WO1988003172A1

WO1988003172A1 - DNA EXPRESSING Fc RECEPTOR PROTEIN

Info

Publication number: WO1988003172A1
Application number: PCT/US1987/002845
Authority: WO
Inventors: Jeffrey V. Ravetch; Jay C. Unkeless
Original assignee: Memorial Sloan-Kettering Cancer Center; The Rockefeller University
Priority date: 1986-10-28
Filing date: 1987-10-28
Publication date: 1988-05-05
Also published as: AU8270187A; JPH01501206A

Abstract

Substantially pure DNA expressing Fc receptor proteins is taught. Additionally, the proteins thus expressed and applications employing these are set forth as well.

Description

DNA EXPRESSING F_c RECEPTOR PROTEIN

FIELD OF THE INVENTION

This invention relates to immunoglobulin receptors. In particular, it relates to nucleotide sequences which express the receptor molecules, the receptor molecules themselves, as well as transformed cell lines which produce the molecules. In addition, methods for producing the protein are taught. Finally, also taught are analogs to organs which are associated with immunoglobulin receptors.

BACKGROUND AND PRIOR ART

For the immune system to achieve the function of protecting the organism against foreign antigens, cooperation between the humoral and cellular pathways occurs via interaction of antibody-antigen complexes with effector cells, mediated by specific antibody receptors, known as Fc receptors. These receptor molecules act in a critical way to mediate binding of antibodies to effector cells, as well as in the regulation of antibody function.

Receptors for the Fc domain of immunoglobulin G, which is the most common class of immunoglobulin, are known to be present on B cells, some T cells, natural killer or "NK" cells, macrophages, and polyrnorphronuclear leukocytes. In this regard see, e.g. ϋnkeless, et al., Adv. Immunol. 31: 247-270 (1981); Springer, et al., Contemp. Top. Immunobiol. 13: 1-31 (1984); Dickler, Mol. Immunol. 19: 1301-1306 (1982).

When immune complexes bind to Fc receptors on neutrophils and macrophages, this triggers cellular responses which include phagocytosis, release of activated oxygen metabolites, and mediators of inflammation such as leukotrienes and prostaglandins, as well as induction of neutral hydrolases. See, e.g. Nathan, et al., N. Eng. J. Med. 303: 622 (1980). Fc receptors have been described on lymphocytes as well; Uhen, et al., Cellular Immunol. 95: 368-379 (1985), where they are presumed to have a role in the modulation of antibody production by B cells.

Fc receptors have been described for all classes of immunoglobulin (IgA, IgD, IgE IgG, IgM), but very little is known about the molecules themselves. Perhaps the best characterized receptors are what are known as the "high avidity basophil/mast cell" IgE receptor (FcE), and mouse macrophage Fc receptor which binds IgG2b/TgGl immune complexes (FcG2b/1R). With respect to murine specimens , studies o f competitive binding of different IgG subclasses (Diamond, et al., J. Exp. Med. 150: 721-726 (1979); J. Immunol. 125; 631-633 (1980); J. Exp. Med. 153: 514-519 (1981), and differential sensitivity to proteases (Unkeless, J. Exp. Med. 142: 1520 (1975); have demonstrated the presence of binding sites for IgG3 (FcG3R), IgG2a (FcG2aR), and IgG2b/G1 complexes (FcG2b/1R). The latter molecule has been described as an integral membrane glycoprotein of 50-60

K daltons, with four sites for N-linked glycosylation. See, in this regard. Green, et al., J. Biol. Chem. 260: 9867-9874

(1985).

Recent work has made it necessary to obtain detailed biochemical characterization of receptors. A monoclonal antibody, 2.4G2 is known which is directed against an epitope present on murine FcG receptors of both macrophages and lymphocytes. Unkeless, J. Exp. Med. 150: 580-596 (1979). While the epitope recognized is known to be present on macrophages and lymphocytes, work by, e.g. Phillips, et al., J. Immunol. 134: 2835-2838 (1985); Baum, et al., J. Exp. Med. 162; 282-296 (1985), and Teilland, et al . , J. Immunol . 134 : 1774- 1779 ( 1985 ) , shows that the isotype specificity of the lymphocyte receptor active with 2.4G2 is broader than FcG2b/1R, which does not bind IgG2a. Also, FcG2b/1R has been identified as an alloantigen related to the known Ml system described by Mark, et al., J. Immunol. 135: 2635-241 (1985), and Hibbs, et al., Immunogen. 22: 335-348 (1985), a locus on chromosome 1 which specifies products on antigen presenting cells and governs a non H-2T cell proliferative response resulting in intense stimulation of the mixed lymphocyte reaction. Four alleles of the locus have been identified (Festenstein Transplantation Proceedings 8: 339-342 (1976), and the Ly-17 antigen, which is known to be identical to FcG2b/1R (Holmes, et al., Proc. Natl. Acad. Sci. 82: 7706-7710 (1985) is either closely linked to or determined by this locus.

Given the differences in isotype specificity between macrophage and lymphocyte receptors, as well as the functional heterogeneity thereof, isolation and characterization of cDNAs of macrophage and T cell clones was undertaken. The result of these experiments was isolation of two genes, one of which exists in two different allelic forms, which express proteins with highly homologous extracellular domains which bind IgG, but which possess different transmembrane and cytoplasmic domains. These genes will be referred to as FcαR, FcβR, and Fcβ₂R hereafter.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the restriction map and sequencing strategy (a) and nucleotide sequence (b) for the gene expressing macrophage FcGα UT = untranslated sequences, S = signal sequence, extracellular domain = E-C, transmembrane domain = TM, and cytoplasmic domain = C. Figure 2 shows homology of FcG protein to MHC class II protein E . A single dot indicates conservative mutation, while two dots indicate identity.

Figure 3 shows the distribution of FcGα mRNA in various cell lines.

Figure 4 parallels Figure 1 in that it shows the restriction map and sequencing strategy (a), and nucleotide sequence of FcGβ₁ cDNA (b).

Figure 5 depicts amino acid alignment of FcGα and FcGβ₁ proteins. Overall, there is 95% homology in the extracellular portion.

Figure 6 shows Southern Blot Analysis of DNAs of inbrid mice, and identifies a polymorphism linked to the FcG gene.

Figure 7 shows the distribution of β, transcripts in different cell lines, and provides evidence for a macrophage β₂ transcript.

Figure 8 shows the restriction map and sequencing strategy (a), and the nucleotide sequence (b) of FcG β₂ cDNA. Figure 9 shows the expression of FcGβ₂ in transfected mouse melanoma cells, via reconstitution of immunoglobulin binding activity.

Figure 10 is a summary of the structure of the IgG2b/G1 FcR cDNA genes obtained from macrophage and T cell lines.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Purification and Amino-Terminal Sequence of FcγR

Fcγ receptor was purified from the S49.1 cell line by a slight modification of the published procedure of-Mellman, et al. J. Exp. Med. 152: 1048 (1980). Cells (1 x 10¹⁰) grown in suspension culture were lysed in 30 ml of 2% Nonidet P-40 in PBS containing 0.2 TIU aprotinin per ml and 5 mM diisopropylfluorophosphate. The 40,000 xG supernatant from the lysate was applied to a 5 ml column of Sepharose 4B coupled with 2 mg of 2.4G2 IgG per ml of resin. The column was washed first with 10 column volumes of 1% NP-40/0.2% sodium dodecyl sulfate in PBS, and then with 10 column volumes of 10 mM octyl-β-D-thioglucoside in PBS. Protein was eluted with PBS containing 50 mM triethylamine, 10 mM octyl-β-D-thioglucoside, pH 11.0 and rapidly adjusted to neutrality with Tris HCl. The protein was acidified with trifluoroacetic acid, applied to a Supelco C8 2 cm column and eluted with a gradient of acetonitrile 0.1% trifluoroacetic acid, as described by Pan, et al.,

J. Chromatog. 297: 13-19 (1984).

The amino terminal 22 amino acids for the S 49 .1

IgG2b/γ1 receptor were determined on 200 pMoles of the above material using a Waters HPLC system and a microsequencing apparatus (Applied Biosysems model 470A) and found to be THDLPKAWKLEPPWIQVLKED. The seven amino acids EPPWIQV were chosen for the synthesis of a corresponding oligodeoxynucleotide based on their relatively low degeneracy. After considering preferred codon usage described by Chen, et al., DNA 4: 365-374 (1982) and the ability of G-T base pairing to form, a 20 nucleotide sequence was synthesized that corresponds to the complementary strand of the sequence encoded above. That sequence is 5' 3'.

Necleotide and Deduced Portein Sequence of the Macrophage FcγRα

The low degeneracy mixed probe described supra was end-labelled to high specific activity with ³²p-γATP and used to screen a mouse macrophage cDNA library constructed to size-fractionated J774 mRNA in the plasmid vector pUC9 (Portnoy, et al., J. Biochem. (1986). A library of 50,000 clones was screened from which 2 positives were identified. The screening involved a hybridization reaction which included 6X NETS, IX Denhardts, 10⁶cpm/ml ³²p-end labelled oligonucleotide (10⁹cpm/Ag) at 45ºC for 16 hours. The filters were then washed in 6X SSC at 25º for 5 minutes, followed by a 40°C wash in the buffer described supra for 1 minute, followed by a 1 minute wash in the same buffer at

45°C. Filters were dried, autoradiographed for 6 hours at

-70ºC with intensifying screens and developed. Positive clones were identified and the filters rewashed at 50°C in

6X SSC for 1 minute and then at 65°C for 1 minute.

Positives melt off differentially at the higher temperature and were subjected to colony purification, plasmid preparation and restriction analysis. The clone with the largest insert (a 1300 base pair Pstl fragment) was chosen for subsequent analysis. The sequencing strategy for this clone is presented in Figure la with the nucleotide sequence and deduced amino acid sequence in Figure 1b. An open reading frame of 782 nucleotides was found, beginning with an ATG at position 64 and terminating at position 846. The predicted signal peptidase cleavage site is indicated by an arrow and is assigned based on the consensus rules for such sequences (Von Heijne , Eur. J. Biochem 133: 17-21 (1983). 19 amino acids, encoded from nucleotides 160-210, are identical to amino acids 3-22 of the S49.1 sequence presented above, with the exception of position 12 which is a glutamic acid residue in S49.1 and an aspartic acid in J774. The discrepancy in the first three amino acids and position 12 resulted from the heterogeneity between the macrophage and T cell proteins, described infra. A 30 amino acid signal sequence is predicted, and is numbered -30 to -1 with a hydrophobic core overlined. Predicted signal peptidase cleavage site is indicated by the arrow between -1 and 1 following Von Heijne, supra. N-linked glycosylation sites are boxed and cysteine residues circled. The deduced protein sequence contains two regions of hydrophobic amino acid residues, overlined in Figure 1b. These regions encode the putative signal sequence (nucleotides 64-153) and a transmembrane anchor sequence (nucleotides 709-769). An extracelluar domain of 185 amino acids is expected for the mature protein which contains 4 potential N-linked glycoslyation sites (boxed in Figure 1b) as well as four cysteine residues which could form 2 intra-chain disulfide bonds. A serine and threonine rich region is encoded from amino acids 155-185, just preceding the transmembrane domain, in which 30% of the residues are represented by these two amino acids. A cytoplasmic domain of 26 amino acids is predicted from this sequence. The primary sequence predicts a molecular weight of 30,040 daltons, which would then be subject to glycoslyation at the four N-linked sites described, and possibly O-linked sites as well.

The extracellular domain described above consists of two internally repeated sequences. Amino acids 25-75 of the predicted mature protein show homology to amino acids 100-155. These homologies cluster around the cysteine residues, suggesting a structural repeating domain. These data suggest that the extracelluar domain consits of a repeated domain defined by the cysteine residues. Comparison of this sequence to the protein sequence databanks revealed significant homology to immunoglobulin molecules, MHC class I and class II proteins, β2 microglobulin and other members of this supergene family. Homology of the extracelluar domain with a rabbit V region has been deomonstrated showing clusters of homology centered about the cysteine residues in both proteins. This homolog suggests that this Fc receptor contains two immunoglobulin-like domains, each consisting of a potential disulfide loop of 42 amino acids within a domain of 70-80 amino acids. The most significant homology we found for this FcR is to the MHC class II protein Eβ, with 32% identity over a 91 amino acid region, as shown in Figure 2a. Random shuffling of these two sequences, using the program rdf (Lipman, et al., Science 227: 1435-1441 (1985), indicated that the optimized alignment shown in Figure 2a is highly significant, at 5 standard deviations above the mean. This homology to E β occurs in the β₂ domain (Figure 2b) which itself is an immunoglobulin-like domain. Homology is also apparent by this analysis in the transmembrane domain of these two proteins. mRNA extracted from a variety of cell lines was analyzed for the presence of message corresponding to the cDNA cloned from the J774 cell line, referred to as the α gene. The procedure used generally followed Chitgwin, et al., Biochem. 18: 5294 (1979), and Lehrach, et al., Biochem. 16: 4743-4751 (1977). Briefly, 1 ug of poly A⁺ RNA was fractionated on agarose-formaldehyde gels transferred to nitrocellulose and hybridized under stringent conditions either with the complete α probe (a) or a probe constructed to the 5' α sequences (b). As can be seen in Figure 3, a broad band of hybridization is detected in the macrophage lines P 388D1, WEHI, 3A, RAW 264.7 and J774. A T cell line S49.1, which reacts with the FcfR monoclonal antibody 2.4G2 contains a higher molecular weight mRNA species. P388 demonstrates two RNA species of equivalent abundance, while WEHI 3A has a major band migrating faster than 18S and a band of much lower abundance migrating slower than 18S. 2.4G2 negative lines CL.7 (fibroblast), L-cell (fibroblast) and L51789 (T cell) do not contain an α transcript. The cell type specificity of expression of the α gene was apparent when a probe was constructed to the 5' sequences of the cDNA and used to examiner the same macrophage and T cell RNAs (Figure 3b). No transcript is detected in the S49.1 cell line with this probe and only a single species is seen in P388. Similarly, the lower abundance species migrating slower than 18S in WEHI 3A is not detected with this probe. Similar results were obtained when a 3' probe was constructed from the α cDNA (Sca-Pst, data not shown). These results show that the T cell line S49.1 and the macrophage-like lines P388 and WEHI 3A contained cross-hybridizing RNA species when probed with the complete probe which are not homologous to the macrophage α transcript on their 5' and 3' ends. Analysis of two other T cell lines EL-4 and K-36 demonstrated transcripts of similar size to that found in S49.1 (data not shown). To identify these transcripts and the genetic basis for their expression, a cDNA library was constructed to size-fractionated S49.1 mRNA and probed with the complete cDNA α probe. Positive clones were identified at a frequency of .1% and were characterized by restriction mapping and DNA sequence analysis.

Figure 4a presents the physical map and sequencing strategy for the T cell transcript, referred to as β₁, and 4b presents the nucleotide sequence and predicted amino acid sequence for this protein. The procedures used were the same as those described supra for obtaining the physical map and sequence of macrophage DNA. A single open reading frame was found, beginning with an ATG at nucleotide 340 and terminating at nucleotide 1326. The predicted molecular weight of this protein before modification is 36,750 daltons. Beginning at nucleotide 427 (position +1) 22 amino acids are encoded which are identical to the determined amino terminal sequence for this protein. The sequence which precedes this N terminus encodes a signal sequence with a characteristic hydrophotic core (overlined), which bears no homology to the signal sequence of the α transcript (see Figure 1b). The extracellular domain has 95% identity with the α sequence, as shown in Figure 5, beginning at amino acid 4 of the β₁ sequence and continuing through amino acid 174. A transmembrane and cytoplasmic domain are predicted for the β₁ sequence, which bear no homology to the analogous domains of the sequence. No sequence homology is found in the 5' and 3' untranslated domains of these two transcripts.

Four additional tryptic peptides were sequenced for the S49.1 FcγR. A 10 amino acid sequence SQVQASYTFK was confirmed at positions 50-59 of the β₁ sequence; the 8 amino acid sequence ISFFHNEK was confirmed at positions 120-127; the 13 amino acid sequence EMGETLPEEVGEY was present at positions 222-235, and finally the 13 amino acid sequence TEAENTITYSLLK was confirmed at positions 271-283.

Since both α and β ₁ sequences were obtained from cell lines derived from Balb/c mice, the 5% sequence variation in the highly conserved extracelluar domain could not arise from allelic variation. To confirm that the β₁ transcript was derived from a second gene. Southern blot analysis of DNA obtained from different inbred strains of mice was used to map the α and β genomic sequences. As seen in Figure 6, DNA cut with Taq 1 and probed with the complete α probe detects a polymorphism associated with this gene, giving rise to three different restriction fragment lengths. This polymorphism resides 3' of the α gene, as determined by reprobing these blots with 5' and 3' probes (data not shown). However, when these same DNA samples are probed with the complete B. cDNA probe (Figure 6b), the 3' polymorphic fragment associated with the α gene is replaced by 2.4 kb non-polymorphic fragment. These data suggest that the α and β; transcripts are derived from different genes, which contain a highly conserved sequence encoding an extracellular domain. Hybridization with both the α and β probes under conditions of low stringency (25% formamide, 10% dextran sulphate, 5X SSC at 40°C; final wash = .2X SSC, .1% SDS 40ºC) revealed an additional cross-hybridizing restriction fragment, perhaps suggestive of a third member of this gene family (not shown).

Expression of the β Gene in T cell and Macrophage Lines

Figure 7a presents the result of RNA blot analysis of macrophage and T cell lines probed with the β₁ cDNA probe. This probe is expected to detect the S49.1 transcript, as well as cross-hybridize to the α transcript in macrophage lines. However, comparing the results of this blot with that in Figure 4a it is clear that a different pattern of transcripts are detected with different abundance. In particular WEHI 3A and P388 demonstrates two transcripts of comparable abundance one of which has the same apparent mobility as the S49.1 transcript. This is confirmed by using a 5' β₁ probe, shown in Figure 7b, constructed to the 5' untranslated region of this transcript in which no homology to the α gene is found. Transcripts are detected in macrophage lines with this probe, demonstrating that the β gene is expressed in macrophages, resulting in a different transcript size in J774, RAW and P388D1 than what is found in the S49.1 T cell line. To investigate these macrophage β transcripts, the J774 cDNA library was screened with both unique and common sequences derived from the β gene. Restriction enzyme mapping of the clones obtained demonstrated that these clones differed from the _β1 transcript of the S49.1 cell line in lacking an Xmn site. The physical map and sequencing strategy for the macrophage β transcript, referred to as β₂ is shown in Figure 8a and the nucleotide sequence and deduced amino acid sequence in Figure 8b. The procedure followed for obtaining these was the same as that given supra. Comparison of the β₁ and β₂ Transcripts; Evidience for a T Cell Specific Splice in the Cytoplasmic Domain

The β₁ and β₂ sequences are identical throughout their length, both for coding and noncoding sequencies, with the exception of a 138 nucleotide insertion found in the β₁ sequence (nucleotides 1066-1204, indicated by the upward arrows in Figure 4b) which occurs after nucleotide 783 in the β₂ sequence. This sequence results in a 46 amino acid insertion in the cytoplasmic domain of the β₁ transcript. This insertion accounts for the larger transcript found in S49.1 as well as in the T cell lines EL-4 and K-36 (data not shown). RNase protection using a T7 RNA polymerase generated radiolabelled RNA probe specific for the β₁ gene (Xmn-Pst, see Figure 4a) detected a 500 nucleotide protected fragment, specific for the β₁ gene, in the S49.1 and k-36 T cell lines and not in the macrophage line P388D1. In addition to the 500 bp protected fragment, a 362 bp fragment was detected in S49.1 and K-36 which comigrated with the P388D1 macrophage protected fragment, consistent with the size expected to be protected by the β₂ transcript. These results suggest that the β gene is transcribed in T cells and macrophages. In T cells, however, a transcript with a 138 bp insertion is found, which is most simply explained by an alternative splicing pathway which gives rise to an additional exon in the T cell FCγRβ. The predicted molecular weight of the β₂ protein is 31,886 prior to any post-translational modification.

Functional Domains of the FcγR; Expression in Transfected Cell Lines

In order to begin to assess the functional role of the structural heterogeneity described for this Fc receptor and to ascertain if more than one polypeptide chain was necessary for ligand binding, Fc receptor negative cell lines were transfected with these cDNA clones. Expression was achieved by cloning the coding sequence of the β₁ or β₂ cDNAs into an expression vector (pcEXV-3) as described by Miller, et al., J. Immunol. 134; 4212-4217 (1985) which utilizes the SV40 early promoter to achieve transcription of the cloned sequences. B78H1 mouse melanoma cells were cotransfected with the plasmid constructions and pGCcos3neo, which confers resistance to the drug G418 (Southern, et al., J. Mol. Appl. Genet. 1: 327-341 (1982). After 10 days in G418 containing medium colonies were screened by rosetting with human erythrocytes conjugated with the monoclonal antibody 2.4G2, as described by Albino, et al., Mol. & Cell Biol. 5: 692-697 (1985). Positive cells were cloned and then tested for Fcγ receptor activity.

Figure 9 presents the results obtained with the β₂ cDNA cloned into the expression vector. Stable lines expressing the 2.4G2 epitope avidly bound sheep red blood cells opsonized with rabbit anti-SRBC, which is diagnostic of Fcγ receptor function. In addition, this binding is blocked in a concentration dependent fashion by the monoclonal antibody 2.4G2, demonstrating the specificity of binding to this receptor. Transfectants which were obtained with the β₁ insert in the expression vector demonstrated the same pattern of binding. Controls with untransfected B78H1 cells, transfected cells in which the FcγR β sequences were in the reverse orientation with respect to the SV40 promoter as well as studies with SRBCs not coated with antibody all gave negative results (data not shown). These experiments indicate that the protein expressed by these cDNA clones is able to be displayed on the cell surface and mediate the binding of antibody-antigen complexes in a specific manner. Experiments with other cell lines, such as mouse L cells or monkey cos cells suggests that the ability of these sequences to specify an Fc receptor is not cell type specific. Two genes have been identified that encode Fcγ receptors. One of these, referred to as α, is expressed in macrophage cell lines and peritoneal macrophages. The second gene, referred to as β, is expressed in both macrophage and in T-cell lines. These genes enclode transmembrane proteins with two repeated N-terminal domains each containing two glycosylation sites, and a potential intrachain disulfide loop of 42-45 amino acids. This predicted structure is consistent with in vivo labeling studies, which have suggested the presence of 4 N-linked glycosylation sites. While the extracellular domains of the γ and β genes are 95% homologous, the transmemb rane and cytoplasmic domains encoded by the two genes are totally different, which suggests that the different functions of lymphocyte and macrophage Fcγ receptors derives in part from different signaling mechanisms.

There is, however, still another level of complexity in this gene system, since there appear to be cell-specific splicing mechanisms that may result in altered protein products. Although the β gene is transcribed in both T cell lines and in macrophages, analysis of the T cell specific transcript (β₁) revealed an additional 138 nucleotides, which result in a 46 amino acid insertion in the cytoplasmic domain of the T cell lines Fcγ receptor. RNase protection experiments have shown that the β₁ transcript is seen in T cell lines, and not in the J774, P388D1 or RAW macrophage cell lines and most probably arises from an alternative splicing pathways for the β gene. The early macrophage lines P388 and WEHI 3A have 3 transcripts of the same apparent mobility as the T cell lines which may suggest that β₁ splicing is developmentally regulated during macrophage maturation. The consequences of the insertion are not understood, but it is possible that the longer cytoplasmic domain in the β₁ FcγR interacts differently with cytoplasmic or membrane proteins involved in signal transduction. The structures of the three transcripts derived from the α and β genes are summarized in Fig. 10.

The sequences obtained for these receptors demonstrate that the Fcγ receptor belongs to the immunoglobulin supergene family, as does the poly IgA receptor, which functions in the transport of IgA across epithelial cells (Mostov, et al., Nature 308; 37-43 (1984). Apart from the overall immunoglobulin homology, several other significant homologies were identified for the FcγR. The extracellular domain of the α and β genes are most homologous to the MHC class II protein E β in its β₂ domain. The poly Ig receptor, by contrast, has little significant homology to the Fcγ receptor described here, suggesting that it is more distantly related to the Fcγ receptor than are class II determinants. The other significant homology detected for the transmembrane domain of the α chain, but not the β chain, is with one of the transmembrane domains of the chain of the acetylcholine receptor. Young et al., Proc. Natl. Acad. Sci. 80: 1636-1640 (1983) and Nelson, et al., J. Clin. Invest 76: 500-507 (1985) have reported ion channel activity in response to binding of ligand to Fcγ receptors. The precise role of the various structural domains of these Fc receptor proteins is under investigation.

Applications of the foregoing, aspects of the invention will be recognized by one skilled in the art. For example, in some individuals with immune disorders, the cause can be traced to cells and organs which do not express the necessary Fc receptor proteins, and therefore do not mount a sufficient or complete response to infections. Artificial organs are contemplated, which either incorporate therein Fc receptor proteins produced in vitro by appropriate transformed cells in culture, or organs which are "seeded", with physiologically acceptable transformed cells which also produce the necessary protein. A second application will be seen in the use of DNA of this invention to obtain human DNA corresponding to the DNA described herein. One may now follow established techniques of DNA hybridization, e.g., to locate and hybridize human Fc receptor protein expressing DNA with murine "probes". The details of such hybridization technologies are known to those skilled in the art and need not be elaborated upon further.

While there have been described what are at present considered to be the preferred embodiments of this invention, it will be obvious to one skilled in the art that various changes and modifications may be made therein without departing from the invention, and it is, therefore, aimed to cover all such changes and modifications as fall within the true spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. Substantially pure nucleotide sequence expressing Fc receptor protein.

2. Nucleotide sequence of claim 1, wherein said receptor protein is Fcγ receptor protein.

3. Nucleotide sequence of claim 2, wherein said receptor protein is Fcγα receptor protein.

4. Nucleotide sequence of claim 2, wherein said receptor protein is Fcγβ receptor protein.

5. Nucleotide sequence of claim 4, wherein said Fcγβ receptor protein is Fcγβ receptor protein.

6. Nucleotide sequence of claim 4, wherein said Fc receptor protein is Fcγβ₂ receptor protein.

7. Nucleotide sequence of claim 3, comprising sequence:

-30

GTAGTTCATCTCCTGAACCTCATCAGACTCTGATCCAG TTCTTGAATGACTTTGGACACCCAGATGTTTCAGAAT 10 20 30 40 50 60 70

GCACACTCTGGAAGCCAATGGCTACTTCCACCAC TG ACAATTCTGCTGCTGTT TGCTTTTGCAGACAGGCAGAGT 85 95 105 115 125 135 145

10

GCAGCTCTTCCGAAGGCTGTGGTGAAACTGGACCCCCCATGGATCCAGGTGCTCAAGGAAGACATGGTGACACTG 160 170 180 190 200 210 220

30 40

ATGTGCGAAGGGACCCACAACCCTGGGAA CTCTTCTAC CCAGTGGTTCCACAACGGGAGGTCCATCCGGAGCCAG

235 245 255 265 275 285 295

50 60 70

GTCCAAGCCAGTTACACGTTTAAGGCCACAGTCAATGACAGTGGAGAATATCGGTGTCAAATGGAGCAGACCCGC 310 320 330 340 350 360 370

80 90

CTCAGCGACCCTGTAGATCTGGGAGTG ATTTCTGACTGGCTGCTGCTCC AGACCCCTCAGCGGGTGTTTCTGGAA 385 395 405 415 425 435 445

100 110 120

GGGGAAACC ATCACGCTAAGGTGCCATAGCTGGAGGAACAAACTACTGAACAGGATCTCATTCTTCCATAATGAA

460 470 480 490 500 510 520

130 140

AAATCCGTGAGGTATCATCACTACAAAAGTAATTTCTCTATCCCAAAAGCCAACCACAGTCACAGTGGGGACTAC 535 545 555 565 575 585 595

150 1 60 170

TA CTGCAA AGGAAGTCTAGG AAGTACACAGCACCAG TCCAAGCCTGTCACCATCACTGTCCAAGATCCAGCAACT

610 620 630 640 650 660 670

180 190

ACATCCTCCATCTCTCTAGTC TGGTACCACACTGCTTTCTCCCTAGTG ATGTGCCTCCTGTTTGCAGTGGACACG 685 695 705 715 725 735 745 200 210 220

GGCCTTTATTTCTACGTACGGAGAAATCTTCAAACCCCGAGGGAGTACTGGAGGAAGTCCCTGTCAATCAGAAAG 760 770 780 790 800 810 820 230

CACCAGGCTCCTCAAGACAAGTGACACCCCATCCATCCTATGGCAAAACATACGATGTTTTGGTGGCACCAGCAA 835 845 855 865 875 885 895

C TTTTCAGCCACACAGCCTTCCTTTGAAAGCAACTTACA AGCAGGCCGGGATGTTTGGTTCTTCAATCACAACGA 910 920 930 940 950 960 970

CTTAGGATCACCAGTTCAAGGCTTGCTGGGTCACACAGAGAGAGTGAGTGCAAGTCTAGCCTGGATAACCCAGTG 985 995 100 5 1015 1025 1035 1045

AGATCCTGGGTTTAGGCGGCTCATCAGGAAAGAGAACCTGTTGCTAATCTCACAAACAAGATGCCTACTGCCCAT 1060 1070 1080 1090 1100 1110 1120

GTGGCCAAAGGAGAGAACAAGGTCCTGGAAGTTGTCCTCTGACCTCCACCATCCACCATGGCAGGTGCACACAAT 1135 1145 1155 1165 1175 1185 1195

AAATTAAAATGTCATGTATATTTTTAAACAAGAGACAGGGGCAGGCTAAGGGTTGATGGCATAG CTGTTATCCAG 1210 1220 1230 1240 1250 1260 1270

TACACATAATGCCCTGGGTTTGACCTCCTATAATAAAGCAAAAAAAAAAAAAAAAAAAAAAA

1285 1295 1305 1315 1325 1335

8. Nucleotide sequence of claim 5 , comprising sequence :

AATGTATGTGAAGGATGAGTGTTCTACTGCTGTTCTCACATATGGCCTAGCTTTTGGTCTACAATCCAACAGTGA

10 20 30 40 50 60 70

GCTGGGGATTGTGATAATAGAGAAACCCAGATGGTGACTGAGGCAAATGACTTCTGAGCTGCGTTGGGGTGAAGT 85 95 10 5 11 5 125 135 145

TTTCCCTCTCTGTACCAGACGTCCAGGTCAGCTACGGCTCCAGCAGAACATGAAGGGAGTTGTTTCTCAGTGTCT 160 170 180 190 200 210 220

AAAAAATTTTCTGTGATTTGAGCTGAATCCAGTTTATTCTGCCCGGGAGGAAGCCTGTGCCTGCAGCTGACTCGCT

23 5 24 5 255 26 5 27 5 28 5 2 95

CCACAGCTGATCGGAATCCTGCCGTTCCTACTGATCCCCATGGAGAGCAACTGGACTGTCCATGTGTTCTCACGG 310 320 330 340 350 360 370

AC TTTG TGCCATATGCTACTGTGGACAG CCG TGCTAAATCTTGCTGCTGGGACTCATGATCTTCCAAAGGCTGTG 385 395 405 41 5 425 435 445

10 20 30

GTCAAACTCGAGCCCCCGTGGATCCAGGTGCTCAAGGAAGACACGGTGACACTGACATGCGAAGGGACCCACAAC 460 470 480 490 500 510 520

40 50

CCTGGGA ACTCTTCTACCCAGTGGTTCCACAATGGCAGCTCCATCCGGAGCCAGGTCCAAGCCAGCTACACGTTT 535 545 555 565 575 585 595

60 70 80

AAGGCCCCA CAGTCAATGACAGTGGAGAATATCGGT GG TCAA TGGAGCACCCGCCTCAGCGTGTAGATCTG

610 620 630 640 650 660 670

90 1 00

GGACTGA TTTGACTGGCTGCTGCTCCAGACCCCTCAGCTGGTGTTTCTGGAAGGGGAAACCATCACGCTAAGG 685 695 705 715 725 735 745

120 130

TGCCATAGCTGGAGGAACAAACTACTGAACAGGATCTCGTTCTTCCATAATGAAAAATCCGTGAGGTATCATCAC

760 770 780 790 800 810 820

140 150

TACAGTAGTAATTTCTCTATCCCAAAAGCCAACCACAGTCACAGTGGGGACTACTACTGCAAAGCAAGTCTAGGA 835 845 855 865 875 885 895

160 170 180

AGGACACTGCACCACTCCAAG CCTGTCACCATCACTGTGGAAG G GCCCCAAGTCC

GC GG C

910 920 930 940 950 960 970

190 200

ACAATTGTTGGCTGCTGTCACTGGGATTGCTGTCGCAGCCATTGTTATTATCCTAGTATCCT TGGTCTATCTCAAG 985 995 1005 1015 1025 1035 1045 210 220 230

AAAAAGCAGGTTC CAGCTCTCCCAGGAAA CCCTG ATCACAGGGAAATGGGAGAAACCCTTCCAGAGGAAGTAGGT

1060 1070 1080 1090 1 100 1 1 10 1120

240 250

GAGTACAGACAACCCTG TGGGC TGAG TGCCTGTCAGCCCAGGGCTCCATCTGGACTGGACCCAACAAGCAGCAGC 1135 1145 1155 1165 1175 1 185 1195

260 270 280

CCATACAATCCTCCTGATCTGGAAGAAGCTGCCAAAACTGAGGCTGAGAATACGATCACCTACTCACTTCTCAAG 1210 1220 1230 1 240 1250 1260 1270

290 300

CATCCCGAAGCCCTGGATGAAGAAACAGAGCATGATTACCAGAACCACA TTTAGTCTCCCTTGGCATTGGGAAAA 1285 1295 1305 1315 1325 1 335 1345

GCAAGCCAGAAAGGCCAGGATCTAGTGTCTCCTGGTCCAAGGGATGCTGTAGATATTAAAGAAA ACATCCAGAGT 1360 1370 1380 1390 1400 1410 1420

CACTTCTGTGAGTCCTGAAACCAACAGACACTACGAGATTGGTTCCCAATGGTTGACTGTACTAATGACTCCCAT 1435 1445 1455 146 5 1475 1485 1495

AACTTACAGCTTCCCAACTCAAGACTCTTCTGCTATCGATCCACACTGCCACTAAAATTAATCAACTTACTGCC

1510 1520 1530 1540 1550 1560 1570

9. Nucleotide sequence of claim 6, comprising sequence:

-29

CTTGCAGCTGACTCGCTCCAGAGCTGA TGGGAATCCTGCCGTTCCTACTGATCCCCATGGAGAGCAACTGGAC TG 10 20 30 40 50 60 70

-1 0

TCCATGTGTTCTCACGGATTTTGTGCCATATGCTACTGTG GACAGCCGTGCTAAATCTTGCTGCTGGGACTCATG 85 95 105 115 125 135 145

10 20

ATCTTCCAAAGGCTGTGGTCAAACTCGAGCCCCCGTGGATCCAGGTGCTCAAGGAAGACACGGTGACACTGACAT 160 170 180 190 200 210 220

30 40 50

GCGAAGG GACCCACAACCCTGGGAACTCTTCTACCCAGTGGTTCCACAATGGGAGGTCCATCCGGAGCCAGGTCC 235 24 5 255 265 275 285 295

60 70

AAG CCAGCTACACGTTTAAGGCCACAGTCAATGACAGTGGAGAATATCGGTGTCAAATGGAGCAGACCCGCCTCA 310 320 330 340 350 360 370

80 90 100

GCGACCCTGTAGATCTCGGAGTGATTTCTGACTGGCTGCTGCTCCAGACCCCTCAGCTGGTGTTTCTGGAAGGGG 3 85 395 405 415 425 435 445 110 120

AAACCATCACGCTAAGGTGCCATAGCTGGAGGAA CAAACTACTGAACAGGATCTCGTTCTTCCATAATGAAAAAT

460 470 480 490 500 510 520

130 140 150

CCGTGAGGTATCATCACTACAGTAGTAATTTCTCTATCCCAAAAFCCAACCACAGTCACAGTGGGGACTACTACT

535 545 555 565 575 585 595

160 170

GCAAAGGAAGTCTAGGAAGGACACTGCACCAGTCCAAGCCTGTCACCATCACTGTCCAAGGGCCCAAGTCCAGCA

610 620 630 640 650 660 670

180 190 200

GGTCTTTACCACTATTGACAATTGTGGCTGCTGTCACTGGGATTGCTGTCGCAGCCATTGTTATTATCCTAGTAT 685 695 705 715 725 735 745 210 220

CCTTGGTCTATCTCAAGAAAA AGCAGGTTCCAGAC AATCCTCCTGATCTGGAAGAAGCTGCCAAAACTGAGGCTG

760 770 780 790 800 810 820

230 240 250

AGAATACGATCACCTACTCACTTCTCAAGCATCCCGAAGCCCTGGATGAAGAAACAGAGCATGATTACCAGAACC 835 845 855 865 875 885 895

254 I

ACATTTAGTCTCCCTTGGCATTGGGAAAAGCAAGCCAGAAAGGCCAGGATCTAGTGTCTCCTG GTCCAAGGGATG

910 920 930 940 950 960 970

CTGT AGATATTAAAGAAAACATCCAGAGTCACTTCTGTGAGTCCTGAAACCAACAGACACTACGAG ATTGGTTCC

985 995 100 5 1015 1025 1035 1045

CAATGGTTGACTGTACTAATGACTCCCATAACTTACAGCTTCCCAACTCAAGACTCTTCTGCTATCGATCCACAC 1060 1070 1080 1090 1100 1110 1120

TGCCACTAAAATTAATCAACTTACTGCCGTTAAGAGA 113 5 1145 115 5

10. Substantially pure Fcγ receptor protein.

11. Substantially pure Fcγ receptor protein of claim 10, wherein said protein is Fcγα protein.

12. Substantially pure Fcγ receptor proteins of claim 10, wherein said protein is Fcγβ protein.

13. Substantially pure Fcγβ receptor protein of claim 12, wherein said protein is Fcγβ₁ protein.

14. Substantially pure Fcγβ receptor protein of claim 12, wherein said protein is Fcγβ₂.

15. Substantially pure protein of claim 11, comprising amino acid sequence:

L L

L L L L

16. Substantially pure protein of claim 13, comprising amino acid sequence

L L L L L L L L L

R L L L L L L

17. Substantially pure protein of claim 14, comprising amino acid sequence:

L L L

L

L L L

18. A method of obtaining substantially pure Fc receptor protein comprising transforming a eukaryotic cell with substantially pure nucleotide sequence expressing an Fc receptor protein selected from the group consisting of Fcα

Fcβ₁ and Fcβ₂ receptor protein culturing said transformed cell under conditions favoring expression purifying of said protein, and said expressed protein.

19. Vector which expresses Fc receptor protein, comprising a substantially pure nucleotide seqiemce expressing Fc receptor protein selected from the group consisting of Fc , Fcβ, and Fcβ₂ ligated into a portion of carrier DNA.

20. Vector of claim 19, wherein said vector is pcEXV-3.

21. Eukaryotic cell line transformed with a foreign nucleotide sequence which expresses an Fc receptor protein selected from the group consisting of Fcα, Fcβ, and Fcβ₂.

22. An immunoglobulin receptor organ analog which comprises a physiologically acceptable cell line of claim 21.

23. A method for obtaining substantially pure human DNA which expresses human Fc receptor proteins comprising contacting a sample containing said substantially pure human DNA with a sample of mouse DNA which expresses mouse Fc receptor protein under conditions favoring hybridization between said mouse DNA and complementary human DNA, treating said sample to separate hybridized DNA from unhybridized DNA and removing said mouse DNA from said hybrid.

24. Method of claim 23, wherein said DNA expresses a receptor protein selected from the group consisting of FCγ, FCγα , Fcγβ ₁, and Fcγβ ₂.

25. Substantially pure human DNA obtained by the method of claim 23.