; the recombinant receptor immobilized on a column (e.g., a Sepharose column or a streptavidin-agarose column by the immoaffinity method of Ausubel et al.) and a solution containing one or more candidate ligands is passed through the column. Such a solution (i.e., such a source of candidate ligands) may be, e.g. , a cell extract, mammalian serum, or growth medium on which mammalian cells have been cultured and into which the cells have secreted factors (e.g. , growth factors) during -culture. A ligand specific for a recombinant receptor is immobilized on the column (because of its interaction with the receptor) . To isolate the ligand, the column is first washed (e.g., with a low salt solution) to remove non-specifically bound proteins, and the ligand of interest is then released from the column (e.g. , by a high salt wash) . The ligand is collected and, if desired, further purified (e.g., by high performance liquid chromatography; see above) . Once isolated in sufficiently-purified form, the protein may be partially sequenced (by standard techniques) . From this partial amino acid sequence, a partial nucleic acid sequence is deduced which allows the preparation of primers for PCR cloning of the ligand gene (e.g., by the method of Ausubel et al. , supra) .

Other Embodiments Using a DNA probe derived from a second transmembrane domain sequence of a G-protein-coupled receptor or the probe of Fig. 1, other neutrophil receptors may be isolated, e.g., from other species. Identification of the ligand which specifically binds to each of these receptors is carried out as described herein.

What is claimed is:

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: the Trustees of Boston University

(ii) TITLE OF INVENTION: METHOD OF ISOLATING LIGANDS WHICH BIND NEUTROPHIL RECEPTORS

(ill) NUMBER OF SEQUENCES: 18

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: IBM P.C DOS (Version 3.30)

(D) SOFTWARE: WordPerfect (Version 5.0)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA :

(A) APPLICATION NUMBER:07/693/440

(B) FILING DATE: April 29, 1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul T. Clark

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER: 04766/006WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070 (B) TELEFAX: (617) 542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TTGATGAAGG ACGACTCGGA CCGGACNCGN CTGGANTAGT AC 42

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 652

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CGATCTTAAG GTGAAACTGT GGCCGTAATG GAAGTAAACG TATGGAATAT GACTGATCTG 60

TGGACGTGGT TTGAGGATGA GTTTGCAAAT GCTACTGGTA TGCCTCCTGT AGAAAAAGAT 120

TATAGCCCCT GTCTGGTAGT CACCCAGACA CTTAACAAAT ATGTTGTGGT CGTCATCTAT 180

GCCCTGGTCT TCCTGCTGAG CCTGCTGGGC AACTCCCTGG TGATGCTGGT CATACTGTAC 240

AGCCGGAGCA ACCGTTCGGT CACCGACGTC TACCTGCTGA ACCTGGCCAT GGCACCTGCT 300

TTTTGCCCTG ACCATGCCTA TCTGGGCCGT CTCCAAGGAA AAAGGCTGGA TTTTCGCACG - 360

CCCCTGTGCA AGGTGGTCTC GCTTGTGAAG GAAGTCAACT TCTACAGTGG AATCCTGCTC 420

CTGGCCTGCA TCAGTGTGGA CCGCTACCTG GCCATTGTCC AGTCTACTCG CACACTGACC 480

CAGAAGCGCC ACTTGGTCAA GTTCATATGT CTGGGCATCT GGGCGCTGTC TCTGATTTTG 540

TCCCTGCCCT TCTTCCTCTT CCGCCAAGTC TTTTCTCCAA ACAATTCCAG CCCGGTCTGC 600

TATGAGGACC TGGGTCACAA CACAGCGAAA TGGTGCATGG TGCTGCGGAT CC 652

«(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Leu Leu Asn Leu Ala Val Ala Asp Leu Phe Met

5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

lie Thr Ser Leu Ala Cys Ala Asp Leu Val Met 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: lie Thr Ser Leu Ala Cys Ala Asp Leu Val Met 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 6:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

lie Thr Ser Leu Ala Cys Ala Asp Leu Val Met 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 7:

V (i) SEQUENCE CHARACTERISTICS:

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Leu Leu Ser Leu Ala Cys Ala Asp Leu lie lie 5 10

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 8; (i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Phe Ser Leu Ala Cys Ala Asp Leu lie lie 5 10

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(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Met Ser Leu Ala lie Ala Asp Met Leu Val . 5 10

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(A) LENGTH: 11

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(D) TOPOLOGY: linear

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(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Leu Ser Leu Ala Cys Ala Asp Leu lie lie 5 10

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(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Leu Ser Leu Ala Cys Ala Asp Leu lie lie 5 10

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(A) LENGTH: 11

(B) TYPE: amino acid (D) TOPOLOGY: linear

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lie Met Ser Leu Ala Ser Ala Asp Leu Val Met 5 10

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(A) LENGTH: 11

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ϊϊe Gly Ser Leu Ala Val Thr Asp Leu Met Val 5 10

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(A) LENGTH: 11

(B) TYPE: amino acid

(D) TOPOLOGY: linear

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(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Phe Ser Leu Ala Cys Ala Asp Leu lie lie 5 10

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(B) TYPE: amino acid (D) TOPOLOGY: linear

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Leu Phe Ser Leu Gly Cys Ala Asp Leu lie lie 5 10

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r Leu Ala Val Ala Asp Leu lie Val 5 10

Claims

Claims 1. A method of determining whether a ligand is capable of binding to a neutrophil receptor, comprising (a) providing a neutrophil-derived DNA clone which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene or with a DNA fragment which hybridizes with the second transmembrane domain-encoding sequence of a G-protein- coupled receptor gene; (b) introducing said DNA clone into a cell which presents substantially no said neutrophil receptor on its surface so that the recombinant protein encoded by said DNA clone is presented on the surface of said cell; (c) contacting said cell or a membrane fragment thereof with said ligand to allow formation of an affinity complex between said cell or membrane and said ligand; and (d) detecting any such complex formation.

2. A method of determining whether a ligand is capable of binding to a neutrophil receptor, comprising ^•*• (a) providing a neutrophil-derived DNA clone which hybridizes with the second transmembrane domain-encoding sequence of a G-protein-coupled receptor gene or with a DNA fragment which hybridizes with the second transmembrane domain-encoding sequence of a G-protein- coupled receptor gene; (b) introducing said DNA clone into a cell which expresses substantially no said neutrophil receptor so that the recombinant protein encoded by said DNA clone is expressed; (c) isolating said recombinant protein; (d) immobilizing said recombinant protein on a solid substrate; (e) contacting said immobilized recombinant protein with said ligand to allow formation of an affinity complex between said immobilized recombinant protein and said ligand; and (f) detecting any such complex formation.

3. The method of claims 1 or 2, wherein the sequence of said second transmembrane domain encoding sequence of a G-protein-coupled receptor gene comprises the sequence: 3' TTG ATG AAG GAC GAC TCG GAC CGG ACI CGI CTG GAI TAG TAC 5' (SEQ ID NO:l).

4. The method of claims 1 or 2 wherein said DNA fragment comprises the sequence of Fig. 1 (SEQ ID NO:2).

5. The method of claims 1 or 2, wherein said cell is a eukaryotic cell.

6. The method of claim 5, wherein said cell is a mammalian cell.

7. The method of claim 6, wherein said cell is a COS cell.

8. The method of claim 2, wherein said solid substrate is a column.

9. The method of claims 1 or 2, wherein said neutrophil receptor is derived from a human.

10. The method of claims 1 or 2, wherein said neutrophil receptor is derived from a rabbit.