FC RECEPTOR FOR IMMUNOGLOBULIN

Abstract: A receptor for the Fc portion of immunoglobulin, derived from mouse or human cells, the receptor exhibiting the ability to bind to the Fc portion of mouse and human immunoglobulin and comprising a transmembrane proteine having about 280-301 amino acids including two substantially regularly spaced pairs of Cys residues and two or four potential N-linked glycosylation sites. The present invention also provides for a nucleotide sequence, gene, cDNA clone or a vector containing same capable of encoding the above receptor.

Description: TITLE: FC RECEPTOR FOR IMMUNOGLOBULIN Th i s i n v en t io n r e l a t e s t o a F C r e c e p t o r f o r immunogαbulin . In one aspect this invention relates to cDNA clones encoding the murine and human Fc receptor for IgG. The murine clones mere sequenced and expression of mRNA and the cel l surf ace Fc receptor investigated. The murine Fc receptor is a 301 amino acid ransmembrane glycoproteiπ with tωo homo l ogous extrace l lu l ar domains that are a l so homo l ogous to members of the Ig superf ami l y. The Fc receptor has 4 sites of N linked glycosylation and a long 94 amino acid cytoplas ic tail. Northern analysis of mRNA, immune complex binding and serological studies of cell lines and t r an s f e c t a n t s demonstrated that the receptor encoded by the cDNA clone binds murine IgG2b and possibly other Ig. Human Fc(gamma)R cDNA clones uiere also isolated and fαund to encode a glycosylated ransme brane molecule that bears striking homology to both the betal and alpha mouse FcR at both the nucleic and amino acid levels. The present invention provides a receptor for the Fc portion of immun og obu 1 in and being derived from ATCC , ATCC or ATCC The present invention provides a receptor for the Fc portion of i m m u n o g o b u 1 i π , the receptor exhibiting the abi l ity to bind to the Fc portion of mouse and human immuπogobulin . The receptor preferably exhibits an ability to bind to antibodies and immune complexes, has a size of 40. -70 kilodaltons, is formed from mRNA of a size of about 1.8, about 2.0, about 1.9, about 1.4 or about 2.4 kilobases, and includes t io su stantially regularly spaced pairs of Cys residues, tuio or four potential N-linked glycosylation sites. The present invention includes a receptor f or the Fc portion of i m m u π α g o b u 1 i n and having at least 40!?, more preferably at least 50 !5 and stil l more preferably at least GΩ% homology ωith the above receptor. In one instance the receptor includes the amino acid In one instance the receptor includes the amino acid sequences : - (a) met leu leu trp thr ala val leu asn leu ala ala gly thr his asp leu pro lys ala val val lys leu glu pro pro trp i1e , (b) glu gin thr arg leu ser asp pro val asp leu gly val ile, and (c) lys gly ser leu gly arg thr leu his gin ser lys pro val thr ile thr val gin gly pro lys, and (d) glu ala glu asn thr ile thr tyr ser leu leu lys his pro glu ala leu asp glu glu thr glu his. In a preferred instance the receptor includes at least one of the amino acid sequeπces:- (a) fist Glu Ser Asn Trp Thr Val His Val Phe Ser Arg Thr Leu Cys His Met Leu Leu Trp Thr Ala Val Leu Asn L.eu Ala Ala Gly. (b) Cys Glu Gly Thr His Asn Pro Gly Asn Ser Ser Thr Gin Trp Phe His Asn Gly Arg Ser Ile Arg Ser Gin Val Gin Ala Ser Tyr Thr Phe Lys Ala Thr Val Asn Asp Ser Gly Glu Tyr Arg Cys . (c) Cys His Ser Trp Arg Asn Lys Leu Leu Asn Arg Ile Ser Phe Phe His Asn Glu Lys Ser Val Arg Tyr His His Tyr Ser Ser Asn Phe Ser Ile Pro Lys Ala Asn His Ser His Ser Gly Asp Tyr Tyr Cys. (d) Leu Pro Val Leu Thr Ile Val Ala Ala Val Thr Gly Ile Ala Val Ala Ala lie Val Ile lie Leu Val Ser Leu Val Tyr Leu . (e) Lys Lys Lys Gin Val Pro Ala Leu Pro Gly Asn Pro Asp His Arg Glu Wet Gly Glu Thr Leu Pro Glu Glu Val Gly Glu Tyr Arg Gin Pro Ser Gly Gly Ser Val Pro Val Ser Pro Gly Pro Pro Ser Gly Leu Glu Pro Thr Ser Ser Ser Pro Tyr Asn Pro Pro Asp Leu Glu Glu Ala Pro Lys Thr Glu Ala Glu Asn Thr Ile Thr Tyr Ser Leu Leu Lys His Pro Glu Ala Leu Asp Glu Glu Thr Glu His Asp Tyr Gin Asn His Ile. (f) N S G P R N L lil L L Q P L T V L L L L A S A D S q A A. (g) C Q G A R S P E S D S I Q lil F H N G N L I P T H T Q P S Y R F K A N N N D 5 G E Y T C. (h) C H S U K D K P L V K V T F F Q N G K S Q F S R L D P T F S I P Q A N H S H S G D Y H C. (i) S P M G I I V A V V I A T A V A A I V A A V V A L I Y C. (j) R K K R I S A N S T D P V K A A Q F E P P G R Q iη I A I R K R Q L E E T N N D Y E T A D G G Y M T L N P R A P T D D K N I Y L T L P P N D H V N S . The present invention also provides a nucleotide sequence, gene, cDNA clone, or a vector for same adapted to encode or to produce a material adapted to encode for the receptor above or a fragment thereof. The present invention also provides a nucleotide sequence, gene, cDNA clone, or a vector for same adapted to encode or produce a material adapted to encode for the leader sequence, extracellular region, transmembrane region or intrace11u 1ar region of the receptor above. The present invention also provides a cDNA clone, gene or nucleotide sequence being incorporated in ATCC ,ATCC or ATCC and capable of encoding for a receptor for the Fc portion of immuπogobulin or a fragment thereof and a material being ATCC ,ATCC , or ATCC A preferred nucleotide sequence, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion of immu n o g o b u 1 in includes the nucleotide sequences:- (a) ATG CTA CTG TGG ACA GCC GTG CTA AAT CTT GCT GCT GGG ACT CAT GAT CTT CCA AAG GCT GTG GTC AAA CTC GAG CCC CCG TGG ATC, (b) GAG CAG ACC CGC CTC AGC GAC CCT GTA GAT CTG GGA GIG ATT, (c) AAA GGA AGT CTA GGA AGG ACA CTG CAC CAG TCC AAG CCT GTC ACC ATC ACT GTC CAA GGG CCC AAG, and (d) GAG GCT GAG AAC ACG ATC ACC TAC TCA CTC CTC AAG CAT CCC GAA GCC TTG GAT GAA GAA ACA GAG CAT. In a preferred instance the nucleotide sequence, cDNA clone, gene or vector containing same includes at least one of the nucleotide sequeπcess- (a) ATG GAG AGC AAC TGG ACT GTC CAT GTG TTC TCA CGG ACT TTG TGC CAT ATG CTA CTG TGG ACA GCC GTG CTA AAT CTT GCT GCT GGG. (b) TGC GAA GGG ACC CAC AAC CCT GGG AAC TCT TCT ACC CAG TGG TTC CAC AAT GGG AGG TCC ATC CGG AGC CAG GTC CAA GCC AGC TAC ACG TTT AAG GCC ACA GTC AAT GAC AGT GGA GAA TAT CGG TGT, (c) TGC CAT AGC TGG AGG AAC AAA CTA CTG AAC AGG ATC TCG TTC TTC CAT AAT GAA AAA TCC GTG AGG TAT CAT CAC TAC AGT AGT AAT TTC TCT ATC CCC AAA GCC AAC CAC AGT CAC AGT GGG GAC TAC TAC TGC. (d) TTA CCA GTA TTG ACA ATT GTG GCT GCT GTC ACT GGG ATT GCT GTC GCA GCC ATT GTT ATT ATC CTA GTA TCC TTG GTC TAT CTC, (e) AAG AAA AAG CAG GTT CCA GCT CTC CCA GGA AAC CCT GAT CAC AGG GAA ATG GGA GAA ACC CTT CCA GAG GAA GTA GGT GAG TAC AGA CAG CCC TCT GGG GGC TCA GTG CCT GTC AGC CCA GGG CCT CCA TCT GGA CTG GAG CCA ACA AGC AGC AGC CCA TAC AAT CCT CCT GAT CTG. GAA GAA GCT CCC AAA ACT GAG GCT GAG AAC ACG ATC ACC TAC TCA CTC CTC AAG CAT CCC GAA GCC TTG GAT GAA GAA ACA GAG CAT GAT TAC CAA AAC CAC ATT TAG. (f) AAT TCC GGT CCC AGA AAC CTG TGG CTG CTT CAA CCA TTG ACA GTT TTG CTG CTG CTG GCT TCT GCA GAC AGT CAA GCT GCA. (g) TGC CAG GGG GCT CGC AGC CCT GAG AGC GAC TCC ATT CAG TGG TTC CAC AAT GGG AAT CTC ATT CCC ACC CAC ACG CAG CCC AGC TAC AGG TTC AAG GCC AAC AAC AAT GAC AGC GGG GAG TAC ACG TGC, (h) TGC CAC AGC TGG AAG GAC AAG CCT CTG GTC AAG GTC ACA TTC TTC CAG AAT GGA AAA TCC CAG AAA TTC TCC CGT TTG GAT CCC ACC TTC TCC ATC CCA CAA GCA AAC CAC AGT CAC AGT GGT GAT TAC CAC TGC. (i) TCA CCA ATG GGG ATC ATT GTG GCT GTG GTC ATT GCG ACT GCT GTA GCA GCC ATT GTT GCT GCT GTA GTG GCC TTG ATC TAC TGC, (j) AGG AAA AAG CGG ATT TCA GCC AAT TCC ACT GAT CCT GTG AAG GCT GCC CAA TTT GAG CCA CCT GGA CGT CAA ATG ATT GCC ATC AGA AAG AGA CAA CTT GAA GAA ACC AAC AAT GAC TAT GAA ACA GCT GAC GGC GGC TAC ATG ACT CTG AAC CCC AGG GCA CCT ACT GAC GAT AAA AAC ATC TAC CTG ACT CTT CCT CCC AAC GAC CAT GTC AAC addition or substitution of amino acids or by chemical or irradiation modification not substantial ly deleteriously affect ing the abi l ity to bind to the Fc portion of immuπogobu 1 in and a nucleotide sequence, gene, cDNA clone, or a vector for same as above and modified by deletion, addition or substitution of πucleαtides or by chemical or irradiation modification not substantially deleteriously affecting the ability to code for a receptor for the Fc portion of immuπogobu 1 in or a fragment thereof. DESCRIPTION OF PREFERRED ASPECTS Fc receptors (FcR) form a ma or groups of cell membrane g 1 copro teiπs involved in homeostasis of the immunological system. Specific receptors for al l immunoglobulin (Ig) classes have been defined and are found on a ωids variety of immune cells - 8 cells and some T cells as ωell as myelαid cells and π o n -h a e m o p o i e t i c cel ls (Hubscher et al , 1971 ; Dickler, 1976; Tsay et al, 1980; Uπkless et al , 1981 ). The principal role of these receptors is to bind (Ig) via the Fc region of the Ig molecule and it is through this interaction that a ωide range of biological effects are mediated. These include phagocytosis of immune complexes by macrophages (Leslie, 1980) and neutrophils (Capron et al , 1984) and direct or indirect regulation of antibody production by membrane bound or soluble Fc receptor (Yodoi et al , 1980; Fridman et al, 1981 ; Kolsch et al , 1983). In the murine system s-f forts have been devoted to the analysis of the receptor for IgG1 /IgG2b ( F c ( g a m m a ) 1 / ( g a mm a ) 2 b ) ωhich is important in the binding of immune complexes (Unkless, 1974; Kurlander et al, 1984). To investigate these receptors further at both the structural and functional level, as ωell as to study its relationship to other FcR, ωe have produced a monoclonal aπti-Ly-17.2 antibody that defines a genetic polymorphism of the Fc(gamma)R genes (Hibbs et al , 1985). This antibody ωas used to immunopurify receptors for ωhich ωe have:- 1. isolated and determined the partial amino acid; 2. isolated and determined the complete nucleotide sequence of cDNA clones termed ρFc24 and pFc113 encoding the betal Fc(gamma)R receptor. 3. Shαωπ by Northern analysis the presence of multiple FcR transcripts in different cell types, these variant transcripts are termed beta2 and alpha. 4. Using the mouse FcR cDNA and oligonucleo ide probes (based on pFc 113 DNA sequence) isolated cDNA clones termed pFC 3.0 , 3.1 and 3.47 encoding human FcRs that are homologous to the mouse FcR. MATERIALS AND METHODS ' Monoclonal Antibodies: The monoclonal anti-Ly-17.2 and Ly-2.1 antibodies have been previously described (Hibbs et al 1985, Hogarth et al 1982). Preparation of F(ab') 2 fragments from purified antibody ωas performed as described (16). The 2.4 G2 antibody has also been described before (Uπkless 1979). Purification of FcgammaR: J774 macrαphage cells (2x10 ) ωere harvested from ascites fluid obtained from (CBAxBALB/c)F1 mice and immediately lysed in cold phosphate buffered saline (PBS) / 0.5 Noπidet P-40 (PBS/0.5% NP40) pH 7.4 containing \ aprotiniπ (Sigma, St. Louis, M0) and 1mM ph eπy 1 methy 1 su 1 fony 1 fluoride (Sigma, St. Louis, M0). An additional 2x10 cells ωere surface labeled ωith I, and pooled ωith the cold lysate. After lysis for 1 hour at 4 C, the lysates ωere clarified and ncubated for 1 hour at 4 α C ωith anti-Ly-17.2 antibody con ugated Sepharose 4B (Pharmacia, Uppsala, Sωedeπ). The immunoabsorbent ωas ωashed three times in 0.SM NaCl, 0.0125M KH 2 P04 » P H 7 - 4 » three times in PBS/0.5 % NP40 pH 7.4 and a further three times in 0.5J5 deoxycho l ate in 100mM Tris-HC l pH 8.0. The immunoab s orb ent ωas packed into a column and the bound material eluted ωith 0.5% sodium dodecyl sulfate (NaDodSO^) in 0.1M triethy1amiπe pH 11.5 and freeze dried. The purity of the Fc gamma R preparations ωas assessed by NaDodS0 ^/ o 1 yacry 1 a id e gel e 1 ectroph ore si s (NaDodSO Λ/PAGE) and proteins ωere detected by Coomassie blue staining or by autoradiogra hy. Protein Sequence Analysis: Protein samples ωere carb o amid o eth 1 at e d and ethanol precipitated prior to sequencin g. Brief ly , samp l es ωere dissol v ed in 50mM boric acid , 0.1 % (ω/v ) SDS and 10mM dithio hreitol pH 8.0 (NaOH) and heated to 60°C f or 1 hour. I o d o a c e t a m i d e ωas added to a final concentration of 22mM and the samples incubated for 15 minutes at ambient temperature in the dark. Ice cold ethanol containing 50mM HCl ωas added and the protein al lowed to precipitate at -20°C for 2 hours. The precipitate ωas co l lected by c e n t r i f u g a i o n , disso l v ed -in C F , C 00 H and sequenced by automatic Edman degradation using an Applied Biosyste s 470A Sequencer (Foster City, CA., USA) Sequencing and ph eπy 1 thiαhy dant oin amino acid identification techniques have previously been described in detail Preparation of FcgammaR Peptides: Fcga maR peptides ωere obtained by digesting samp les of affinity purif ied, carboxamidome thy 1 at ed Fc gamma R ωith either S Λ uu.Ef.us_ V8 protease (Miles Laboratories) (Hibbs et al 1986 ) lysiπe-C proteinase (according to manuf acturers ins tructions , Boehringer Mannheim, Mannheim, FRG) or cyanogen bromide ( C N B r ) . P e p t i d e s ω e r e p u r i f i e d b y r e v e r s e p h a s e ch rαma t αgr ap h y us ing a Ph armac ia f a s t protein l iquid chroma tograph y (FPLC). The proteinase digested material ωas applied to a Pep RPC HR5/5 (C2/C18) reverse phase column (Pharmacia) ωhi le a Pro RPC HR 5/10 (C1 /C8) rev erse phase co l umn ( P h a rmac ia ) ωa s u s e d t o p urif y CN B r p ep tid e s . Pεptides ωere eluted ωith ascending linear acetonitrile gradients using 20mM ammonium f ormate as a buff er and an absorbaπce trace at 214πm to detect peptide peaks. Selected peak f ractions ωere then r e c h r o m a t o g r a p h e d on the same column again using linear gradients of acetonitrile but in this case containing 0.1 !? ( v/v ) unbuf fered C F , c 00 H to promote ion suppression. P r e p a r a t i o n o f O l i g o n u c l e o t i d e P r o b e s : O l igodeox ynuc l eotide prob es ωere sy nthesized by the phosphor amidi te method using an Applied Biosystems 380A DNA Synthesizer. The oligonucleotide probes ωere purified from crude mixtures by reverse phase HPLC and ωere radiolabeled 8 ωith [ g amma 32 P ] A TP and T4 p o 1 y n u c 1 e o t i d ε kinasε to 2x10 dpm/ug (Maniatis et al 1982). Library Screening: The cDNA library used in the murine study ωas kindly provided by Drs N. Gough and A. Dunn (Ludωig Institute for Cancer Research, Melbourne, Australia) and ωas constructed using poly A + mRNA from the FcR + my e 1 omonocytic cell line liiEHI 3B (Gough et al, 1985). The cDNA ωas GC tailed into the SacI site of the pJL3 vector, ωith the result that all cDNA inserts are flanked by EcoRI sites. 01 i g od e oxyπuc 1 eotid e probes (Tables 2,3) ωere constructed using the phosphoramidite method (lilinnaker and Dαrper, 1982) and ωere complementary to mRNA for use in Northern analysis. The cDNA library ωas screened ωith end labeled oligonucleotide probes using T4 polyπuc.leotide kiπase to >10 dpm/ug DNA. Hybridisation and ωashing conditions ωere as previously described (Hibbs et al., 1986) . Northern Blot Analysis of ' RNA: Poly A + mRNA ωas obtained from lysates of in vitro grown mouse cells and normal human spleen . PolyA + mRNA (5ug of polyA + or variable amounts of total mRNA) ωas denatured and ε 1ectrophoresed in 1% agarose gels formaldehyde, transferred to nylon membrane and hybridised overnight (Maniatis et al, 1982). Hybridisations ωith the cDNA probe ωere performed in 5xSSPE, 0.1% SDS, 50% formamide, 0.125% skim milk powder and 1ug/ml degraded DNA, at 42°C for 16 hrs. The filters ωere then ωashed twice in IxSSPE at room temperature, then tωice in 0.2xSSPE at 50°C for 16 hrs. Hybridisations ωith the o1 igodeoxynuc1eotide probe ωere performed in 5xSSPE, 6% SDS, 10x Deπhardts and 1ug/ml degraded salmon sperm DNA at 42 α C. Filters ωere ωashed tωice in 2xSSPE at room temperature then tωice in IxSSPE at 42°C. The cDNA probe ωas labelled by nick translation and unincorporated label removed on a spinning sepharose G50 column (Maniatis et al, 1982). The o1igodeoxynuc 1 eotidε probe ωas end labelled ωith 32 P- ATP for 1 hour at 37 α C using T4 po1ynuc1eotide kiπase then added directly to the hybridisation mix (Maniatis et al, 1982). Southern Analysis: DNA ωas prepared from C57BL/6 mouse spleen, human thymus and peripheral blood (Maniatis et al, 1982); 20ug of DNA digested ωith appropriate restriction e n z ym e ac c ord in g t o t h e m a nu f ac tu r er ' s in st r uc t io n s (Pharmacia , Uppsa l a, Sweden) ωas e 1 e c t r o h α r ε s e d in a 0.5% agarose gel , transferred to a nylon membrane and hybridised in 5xSSC, 0.1 % SDS, 50% fαrmamide, 20mM phosphate buffer pH 6.8 and 0.125% skim mil k pαωder ov ernight ωith randomly primed or nick translated cDNA at 55°C. Filters ωere ωashed 4 times in 1 xSSC, 0.1 % SDS, 0.125% skim milk powder then t ω i c e i n 0.2 x S S C , 0.1 % S D S a t 55 ° C , d r i e d a n d a u t o r a d i o g ra p h e d . Southern anal ysis ωas performed on cDNA clones pFc24 , pFc113 , HFc3.47 , HFc3.1 and HFc3.0 fol lowing EcoRI digestion, e 1 ec t ro p hore s is on 1 % agarose gels followed by transfer to nylon membranes (Hibbs et al , 1986). Filters containing pFc24 or pFc113 ωere probed as described (Hibbs et al , 1986). Filters containing HFc3.1 HFc3.0 or HFc3.47 ωere probed ωith nick trans l ated cDNA or end l abel l ed oligonucleotides by hybridisa ion at 35° in 20% formamide, 5xSSC , 0.1 % SDS , 0.125% skim milk poωder f or 16 hours , then ωashed in 1 xSSC at 35°C. Detection of FcR by EA rosetting : Sheep erythrocytεs ωεre washed f our times in norma l sa line. Packed cel l s (50ul) were added to 4ml of t r in i t rob en z eπε sulphonate (12.5 mg /m l i n ph o sp hat e b u f f e red sa l ine (P BS ) ) a n d th e pH adjusted to pH 7.2. The mixture ωas incubated at room temperature for 20' then washed three times. The cel ls were resuspended in 20m l of anti-TNP antibody (K1 , IgG2b or A3 -IgG1 ; L op e z et a l . , 1 983 ) αr r ab bit I g G anti sh eep erythrocytes at a s u b h a e m a g g 1 u t i n a t i n g dilution to giv e IgG2b-EA, IgG1 -EA or rabbit IgG-EA After a 20' incubation at r oom temp era ture , th e c e l l s were wa sh e d twice an d resuspended to 1 % in PBS + 5% BSA and l OmM azide. EA rosetting for detection of FcR was then performed using the sensitized erythrocytεs (Parish and Haywood, 1974). Cell lines: The cel l lines used in this study were the BALB/c macrophage tumour J774, BALB/c yel αmonocytic cel l liπε lii E H I 3B , and the AKR thymαma K36. Cel l line were maintained in vitro in Dulbecco's modifiεd Eagle's medium or RPMI 1640 both supplemented with 10% fetal calf serum. Serological Detection of Ly-17 (FcR) expression: Cell lines ωere tested for FcR expression using a monoclonal anti-Ly- 17.2 antibody and rosetting ωith sheep anti-mouse Ig coated erythrocytes (Hibbs et al., 1985; Parish and McKeπzie, 1978 ) . DNA sequencing! The cDNA inserts of pFc24, pFc113 were sequεπced by either chemical cleavage described by Maxam and Gilbert (1980) or Saπgεr di-deoxy sequencing (Saπger et al., 1977) after subcl oπiπg into M13mp8 , M13mp9, or M13mp18 according to the sequencing strategy outlined in Fig. 3A. The cDNA inserts of the human FcR clones were also sequeπced by the dideoxy nucleotide method after subcloπing fragments into M13 vεctors. Isolation of Human FcR cDNA: A human monocyts library ( f rom the h uma n TH P -1 c e l l l in e ) construct ed in t he lamdagtI O vector was usεd to isolate human Fc(gamma)R cDNA. Dual screenings were performed using both the mouse betal cDNA clone and a pool of oligonucleotide probes constructed from the nucleotide sequence of the mouse betal Fc(gamma)R cDNA. This poo l consisted of three unique probes which co rre sponded to nuc l eotidεs 1 37- 185 in the ous ε ( N - terminus), nucleotides 545-574 (second extracel lular domain) and nucleotide 956-1000 ( C-t ermiπu s ) . After four rounds of screen in g , se v era l c l ones ( HFc3.1 , 3.0 , 3.47) wh ich hybridised ωith both the murine cDNA and o l igonucleotide probe pool were isolated and further characterised. Phage DNA was prepared from these. Southern hybridisation analysis of EcoRI digested l ambda DNA confirmed that the murine betal Fc(gamma)R cDNA probe hybridised with cDNA inserts from all clones. Probing southern bl ots of EcoRI digsstεd HFc3.1 , 3.0 , 3.47 inde endently with each of the three murine oligonucleotide probes , showed that onl y the mouse N-terminal probe hybridised to thε insεrts from HFc 3.1 , 3.0 and only the mousε C-termiπal probe hybridised to the 1.9kb insert from HFc3.47. In addition, DNA inserts from HFc3.1 , HFc3.0 and HFc3.47 cross-hybridised. The DNA inserts were purified and subclαπed into the vector pJL4 to give the recombinant plas ids. RESULTS AND DISCUSSIONS Reference is made to the Figures of drawings as follows : FIGURE LEGENDS Figure 1. One dimensional NaDodS0 /PAGE analysis of the IgG FcR of J774 macrophage cells. Sizes are given in kDa. (A) Immunoprecipitation of Fc gamma R from surface iodinated J774 cells. Immunoprecipitations were performed ωith anti-Ly-2.1 F(ab')2 (Lane 1 ) or ωith aπti-Ly-17.2 F(ab')2 (Lane 2). (B) a D o d S 04 / P A G E and Coomassie staining of a typical preparation of affinity purified FcR used for protein sequencing and peptide generation. Lanes 1-6 represent successive fractions of eluted material from the affinity column (C). Autoradiogram of NaDαdSO /PAGE of affinity purified 125I-1 abe 11 ed FcR. Lanes 1-6 are as for B, above. Figure 2. Southern blot analysis of pFc24 and pJL3. One microgram of pFc24 (lanes B,D,F) or pJL3 (lanes A,C,E) was digested with EcoRI and electrophoresed in 1% agarose gsl. The DNA was transferred onto nitrocellulose and hybridised with [32P] labelled probe 1 (lanes A,B), [32P] probe 2 (lanes G,D) and [32P] probe 3 (lanes E,F). Hinfl fragments of pBR322 DNA were used as markers to estimate the size of the cDNA insert of pFc24 (shown in kb). The relative position ' of pJL3 is also indicated (5.49kb). Figure 3. (A) Partial restriction map and sequencing strategy for cDNA inserts of clones pFc24 and pFc113. Restriction enzymes sites shown are A, Alul; B, BamHI; E, EcoRI; S, Sau3A1 ; and V, PvuII. The EcoRI sites shown are present in the polycloπing site of the pJL3 vector. The shaded area represents sequence encoding the signal sequεnce and coding sequence for the mature protein is located within the open box. Sequence from 5' and 3' untranslated regions is indicated by thε solid line ωhilε unsεquencεd untranslatεd rεgions arε represεnted by the dashed line. Sequence ωas obtained across all restriction sites except for the PstI sites and both strands of the coding region ωere entirely sequenced. (B) Nucleotide and deduced amino acid sεquεπce of thε mousε FcR eπcod ' ed by p F C 113. Amino acids are numbered above the line in decades commencing at the amino terminal Thr deduced by protein sequencing (Hibbs et a l , 1986). Nucl eotides are numbere d a t the end of t h e l ine a nd the 5 ' an d 3 ' untrans lated regions are shoωπ in cl osed up typε. The signa l sequence is numbered from residue -29 to -1 and the transmembrane region (Tm) is underlined by a broken line. The sequences underlined ωith a so lid 1 i n-e correspond to sequences identical to the amino terminal sequence (NH2) and to amino acid sequence of peptides (L9 , V17 , 2V16 , 2V8 , CNBR, L5, L3, L4, V10, V11 ) isolated from the immunopur if ied FcR (Hibbs et al., 1986). Other notation is as fo l lows: o, first Cys in each domain; 0 second Cys in each domain; N- liπkεd glycosylation sites. The nucleotide sequence of pFc24 is embodied ωithin the cDNA insert of pFc113 from nucleotides 61 to 1023. Figure 4 Amin o acid s equen c e c omp ariso n of Fc(gamma)R domains (amino acids 5-116 for domain 1 and amino acids 118-175 for domain 2) to each other and to Ig related molecules. Identical residues are boxed and a "-" indicatεs a break in sequence f or alignment purposes. (A) The FcR domains ωere aligned ωith each other to givε the optimal score using the ALIGN program ωith a matrix bias of +6 and brsak penalty of 6 (see text). (B) Alignment of amino acid sequences arou.πd the first Cys residue of both FcR domains ωith Cys residues of the corresponding first Cys of domains of Ig and Ig related molecules. Amino acids common to FcR domains and other sequences are boxed ; V a , M OPC 104E (Apella, 1977); Vk, MOPC 1 9 (Nishioka and Lεder, 1980); T4, L3T4 (Classon εt al , 1986; Tαurvieil le et al , 1986; G. Clark, N. Deacon Personal Communication); N-CAM, Neural Cell Adhesion Molecule (Hεmperly et al, 1986); Poly IgR, Poly Ig Receptor (Rostov et al , 1984). (C) Alignment of sequences around the second Cys of FcR domains ωith the second Cys of domains of Ig and Ig related molecules as in C above except Vk ωhich is derived from an antidigoxiπ antibody (Nov otπy and Margol ies, 1983). Common residues are boxed as abo v e. (D) Alignment of lambdaJ regions from human (Engelhard and Hilschmann, 1975) and mouse (Apel la, 1977) , ωith J-like sequences from the FcR domains. Figure 5 Analysis of FcR mRNA transcripts in mouse c e l l l in e s . ( A ) N o rt h e r n b l ot o f R N A f r om F cR - e r y t h r o 1 e u k a εmi a cel ls F4N (a) or FcR+ Ul E H I 38 cells (b) probed ωith 840 base pair Bam HI - Eco RI fragment of pFc24 (Eco RI site of the vector pαlycloπing site). (B) Northern blot of poly A+ RNA from FcR+ cel l lines, Ul EH I 3B (a); J774 macrophage cells (b); K3S T ly phoma (c); and FcR- cells F4N (d), probed ωith an oligodeoxynuclεotidε corresponding to nucleotides 542-674 in pFc113. The position of 28S and 18S rRNA is indicated. Figure 6 Southern analysis of the FcR gene. Spleen DNA from C57BL/6 mice ωas digestsd ωith (a) Hindlll, (b) EcoRI or (c) PstI and probed ωith tha 840bp BamHI-EcoRI f ragment of pFc24 (see Legend Fig. 3). Molecular size- markers in Kb (Hind III digested lamdaphage DNA) are indicated. Figurε 7 Southern blot analysis of human FcR clones HFc3.1 (a) and HFc3.47 (b). HFc3.1 or HFc3.47 DNA ωas digested ωith EcoRI and electrophoresed in 1 % agarose gel, transferred to nylon membranes as per the materials and methods and hybridised ωith A, pFc24 cDNA; B, probe 1.5; C, probe 1.10; D, probe alpha or E, probe 1.6. The position of the 1.5kb cDNA insert of HFc3.1 and of the 1.9kb insert of HFc3.47 is indicated on the right hand side of each figure. Figure 8 Nucleotide and predicted amino acid sequence of the human FcR encoded by HFc3.1 Nucleotides are numbered every decade and the translated sequence is found above the nucleotide sequence. Amino acids are numbered above the line and number 1 indicates the N-termiπal residue. The incomplete signal sequence is underlined by a broken line to residue -1. The tωo glycos la ion sites arε marked by stars and the single hydrophobic trans e brane region is underlined by a solid line. Cystεine residues invol ved in disulphide bonding are circled. The "iπtron- like" sequence is located betωeen the vertical arrows. Figure 9 Homology of the human FcR encoded by HFc3.1 cDNA with the mouse alpha and betal Fc(gamma)R. Alignment of thε nucleotides of the human FcR with both mouse alpha (A) and betal (B) Fc(gamma)R. Breaks (indicatεd by dashes) in thε sequence have been introduced to optimize the alignment. Amino acid residues common to thε human FcR επcαdεd by HFc3.1 and the alpha FcR (A) or betal (B) are shown by asterisks. The human (HFC3.1 ) , mouse alpha or beta FcR leader sequence, extracellular domain-, transmembrane domain and cytop lasmic domains are indicated by the sequences betωeeπ the vertical arrows. The cysteine (C) residuεs ωithiπ the extracellular domains (involved in S-S bonds) arε idεπtifiεd by thε solid circles. Figure 10 Partial πucleotidε sequence and predicted amino acid sequence for HFc3.47 cDNA derived from tωo non- overlapping HFc3.47 fragments and identification of homologous regions in thε beta (1 or 2) FcR of thε mouse. The HFc3.47 nucleotide and amino acid sequences are shown as thε upper sequences in A and the lower sequences in B. Diamonds indicate nucleotidε identities where as asterisks indicate amino acid identities. An "X" in the sequence indicates an unknown residue. Figure 11 Nucleotidε and predicted amino acid ' sequence of the human FcR encoded by HFc3.0. Nucleotides are numbered every decade and the translated sequence is f ound abov e the nuc l eotidε sεquεπce. Amino acids are numberεd above the line and number 1 indicates the N- terminal residue. Thε incomplete signal sequence is underlined by a broken line to residuε -1. The two glycosylation sitεs are marked by stars and the single hydrophobic transme brane region is underlined by a solid line. Cysteine residues involved in disulphide bonding are circled. Figure 12 Homology of the human FcR encoded by HFc3.0 cDNA with the mouse alpha and betal FcR. Alignment of the nucleotides of the human FcR with both mouse alpha (A) and betal (B) FcR. Amino acid residues common to the human FcR encoded by HFc3.0 and the alpha FcR (A) or beta 1 (B) are shown by astεriks. The human (HFc3.0) mousε alpha or beta FcR l eader sequence, extrace l l u l ar domain , trans embrane domain and cytoplasmic domains are indicated by the sequences betωεeπ the vertical arrows. Thε cysteine (C) residues within the extracellular domains (involved in S-S bonds) are identified by the solid circles. Figure 13 Northern blot analysis of total RNA isolated from normal human spleen and probed ωith the cDNA insert from HFc3.1. The 28s and 18s ribαsomal RNAs are indicated. A, 20ug RNA loaded; B, 5ug RNA loaded; C No RNA. Figure 14 Southern blot analysis of the human FcR gene. Human thymus DNA (A) and human peripheral leukocyte DNA (B) was digested with 1 , Hind III; 2, PvuII; 3, EcoRI and 4, PstI and probed ωith thε cDNA insert from HFc3.1. Molecular size markers (Hindlll digested RNA) are indicated. Isolation of full length cDNA clone: The F c ( g am a ) R has been purified to homogeneity using the anti-Ly-17.2 antibody (Fig. 1 ). Protein and peptide sequencing studies have enable one third of the molecule to be sequeπced (Table 1 ). Consensus o 1 igαde oxynuc 1 eot ide probes constructed from the protein sequence (Table 2) were used to isolate a cDNA clone (pFc24). Southern analysis established that the cDNA insert of pFc24 reacted with the probes (Fig. 2). Maxam- Gilbert sequencing of the 5' and 3' ends of pFc24 (Fig. 3A,B) indicated it that it was not full length since no entire leader sequence or iπframe termination codon ωas apparent at the 5' and 3' ends respectively. A full length cDNA cloπε, pFc113 (Fig. 3) ωas isolatεd by reprαbiπg thε uJEHI 3B library ωith o 1 ig odεαxynuc 1 eotide probes constructed from the nucleotide sequence of the 5' and 3' ends . of pFc24. These probes corresponded to nucleotides 140-187 (probe 1.5) or 959-1000 (probe 1.6) (Fig. 3B, Table 3) and only clones to ωhich both probes hybridised ωere subsequently Isolated. The cDNA insert of pFc113 contains approximately 2.0 kilobase pairs whereas that of pFc24 has only 962bp but is entirely embodied in pFc113 between nucleotides 61 and 1023. Nucleotide and amino acid sequence: The amino acid sequence predicted from thε nucleotide sequence of pFc113 (Fig. 38) indicates that mature FcR is a membrane molecule composed of 301 amino acids and is syπthesised ωith a 29 amino acid leader sequence. The amino acid sequεncεs of 10 of 11 peptides ωε had prev iousl y sequenced from immunopurif ied FcR were found encoded by pFc113 cDNA (Fig. 3B, Hibbs et al, 1986). Miscalling of several amino acid residues during sequencing of the peptides accounts for the differences between the sequεncε of peptides L5 and CNBR and the predicted sequence of these regions from- the cDNA clone. Also the CNBR peptide is preceded by a Trp not the predicted Met but it is known that CNBR will cleave on the C terminal side of Trp residues in acid solutions (Ozols et al, 1977). Several other important obsεrvations can bε made from the predicted amino acid sequεnce. Firstly, a single transmembrane rεgiαπ of 28 amino acids εxtεπds from Lεu180 to the sequence Lys209 -Lys210 -Lys211 and separates the 94 amino acid l ong cytαp l asmic region from the 179 extracellular amino acids. Secondly, the extracellular portion of the FcR molecule contains two regularly spaced pairs of Cys residues, the first pair Cys 28 and Cys 70 separated from each other by 41 amino acids and the second pair Cys 109 and Cys 153 separated by 43 amino acids. This regular arrangement suggests the e racellular portion may be organised into two disulphidε bonded domains (see hαmologies below). Thirdly, four potential N-linked glycosylation sites are present in the ex racellular region of thε receptor, with two glycosylation sites located within each of the putative domains. These potential glycosylation sites have been verified as being authentic sites of carbohydrate attachment (Green et al-, 1985 and see "Additional Structural Features of Murine FcR" below.) Hαmologies of Murine FcR: Comparison of the amino acid sequεncεs within thε FcR molecule (assessed using the Dayoff ALIGN programme, Dayhoff et al, 1983) indicated that there was a significant degree of internal homology within thε extracellular domains (Fig. 4A). Using the mutation data matrix, scoring is based on the extent of mutation required for aminα acid substitution in established protein families. The score for the optimal alignment is then represented as the number of standard deviations by ωhich the maximum score for the alignment of two sequences exceeds the score for a large number of alignments after randomization of the original sequences (Dayhoff et al, 1983). Arbitrary setting of the boundaries of these domains around the pairs of Cys residues showed that alignment of amino acids 5-86 (FcR, domain 1 ) ωith amino acids 88-175 (FcR, domain 2) gave 29% identical residues ωith an ALIGN score of 7.1 SO i.e. the q probability of such an alignment occuriπg by chance is >10 and implies thε tandem duplication of a single domain. Such repeated domain structures are evident in Ig and related molecules that comprise thε Ig supεrfamily (Williams 1985). To further examine possible ho ologies to other molecules ωe undεrtook computer searches of a number of nucleic acid and protein data bases. These rev eal ed the FcR domain 1 ωas most homologous to murine Class II antigens in particular to the Ig like beta2 domain of I-Ebeta and ga v e a highl y significant ALIGN score of 8.3SD. In addition to the homo l ogy ωith C lass II antigens, other features of the FcR domains indicate their relatedπess to Ig superf amily mεmbers. The homology to members of the Ig supergεnε family is sεeπ principally around thε Cys residues. The sequences surrounding cysteine residues 70 and 153 were shown to be highly representative of the consensus sequence (Gly-X-Tyr- X-Cys) around thε d i s u 1 p h i d e - b o n d e d Cys residue in Ig V - region domains. In addition , the sequences f lanking Cys residuεs 28 and 109 were indicative of the di su 1 ph ide-b on d ed Cys near thε ami no -ter inus of thε V-rεgioπ of Ig chains and other Ig related molecules. Furthermore, the Trp residue located 13 residues downstream from Cys 28 and a Phe residue 13 residues downstream from Cys 109 are commonly observed in thi s p osition of I g - l ik e s tru ctur e s. Th e I g r e l at ed mo l ecu l e s Thy-1 and CD 4 , wh ich a l s o p o s se ss these characteristics have been shown ε p er im en t a 11 y to contain intrachaiπ disu lphidε bonds (Wi l l iams and Gagπoπ , 1982 ; C l asson εt al , 1986a). I t i s c l e a r t h e r e f o r e t h a t t h e r e c e p t o r f o r immunoglobulin shows a common evolutionary ancestry with its ligand but comparison of entire Ig and FcR domains shows a low overall homology indicating that they must have diverged relatively early in evolution. The high degree of homology ωith MHC Class II molecules is interesting in the light of the physical association of some FcR ωith Class II molecules on the cell surface (Oickler and Sachs, 1974). In addition to ths poly Ig receptor (see above), we also sought homology to other IgG binding- molecules. No homo l ogy of FcR domai n s to S t ap h y l o cocca l pro te in A (Sjodah l , 1977) ωas f ound. The Fc(gamma)R receptor described herein is highly homologous to other betal Fc(gamma)R except for a stretch of 9 amino acids in the cytoplasmic tail from Gly241 to Pro249. The discrepancies between the π uc 1 e o t ide/amiπo acid sequences shoωn in Fig. 3B may have arisen by the omission of three nucleotides. The nucleotidε and predicted amino acid sequence of the betal form of the Fc(gamma)R therefore is that shoωπ in Fig. 3B. In addition, another form of the Fc(gamma)R is highly homologous to the FcR described here -almost certainly being a splice variant. Thε splice sitε occurs in the region encoding thε intracellular domain indicating that the betal and beta2 forms of thε FcR havε identical extracellular domains. A third form of the FcR has also been identified by cDNA cloning studies and designated the alpha FcR The mature alpha protein shoωs a little variation from the receptor described herein ωith approximately 7% variation in amino acid content of the ligand binding domains. All major structural features in the domains of the three variants (alpha, betal , beta2) are conserved, i.e. (4 N-linked carbohydrate side chains, 2 pairs of cysteine residues). Thus molecular analysis of Fc(gamma)R clearly indicates that murine FcR are a family of highly homologous protεins. It should also be πotsd that the Ig binding proteins - protein A and the FcR described herein all have repeated ligand binding domains ωhich may be necessary for stability and specificity of ligand binding. Additional Structural Features of Murine FcR: ϋJe have demonstrated that ad acεnt pairs of cystsine residuεs in purif ied Fc(gamma)R are in vol ved in disu l phide bonding. Protein was purif ied b y a f f inity chroma togra ph y th en digested ωith proteolytic enzymes either before or after reduction of disulphide bonds ωith dithiothreitol. Digests ωere then fractionated by reversed-phase ch romat o gr a p hy . P e pti d e s p resent i n th e dig e st s o f unreduced Fc(gamma)R but absent from the digested reduced Fc( gamma ) R ωere s e que π cεd a nd shown t o- co rresp ond to d i s u 1 ph ide -b oπd ed peptides. The results obtained indicated that Cys 28 is disulphide-bonded to Cys-70 and that Cys-109 and 153 are involved in disulphide bonding. Extensive peptidε sequencing has also determined that f our po s s ib l e N- l i nk ed g l c yo sy l at ion s it e s in mou se Fc(gamma)R are in fact authentic sites of carbohydrate addition. This ωas judged by the absence of an asparagine in the expected position in the peptide sequence. Expression of mRNA: Analysis of FcR expression in cell lines ωas then perf ormed by Northern, b lotting. Tωo mRNA transcripts ωere evident ωheπ probing poly-A + mRNA from the UI E H I 38 cel l l ine , ωith nick trans lated cDNA (Fig. 5A). These transcripts ωere absent f rom the FcR " cel l l ine F4N. Sinc e th ere is FcR h etero genity ωith r espect to both specificity and expression in cell lineages (Dickler, 1976; Uπkless et al , 1981 ; Teil laud et al , 1985; mRNA from cel l lines of different lineages ωas examined (Fig. 5B) using an o 1 i g o d e o x y n u c 1 e o t i d e probe (probe 1.10) (corres onding to nuclεotides 545-574) that hybridised to both transcripts in UJEHI 3B cel ls (Fig. 58, track a). Northern blots showed that while both transcripts ware present in the y e 1 o m o n o c y i c cel l line WEHI 3B, the lower Mr species was predominant in J774 macrophage cells (Figs 5B, track b) and was completely absent from the FcR + T lymphoma K3B, where only the higher Mr sp ec ies cou l d b e d e tect ed. Th e pr ese nc e of mu l t ip l e Fc(gamma)R mRNA transcripts (termed alpha, betal and beta2, betal being identical to that encoded by pFc113 described herein) in different cell types ωas also noted. Rel ationship of mRNA transcripts and surf ace FcR : To estab l ish the re lationship between mRNA transcription and surface Fc(gamma)R expression, we investigated the presence o f mR N A t ra n scr ip t s b y N o r th er n a n a l ys i s u s in g oligonucleotide probes specific for the be al , beta2 and alpha transcripts (Table 3) and compared this ωith immune complex binding (using rabbit IgG and various IgG isotypes) and whεthεr thε cells wεre Ly-17.2+ αr 2.4G2+ at the surface (Tab le 4). The results iπdicats that the betal Fc(gamma)R is the receptor for IgG1/2b, heterologous IgG and possibly other Ig since cells which express only the bεtal receptor only can bind IgG1/IgG2b as ωell as rabbit IgG coated erythrocytes (Table 4). In addition, K36 cells have thε Ly-17.2+ and 2.4G2+ molecules which have been shown to be identical and are epitopεs on thε Fc(gamma)R molecule (Unkless, 1979; Hibbs et al, 1985; Holmes et al, 1985). Furthermore, antibodies to thεse molecules completely inhibit the binding of IgG1/2b and rabbit IgG complexes to the cell surfaces. Thus the betal variant must code for molecules which have Fc binding ability and the epitopes detected by thε 2.4G2 and Ly-17.2 antibodies. Both the Ly-17.2 and 2.4G2 epitopes are clearly present on the betal and beta2 molecules since both antibodies were used tα purify thεse molecules for amino acid sequencing in this study. Finally, the pFc113 cDNA (in the pKC3 vector) described herein was transfected into FcR negative LTA-5 cells resulting in expression of FcR (Ly- 17.2) on the cell surface (Table 4) indicating that this clone encodes an immunoglobulin binding FcR. The interactions of thε alpha and bεta2 receptors ωith antibody and immune complexes remains to be precisely determined. ϋJEHI 3B and J774 cells both express alpha and betal mRNA transcripts but only J774 cells express bεta2 mRNA although both bind IgG1/2b and rabbit IgG complexεs and are Ly-17+ (Table 4). Whilst cDNA expression experiments ωill be πeεdεd to define the specificity of thεse receptors, it is likely that the alpha, betal and beta2 receptors ωill have the same binding properties of immune complexes. Southern Blot Analysis: Southern hybridisation studies ωere performed tα determine ωhether the heterogeneity observed at the mRNA and protein levels was also apparent in the genome (Fig. 6). Probing of murine spleen DNA digested ωith Hind III or Pst I (Fig. 6, tracks a,c) generated tωo fragments tα ωhich the cDNA hybridised. Digestion ωith Eco RI (track b) also produced tωo major and several other ωεakly hybridised fragments. Thus it appears likely that only a single, or feω, copies of a highly coπssrved gene are present in the genome. The heterogeneity of receptors shoωπ by serolαgical studies, together ωith the presence of multiple mRNA transcripts indicates the receptors arε likely to bε a family of homologous proteins. Isolation of Human Fc(gamma)R cDNA Clones: Since human Fc can bind mouse and human immunoglobulin and have many similarities ωith mousε FcR, (Dickler, 1976; Anderson and Abraham,. 1980, Kulczycki et al, 1981 ; Perussia et al, 1983; 'Dorrington and Klein, 1983), it is likely that there is a high degree of structural and functional homology ωhich conserved at the nucleic acid and amino acid levels. Thus ωe used the mouse cDNA pFc24 and a combination of oligonucleotides, probes 1.5, 1.6, 1.10 (Table 3) to screen a library constructed from mRNA isolated from human acute monocytic leukaemia cells THP-1. Several clones were isolated including HFc3.0, HFc3.1 and HFc3.47. The results (Fig. 7) demonstrated that, 1. pFc113 cDNA hybridised to the cDNA inserts of HFc3.1 and HFc3.47; 2. probe 1.5 hybridised to HFc3.1 cDNA but not to HFc3.47; 3. probe 1.10 hybridsεd weakly to HFc3.1 cDNA but not to HFc3.47; 4. probe alpha did not hybridise to either clone and 5. probe 1.6 hybridised to HFc3.47 but not HFc3.1. Nona of the probes, except probe 1.6, hybridised tα the lambda arms - the hybridisation of probe 1.6 to the lambda arms was due to incomplete digestion prior to Southern transfer. The likely homology of the HFc3.1 and HFc3.47 cDNA clones to the mouse cDNA clones was then confirmed by nucleotide sequencing. Characterisation of Human FcR cDNA: The human cDNA inserts were subcloned into plasmid vectors. The complete nucleotide sequεnce of a cDNA encoding the human Fc(gamma)R and its predicted amino acid sequence is shown in Fig. 8. Cloπε HFc3.1 contains sequence encoding the mature Fc(gamma)R protein and most of the leader sequence, as ωell as the entire coding sequεπcε. The high degree of homology, seen both at the nucleotide and amino acid level, between the mouse FcR sequences and the sequence of HFc 3.1 , confirms that this clone encodes human FcR. The complete amino acid sequence of the human FcR, aligned with the sequences of mouse alpha and betal FcR is shown (Fig. 9A,B). Breaks have been introduced to optimize the alignment. The incomplete leader sequence of the human FcR encoded by HFc 3.1 is highly homologous to the leader sequence of the mouse alpha FcR (56% conservation of amino acids) (Fig. 9A) but bears no homology to the mouse betal Fc(gamma)R leader sequence (Fig. 9B). The N-termiπus of the human Fc ( g am a ) has been predicted on the basis of homology tα the mouse alpha Fc(gamma)R N-terminal sequence. The region between the N-termiπus and the first cysteine residue is the most highly conserved region between the tωo species, showing 71 and 73 percent amino acid homology ωith the murine betal and alpha Fc(gamma)R respectively (Fig. 9A,B). There is also a high level of amino acid conservation in the remaining extracellular portion between FcR of mouse and man. Like the mouse, the extracellular region is dividεd into two dis u 1 h id ε -bond ed domains: the first pair of cysteine residuεs being separated by 41 amino acids and thε second pair by 43 amino acids. Both disulphide bonded domains bear striking homology tα tha mouse betal and alpha Fc(gamma)R. Amino acid sequence comparison of the first domain shows approx. 56 percent conservation between the human FcR and both mouse alpha and beta Fc(gamma)Rs (Fig. 9A,B). Similarly, there is approx. 56 percεnt conservation of amino acids in the second domain between the human Fc(gamma)R and mouse alpha and beta FcRs. Tωo potential N-linked glycosylation sites are presεπt in thε extracellular region of the human FcR (one in each of the domains) (Fig. 8) and correspond to tωα of the four sites present in both mouse alpha and betal Fc(gamma)Rs (Fig. 3B). The human Fc(gamma)R has a transmembrane region of 28 amino acids extending f rom residue 219 to the hydrophilic stop transfer sequence Arg (245) - Lys (246) - Lys (247) - Arg (248) (Fig. 8). This transmembrane sεquence is highly homologous to the mouse betal F c ( g a m m a ) (50 percent amino acid h α m o 1 o g y ) , b u shoωs no homology to the transmembrane sequence of the mouse a l pha Fc( gamma)R recεptor (Fig. 9A,B). An in frame termination codon is found at nucleotide 1040 yielding a 75 amino acid i n t r a c y t o p 1 a s m i c domain. Comparison of the cytαp lasmic domains of both mousε Fc(gamma)Rs ωith thε human Fc(gamma)R shoωs little idsπtity of either nucleotides or amino acids (Fig. 9A,B). As well as thε very loω level of homology observed between mouse and human Fc(gamma)R cytoplasmic domains, another clear difference betωeεπ the mouse and human FcR sequences ωas apparent. The human FcR sequence contained an additional 117 nucleotides ωhich result in an insertion of 39 amino acids between thε tωo extracellular domains (Fig. 8). This sequence is absent from mouse FcRs (Fig. 3B, 9A,B) and most likely rεpresents an iπtron sequence. This is inferred from the sequencing of murine genomic clones. The striking homology of this human Fc(gamma)R to both alpha and betal murine Fc(gamma)Rs in the e tracel lular domain may ref lect the fact that human FcR bind mouse IgG and mouse FcR bind human Ig. The cytoplasmic domain of the human HFc3.1 FcR is totally unique and bears no resemblance to either mousε alpha, bεtal or bεta2 iπtracellular domains. This difference could mean thε presence of a homologous product in the mouse ωhich has yet to be detected or may reflect th ' e evolutionary divergence of the human and mouse proteins in a region where there may be a high rate of mutation. Partia l nucleotidε sequence of HFc3.47 has a l so revealed homology with the mouse FcRs (Fig. 10). Two non- overlapping fragments were sequenced and the first fragment (Fig. 10A) re vealed 80% nuc l eotidε homo l ogy with the corresponding sequence in betal FcR (nucleotides 629-733). This sequence is present in beta2 and a similar sεquence in alpha. The second fragment shared 72% nucleic acid homology ωith nucleotides 959-1015 of the betal FcR, this sεquence also being present in thε beta2FcR but not alphaFcR. An additional variant sεquεncε ωas also obtained from thε THP-1 library HFc 3.0, the sεqueπce of ωhich is shoωπ in Fig. 11. Like HFc 3.1 , HFc 3.0 sncodεs a protein highly homologous to the mouse alpha and beta FcR receptors (Fig. 12A,B). The nucleotidε sεqueπce HFc 3.0 is identical tα HFc 3.1 ωith the exception of a large segment (nucleotides 338- 455 in HFc 3.1 ) betωeen the two disulphide bondεd domains which has bεεπ deleted in HFc 3.0. The protεiπ encoded by HFc 3.0 has an extracellular ligand binding region consisting of two disulphide bondεd domains εach with a site of attachment for N-linkεd carbohydrate. In addition, thε encoded protein has a 28 amino acid transmembrane rεgion and 75 amino acid cytoplasmic tail. Thε extracellular region shows an overall homology of 67% identical amino acid residues and the transmembrane region has 14 of 28 amino acid identities with thε mouse betal and beta2 transmembrane region (Fig. 12A.B). Expression of Human FcR mRNA: Northern blots were performed to analyse FcR expression in normal human spleen. Two mRNA transcripts were apparent after probing total mRNA from normal human spleen ωith the cDNA insert from pHFc 3.1 (Fig. 13). The presence of at least tωo hybridising mRNA species in human spleen proba.bly indicates that, likε thε mouse, there are multiple human FcR proteins arising from eithεr one or more genes. DNA Analysis: Southern analysis of human geπomic DNA from thymus and peripheral blood leukocytes (PBL) (Fig. 14) demonstrated that identical restriction fragments ωere present in thymus and PBL DNA ωhen digested ωith the same restriction enzyme. This indicates that since thymocytεs arε mostly FcR- and PBLs mostly FcR+, that thε FcR genε is not rearranged. In addition since there ωerε only a fεω major hybridising restriction fragments it is likεly that there is a single αr feω copies αf a highly conserved gene in the human genome. Since human FcR can bind mouse and human immunoglobulin and have many similarities with mouse FcR, (Dickler, 1976; Anderson and Abraham, 1980, Kulczycki εt al, 1981 ; Perussia et al, 1983; Dorrington and Klein, 1983), it is likεly that there is a high degree of structural and functional homology which conserved at the nucleic acid and amino acid levels. Thus we used the mouse cDNA pFc24 and a - c o m b i n a t i o n αf oligonucleotides, probes 1.5, 1.6, 1.10 (Table 3) to screen a library constructed from mRNA isolated from human acute monocytic leukaemia cells THP-1. Several clones were isolated including HFc3.1 , 3.0 and 3.47. Southern analysis of EcoRI digested DNA prepared from each of these clones showed that the mouse betal cDNA (from pFC24) hybridised to the cDNA insert of each HFc clone, i.ε. HFc3.0, HFc3.47, HFc3.1. The oligonucleotide probe 1.5 hybridised to the cDNA insert of HFc3.1 and 3.0 and the oligonucleotide probe 1.6 to the cDNA insert of HFc3.47. Thε cDNA inserts were purified and subcloned into M13mρ8 and M13mp9 bactεriophagεs and sequenced by thε didεoxyπuc 1 εotide method according tα the strategy outlined in Fig. 7a. After sequencing, these clones showed homology with the mouse Fc(gamma)R and indeed identify thε Fc(gamma)R. This strategy and similar could be used to isolate the Fc(gamma)R of any species. IMPORTANCE OF DIFFERENT PARTS OF THE SEQUENCE The amino acid sequence is divided into several parts (Figs. 3B , 8 , 9 , 11 , 12 ): - Leader sequence Extracellular Region Transmembrane Region I n t r ac e 11 u 1 a r Rεgioπ (Cytoplasmic Region) The Leader Sequεnce. The leader sequence of 29 amino acids in the betal FcR alloωs translocation of the nascent protein across the eπdoslasmic reticulum. The key point of this sequence is its hydrophobic nature (required for membrane insertion) and substitution, addition or deletion of one or mαrε hydrophobic (non-polar) amino acids would not substantiall 1 alter this fuπctiαn (see list later). 2 Extracellular Region. 3 Thε extracellular region (FCR) consists of 179 amino 4 acids; their function is to act as the receptor for the Fc 5 piece αf Ig molecules. This binding site has not yet been 6 iden ified but comments on the nature of thε sequence are 7 rεlεvant. (a) The FcR is divided into tωo domains by 8 cysteine residuεs, Domain 1 : Cys 28 - Cys 70; Domain 2: Cys 9 109 - Cys 153. Both of thεse domains may have FcR activity 10 as they are highly homologous. ϋJ ith in each domain all or 11 most of thε amino acids arε likεly to be involved in Ig 12 binding as thεy arε conserved - thε tωo domains arε 13 conserved in man and mousε. (i) Thus when the murine 14 sequences arε compared for internal homology (i.e. domain 1 15 and domain 2) they are very similar, (ii) Further, when 16 domain 1 of mouse is compared with that of man, each contain 17 43 amino acids, of these 24 are identical and in thε 18 identical position, αf the 19 differences 8 arε conservative 19 changes in that only 1 πucleαtidε has been changed, and of 20 the remaining changes, 6 of the 19 are in the same amino 21 acid group (seε below) and 13 are in diffεrεπt groups. 22 Thus the whole sequence in the domain or minor 23 variations thereof are most likely to be involved in Ig 24 binding. 25 The amino acids substituted belong to the same groups 26 and ωould thεrefore not subs antially alter the tertiary 27 structure αf the molecule and the groupings of amino acid 28 are : - 29 (i) non-polar amino acids : A , V , L , I , P , F , M , ϋJ. 30 (ϋ) basic side chain : K,R,H 31 (iii) acidic side chain : E,D 32 (iv) polar side chain : G ,N ,Q,C,S,T ,Y 33 A, ala = alaπiπe; C, cys = cysteine; D, asp = aspartic 34 acid; E, glu = gluta ic acid; F, phe = phen 1 a1 anine; G, gly 35 = glycinε; H, his = histidiπε; I, ilε = isoleucine; K, lys = 36 lysine; M, met = methioπine; N, asn = asparaginε; P, pro - 37 proliπε; Q, gin = glutamine; R, arg = arginine; S, ser = 38 seriπε; T, thr = threonine; V, val = valinε; J , trp = tryptophan; Y, tyr = tyrosinε. Transmembrane Region. This consists of 28 amino acids and extends from Leu 180 - Lys 209 in mouse betal FcR. The key feature of this is the hydrophobic nature of the sequence is requirεd as this portion interacts ωith the hydrophobic cell membrane and anchors the molecule in the cell membrane. Because of the hydrophobic nature, it follows that minor alterations of sequence - substitution of one non-polar amino acid by another, ωould not alter the essential function of the transmembrane region. It shou ld be noted that such hydrophobic (non-polar) amino acids arε alanine (A); valine (V); lεuciπε (L); isoleucine (I); proline (P); p eny1a 1anine (F); methionine (M) and tryptophan (W). Intracεlluar Rεgioπ. The intracel lul ar region is in v o l v ed in signa l transmission - obtained ωheπ thε Fc of Ig binds to the FcR. The modε of signa l transmission is unknown. A usefu l sequεnce of the FcR i s described ; it is l ikel y that variations of this sequence, obtained by deletion αf parts of the whole sequence, could also function in signal ransmission. Thε region of the betal receptor that is spliced out in beta2 is important in cells expressing these molecules. Variants on the materials the subjεct of this specification are possible:- A. Sequence Variation The nucleotide sequences encoding the receptor can be variablε:- 1. Because αf the degeneracy of the genetic code nucleotide change does not necessarily bring about a change in the amino acid encoded, e.g. thε codon GUU specifies a valiπβ residue as do the codons GUC, GUA, GUG each being differεnt by a single nucleotide. 2. Two or three nucleotide changes can give rise to thε same amino acid, e.g. codons UUA, UUG, CUU, CUC, CUA, CUG all encode Leucine. Codons AGU, UCC, UCU, UCA, UCG encode seriπe. 3. Changing one or two nucleotides may result in a conservative amino acid change unlikely to greatly affect the function αf the protein, e.g. codon UUG specifies leucine and AUU specifies isoleucine. Also UGG specifiεs tryphophaπ and UUU specifies phenylalanine - al l conservative changes. 4. Allelic variations. Variations in nucleotide sequence and amino acid sequences of the encoded protein as well as resultant may occur between individual members of thε same species. These variations arise from changes in the nucleotidε sequences encoding the protein. Thus differεπt forms of the same gene (called allεlεs) give rise to protein of slightly different amino acid sequεnce but still have the same function. 5. Variation can occur as the result of differential mRNA splicing where one genε composed of many different segments (exαπs) αf coding sequence - DNA encoding thε mature protεiπ - gives rise to a RNA that is spliced such that the portion of thε RNA derivεd from certain exons are rεmovεd. Selection of εxons is diffεrent in different cell typεs, ε.g. thε betal and beta2 forms αf FcR or the alpha and alpha' forms of Ly-2. 6. Proteins ha v ing thε same function , e.g. immunoglobulin binding, -may arise from related genes. Many protein gene families ha v ε been described , ε.g. im un og 1 αbu 1 in s which have nucleotide and amino acid sequence variation but retain their primary function of antigen binding. Such homologous proteins arε encoded by homologous genεs. These genes arise by duplication αf one original gene or by genε cαnvεrsioπ. 7. Variation may be in entionally introduced by:- (a) Mutating cloned cDNA or genomic DNA by point mutation, rearrangement or insertion of relatεd or unrelated DNA into thε cDNA or gεnomic cloπεs εncoding the functional protein. Such mutatεd (variant) clonεs can be used to generate variant proteins or pεptides which in the context of this specifica ion may havε Ig binding function. (b) By enzymatic cleavage of the protein (from sithεr in vitro synthesis or normal cell synthesised protein) with or without repair/rearrangement of thε clεavage products. (c) By chemical modification. (d) By irradiation. B. Use of portions αf the FcR protein. The present invention also includes the use of segments of the FcR protein (peptides) and variant peptides synthesised or genetically engineered that have the capacity to bind Ig. The FcR protein and its variants extend from thε first mεthioninε in the leader sequence to the most C terminal amino acid of thε protein. C. Isolation of other FcR. Other FcR ωithin and betωeεπ species that are homologous at thε nucleic acid, protein and functional levels. Because of substantial sequence homologies, thε cDNA clonεs described herein ωould enable the isolation of related sequences encoding FcR from man, the mouse and other species. The Principal Novelty of the Invention includes but is not limited to: (i) Description of a molecule, the FcR, ωhich has the capacity to bind the Fc piece of Ig. (ii) Thε structural arrangement of thε 2 extracel lular domains ωhich shαω homology to εach othεr in both man and mouse, (iii) The similarity αf these domains in man and mouse. (iv) Differences in the intraca11u 1ar rεgions in man and mouse; indicating that signals can be transmitted by different structures. (v) cDNA and other DNA and RNA material encoding for the receptor. (vi) Various vectors including materials. Table 1. The N-termiπal sequεnce of thε Fc receptor and amino acid sequences of Fc recεptor peptides. Peptide Sequεπcε NH2-terminal THDLPKAVVKLEPP L3 KGSLGRTLHQSK L4 KPVTITVQGPK L5 KSVRHHYSS-FSIPK L9 KAVVKLEPP-JIQLVK V4 ELSTTGGNSG(S)P(V)(K)N V8 EQTRLSDPVDLGVI V10 ENTITYSLLKHPE V11 EAENTITYSLLKHPE V16 THDLPKAVVKLEP— IQV V17 THDLPKAVVKLEPPIiJIQV CNBr-1 MRNKHLNRIVFL(Q/T)N(Y)(K) "-" indicates an uπassigned residue ( ) indicates an uncertain assignment. Table 2. Amino Acid Sequences of lysiπe-C pεptidεs of thε murinε Fc receptor and corresponding nucleotide sequence of oligonucleotide probes. Oligonucleotide probes ωεre constructed on the basis of codon usage frequencies and synthesised to be complementary to mRNA. L3 (Lys)-Gly-Ser-Leu-Gly-Arg-Thr-Leu-His-Gln-Ser-Lys Probe 1 3' UUC-CCU-AGG-GAC-CCU-UCU-UGG-GAC-GUG-GUC-AGG-UUC 5' L4 (Lys)-Pro-Val-Thr-Ile-Thr-Val-Gln-Gly-Pro-Lys Probe 3 3'- UUC-GGU-GAC-UGG-UAG-UGG-GAC-CUC-CUU-GGU-UUC 5' L5 (Lys)-Ser-Val-Arg-Tyr-Gly-Gly-Tyr-Ser-Ser-Ser-Phe-Cys-Ile-Pro-Lys Probε 2 3'UUC-AGG-CAC-UCU-AUG-CCU-CCU-AUG-AGG-AGG-AGG-AAG-ACG-UAG-GGU-UUC 5' Table 3. Oligodεoxynucleotide probεs used in this study Name Sequεncε* Cαrrεsponding sεquεnce in pFc113 cDNA Probe 1.5 5ΑCGGGGGCTCGAGTTTGACCACAGCCTTTGGAAGATCATGAGTCCCAG3' 135-182** -Probe 1.6 ■ 5'TTCGGGATGCTTGAGGAGTGAGTAGGTGATCGTGTTCTCAGCCTC3' 956-1000** Probe 1.10 5'GTGGTTGGCTTTGGGGATAGAGAAATTACT3' 545-574** Probe alpha 5'AGGGAGAAAGCAGTGTGGTACCAGAC3' Probe betal 5'CTGTCTGTACTCACCTACTTCCTCTGGAAG 821-850**, Probe bεta2 5'AGGAGGATTGTCTGGAACCTGCTT3' * Sεqueπces complεmεntary to mRNA ** Sεquεncεs from this paper and Hibbs et al 1986 Table 4. Comparison of Immune Complex Binding and FcR mRNA synthesis Cell line FcR mRNA* %EA rosettes** %Ly-17+ cells 1 (Type) variant gammal/ gamma2b Rabbit Ig K36 betal >99 >99 >99 (T lymphoma) ϋJEHI 3B alpha, betal >99 >99 >99 (myelomonocytic) J774 alpha, betal , beta2 >99 >99 >99 (macrophage) TransfectantsH- NT NT NT 70 * alpha, bstal , beta2 mRNA transcripts detectεd by Northern analysis using specific oligonucleotide probes (Table 1). ** % EA rosetting cells dεtectεd using IgG2b monoclonal anti-TNP aπtibodiεs or rabbit IgG anti sheεp εrythrocytes (see Materials and Methods). ° determinεd by shεep anti-mouse Ig rosetting with anti-Ly-17.2 monoclonal antibody. Background levels of rosettε formation were determined using an irrelevant antibody and were <5 % . These cells are also tested with 2.4G2 antibody and show identical reactions to the Ly-17.2 antibody. NT = not tested. + The Pst-1 fragment of the pFc113 cDNA insert was subcloned into pKC3 and transfected into LTA-5 cells using the CaCl 2 method. References Apεlla, E. Proc. Natl. Acad. Sci. USA 68: 590-594, 1971. Andεrson, CL. and Abraham, G.N. J. Immunol. 125: 2735- 2741 , 1980. Caproπ, M., Speigelberg, H.L., Prin, L., Bennich, H., Butterworth, A.E., Pierce, R.J., Quassi, M.A. and Capron, A. J. Immun. 132: 462-468, 1984. Classαπ, B.J., Tsagaratos, J., McKenzie, I.F.C. and ϋJa 1 k ε r , I.U. Proc. Natl. Acad. Sci. USA 83: 4499-4503, 1986a. Classαn, B.J., Tsagaratos, J., Kirszbaum, L., Maddox, J., Mackay, C.R., Brandon, M.R., McKenzie, I.F.C. and Walker, I.D. Immuπogenetics 23: 129-132, 1986b. Dayhoff, M.O., Barker, W.C. and Hunt, L.T. Methods in Eπzymol. 91 : 524-545, 1983. Dickler, H.B. Adv. Immun. 24: 167-215, 1976. Dickler, H.B. and Sachs, D.H. J. Exp. Med. 140: 779-796, 1974. Dorringtoπ, K. and Klein, M. In Froesε, H. and Paraskεva, F. (Eds). Receptors and Ligands in Iπtracel lu lar Co munications, Vol. 2. Structurε and Function of Fc receptors, Marcel Dekkεr, New York, 1983 pp Engelhard, M. and Hilschmanπ, N. Ho pp e -S εy 1 er 's Z. Physiol. Chem. 356: 1413-1444, 1975. Fridman, W.H., R abou rdin-Comb e , C, N eau p or t -S an t es^, C, and Gisler, R. Immun. Rev. 56: 51 -88, 1981. Gough, N.M., Metcalf , D., Gough, J., Grail, D. and Dunn, A. EMB0 Journal 4, 645-653, 1985. Green, G.A., Plutner, H. and Mεl lman, I. J. Biol. Chεm. 260: 9867-9874, 1985. Hεmpεrly, J.J., Murray, B.A., Edεlman, G.M. and Cunningham, B. Proc. Natl. Acad. Sci. USA 83: 3037-3041 , 1986. H ibbs , M .L., Hoga rt h , P . M . a n d M cK εn z i e , I .F .C . Immunogεnet ics 22: 335-348, 1985. Hibbs, M.L., Walker, I.D., Kirszbaum, L., Chambers, G.W., Pietersz, G.A., Deacon, N.J., McKenzie, I.F.C. and Hogarth, P.M. Proc. Natl. Acad. Sci. USA 83: 6980-6984, 1986. Holmes, K.L., Palfree, R.G.E., Hammerling, U. and Morss, H.C. Proc. Natl. Acad. Sci. USA 82:7706-7710, 1985. Hogarth, P.M., Edwards J. McKenzie I.F.C, Soding J.N. and Liew F.J. (1982) J. Immol 46 135-144. Hubscher, T. and Eiseπ, A.H. Int. Arch. Allεrgy Appl. Immun. 41 : 689-699, 1971. Humes , J.L., Biπger , S., Ga l a v age , M., Kuεh l , F.A., Wightman, P.D., Dahlgreπ, M.E., Davies, P. and Bonney, R.J. J. Immun. 124: 2110-2127, 1980. Hunkapillar M.W., Heωick R.M., Drεyεr W.J. 9 Hoodie (1983) Mεthods Enzymol 91 , 399-413. Kolsch, E., Haubeck, H. and Schuler, W. In Froese, A. and Paraskεvas, F. (Eds) Rεcεptors and Ligands in I π t ere e 11 u 1 a r Communication , V ol . 2. Structure and Function of Fc Receptors, Marcel Dekkεr, Neω York, 1983, pp 215-231 , 1983. Kulczycki, A., Solanki, L. and Cohen, L. J. Clin. Invest. 68: 1558-1564, 1981. Kurlaπder, R.J., Ellison, D.M. and Hall, J. J. Immun. 133: 855-862, 1984. Leslie, R.G.Q. Eur. J. Immun. 10: 323-346, 1980. Lopez, A.F., Strath, M. and Sanderson, C.J. Immunology 48: 503-509, 1983. 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A deposit αf the material referred to herein as pFc24, pFc113, HFc3.0, HFc3.1 and HFc 3.47 ωas made ωith ATCC on or about 29th M ay, 1987 and ωith Dr. George Hodges of the Cancεr Iπstituts (also known as thε Pεtεr McCallum Clinic) αf 481 Litt l e Loπsda l e Street, M e l bourne , V ictoria , Australia under terms and conditions pεrmitting access to members of- the public. RECEIPT I, Dr. George Hodgson, of the Cancer Institute, (also known as the Peter MacCallum Clinic) of 481 Little Lonsdale Street, Melbourne, Victoria, Australia, have received on 29th May, 1987 a deposit of material identified as HFc 3.0, HFc 3.1 and HFc 3.47 and pFc24 and pFcll3 from Professor Ian McKenzie of the University of Melbourne in connection with an about to be filed PCT Patent Application based on Australian Patent Application No. PH 6166 filed 29 May, 1986. I agree to keep and maintain that material and to make it available to the Public under terms and conditions no more onerous than are required under the Budapest Treaty or the terms of the ATCC in circumstances in which material needs to be made available to the public in consequence of or in support of the filing of a Patent Application. Si n ned Peter MacCallum Research Laboratori Dated ..9, .T.. ..7. $. 1.

Claims: THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. A receptor for the Fc portion of immu n o g ob u 1 i n and being derived from ATCC , ATCC or ATCC 2. A receptor for the Fc portion of immunogobu 1 iπ , the receptor exhibiting thε ability tα bind to thε Fc portion of mouse and human immunogαbulin . 3. A receptor as claimed in claim 2, and exhibiting an ability to bind to antibodies and immune complexes. 4. A receptor as claimed in claim 2, and having a size of 40-70 kilodaltαns. 5. A receptor as claimed in claim 2, and formed from mRNA of a size of about 1.8, about 2.0, about 1.9, about 1.4 or about 2.4 kilobases. 6. A receptor as claimed in claim 2 comprising about 280 to 301 amino acids including tωo substantially regularly spaced pairs of Cys residues, two or four potential N-linked glycosylation sites. 7. A receptor for the Fc portion of i muπogobu 1 in and having at least 40 , more prεferably at least 50$ and still more preferably at least 60$ homology with the receptor of claim 1 or claim 2. 8. A receptor for the Fc portion of immu π o gα b u 1 iπ and including the amino acid sequences :- (a) met leu leu trp thr ala val leu asn lεu ala ala gly thr his asp leu pro lys ala val val lys leu glu pro pro trp ile , (b) glu gin thr arg leu ser asp pro val asp lεu gly val ile, and (c) lys gly ser leu gly arg thr lεu his gin ser lys pro val thr ile thr val gin gly pro lys, and (d) glu ala glu asn thr ile thr tyr ser leu leu lys his pro glu ala leu asp glu glu thr glu his. 9. A receptor for the Fc portion αf immuπ αg ob u 1 iπ and including thε amiπα acid sequεπcε:- Met Glu Ser Asn Trp Thr Val His Val Phe Ser Arg Thr Leu Cys His Met Leu Leu Trp Thr Ala Val Lεu Asn Lεu Ala Ala Gly. 10. A receptor for the Fc portion of immuπ o go b u 1 in and including the amino acid sequence:- Cys Glu Gly Thr His Asn Pro Gly Asn Sεr Ser Thr Gin Trp Phe His Asn Gly Arg Ser -He Arg Ser Gin Val Gin Ala Sεr Tyr Thr Phe Lys Ala Thr Val Asn Asp Ser Gly Glu Tyr Arg Cys. 11. A receptor far the Fc portion of immun o g o bu 1 in and including the amino acid sequence:- Cys His Ser Trp Arg Asn Lys Lεu Leu Asn Arg Ile Ser Phe Phe His Asn Glu Lys Ser Val Arg Tyr His His Tyr Sεr Sεr Asn Phe Ser Ile Pro Lys Ala Asn His Ser His Ser Gly Asp Tyr Tyr Cys . 12. A receptor for the Fc portion of i mm u n o g α b u 1 i n and including the amino acid sequencε:- Leu Pro Val Leu Thr Ile Val Ala Ala Val Thr Gly Ile Ala Val Ala Ala Ile Val Ile Ile Lεu Val Ser Leu Val Tyr Leu. 13. A receptor for the Fc portion of immuπogobuliπ and including the amino acid sεquεncε:- Lys Lys Lys Gin Val Pro Ala Leu Pro Gly Asn Pro Asp His Arg Glu Met Gly Glu Thr Leu Pro Glu Glu Val Gly Glu Tyr Arg Gin Pr.o Ser Gly Gly Ser Val Pro Val Ser Pro Gly Pro Pro Ser Gly Leu Glu Pro Thr Ser Sεr Sεr Pro Tyr Asn Pro Pro Asp Leu Glu Glu Ala Pro Lys Thr Glu Ala Glu Asn Thr- Ile Thr Tyr Ser Leu Leu Lys His Pro Glu Ala Leu Asp Glu Glu Thr Glu His Asp Tyr Gin Asn His Ile. 14. A receptor for the Fc portion of immunogα-bu'lin and including the amino acid sequεπce:- N S G P R N L W L L Q P L T V L L L L A S A D S Q A A. 15. A receptor for thε Fc portion of immuπogαbulin and including the amino acid sεqueπce:- C Q G A R S P E S D S I Q W F H N G N L I P T H T Q P S Y R F K A N N N D S G E Y T C 16. A receptor for the Fc portion of i m mu n o g o b u 1 in and including the amiπα acid sequencε:- C H S W K D K P L V K V T F F Q N G K S Q K F S R L D P T F S I P Q A N H S H S G D Y H C. 17. A receptor for thε Fc portion of i m m u n o g o b u 1 in and including the amino acid sεquence:- S P M G I I V A V V I A T A V A A I V A A V V A L I Y C. 18. A receptor for thε Fc portion of immuπ o g ob u 1 in and iπcludiπg the amino acid sequeπce:- R K K R I A N S T D P V K A A Q F E P P G R Q M I A I R K R Q L E E T N N D Y E T A D G G Y M T L N P R A P T D D K N I Y L T L P P N D H V N S N N. 19. A nucleotide sequence, gene, cDNA clone, or a vector for same adapted to encode or to produce a material adapted to encode for the receptor of any preceding claim or a fragment thereof. 20. A nucleotidε sequence, gene, cONA clone, or a vector for same adapted to encodε or producε a material adaptεd to επcode for the leader sεquεπce, extracellular region, transmembrane region or intracε11u1ar region of the receptor of any one of claims 1-18. 21. A cDNA clone, genε or nucleotide sequence being incorporated in ATCC ,ATCC or ATCC and capable of encoding for a rεcεptor for the Fc portion of immunαgobu1 in or a fragment thereof. 22. A material being ATCC ,ATCC , or ATCC 23. A nucleotide sequence, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion of immuπogobu1in and including the nucleotide sequεncεs: - (a) ATG CTA CTG TGG ACA GCC GTG CTA 'AAT CTT GCT GCT GGG ACT CAT GAT CTT CCA AAG GCT GTG GTC AAA CTC GAG CCC CCG TGG ATC, (b) GAG CAG ACC CGC CTC AGC GAC CCT GTA GAT CTG GGA GIG ATT, (c) AAA GGA AGT CTA GGA AGG ACA CTG CAC CAG TCC AAG CCT GTC ACC ATC ACT GTC CAA GGG CCC AAG, and (d) GAG GCT GAG AAC ACG ATC ACC TAC TCA CTC CTC AAG CAT CCC GAA GCC TTG GAT GAA GAA ACA GAG CAT. 24. A nucleotide sequεπcε, cDNA clone, gεne or vector containing same and capablε of encoding for a rεcεptor for thε Fc portion of immuπ o gob 1 in and having thε nucleotide sequence :- ATG GAG AGC AAC TGG ACT GTC CAT GTG TTC TCA CGG ACT TTG TGC CAT ATG CTA CTG TGG ACA GCC GTG CTA AAT CTT GCT GCT GGG. 25. A nucleotide sequence, cDNA cloπε, gene or vector containing same and capable of encoding for a receptor for the Fc portion of im m u π o g ob u 1 i n and having the nucleotide sequence :- TGC GAA GGG ACC CAC AAC CCT GGG AAC TCT TCT ACC CAG TGG TTC CAC AAT GGG AGG TCC ATC CGG AGC CAG GTC CAA GCC AGC TAC ACG TTT AAG GCC ACA GTC AAT GAC AGT GGA GAA TAT CGG TGT. 26. A nucleotide sequence, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion of i mm u π o g o b u 1 in and having the nucleotide sequence:- TGC CAT AGC TGG AGG AAC AAA CTA CTG AAC AGG ATC TCG TTC TTC CAT AAT GAA AAA TCC GTG AGG TAT CAT CAC TAC AGT AGT AAT TTC TCT ATC CCC AAA GCC AAC CAC AGT CAC AGT GGG GAC TAC TAC TGC. 27. A nucleotidε sequence, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion αf im mu n α g ob u 1 i π and having the nucleotide sequεnce : - TTA CCA GTA TTG ACA ATT GTG GCT GCT GTC ACT GGG ATT GCT GTC GCA GCC ATT GTT ATT ATC CTA GTA TCC TTG GTC TAT CTC 28. A nucleotide sequεnce, cDNA clone, gene or vector containing same and capable of encoding for a receptor for thε Fc portion of im m u n α g o b u 1 in and having the nucleotide sequence : - AAG AAA AAG CAG GTT CCA GCT CTC CCA GGA AAC CCT GAT CAC AGG GAA ATG GGA GAA ACC CTT CCA GAG GAA GTA GGT GAG TAC AGA CAG CCC TCT GGG GGC TCA GTG CCT GTC AGC CCA GGG CCT CCA TCT GGA CTG GAG CCA ACA AGC AGC AGC CCA TAC AAT CCT CCT GAT CTG GAA GAA GCT CCC AAA ACT GAG GCT GAG AAC ACG ATC ACC TAC TCA CTC CTC AAG CAT CCC GAA GCC TTG GAT GAA GAA ACA GAG CAT GAT TAC CAA AAC CAC ATT TAG. 29. A nucleotide sequnce, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion of imm uπ o g αb u 1 in and having the nucleotide sequence : - AAT TCC GGT CCC AGA AAC CTG TGG CTG CTT CAA CCA TTG ACA GTT TTG CTG CTG CTG GCT TCT GCA GAC AGT CAA GCT GCA. 30. A nucleotidε sequnce, cDNA clone, gene or vector cαntaiπing same and capable of encoding for a receptor for the Fc portion of i muπ o g o b u 1 in and having the nucleαtidε sequεnce : - TGC CAG GGG GCT CGC AGC CCT GAG AGC GAC TCC ATT CAG TGG TTC CAC AAT GGG AAT CTC ATT CCC ACC CAC ACG CAG CCC AGC TAC AGG TTC AAG GCC AAC AAC AAT GAC AGC GGG GAG TAC ACG TGC. 31. A nucleotide sequncε, cDNA clone, genε or vector containing same and capable of encoding for a receptor for the Fc portion of immuπ o gob u 1 iπ and having thε nucleotide sequence : - TGC CAC AGC TGG AAG GAC AAG CCT CTG GTC AAG GTC ACA TTC TTC CAG AAT GGA AAA TCC CAG AAA TTC TCC CGT TTG GAT CCC ACC TTC TCC ATC CCA CAA GCA AAC CAC AGT CAC AGT GGT GAT TAC CAC TGC 32. A nuclεotide sequnce, cDNA clone, gene or vector containing same and capable of encoding for a receptor for the Fc portion αf im mu π o g αb u 1 in and having the nucleotide sequεnce : - TCA CCA ATG GGG ATC ATT GTG GCT GTG GTC ATT GCG ACT GCT GTA GCA GCC ATT GTT GCT GCT GTA GTG GCC TTG ATC TAC TGC. 33. A nucleotide sequnce, cDNA clone, gene or vector containing same and capable of encoding for a receptor for thε Fc portion of immuπ o g o b u 1 iπ and having thε nucleotide sequεncε : - AGG AAA AAG CGG ATT TCA GCC AAT TCC ACT GAT CCT GTG AAG GCT GCC CAA TTT GAG CCA CCT GGA CGT CAA ATG ATT GCC ATC AGA AAG AGA CAA CTT GAA GAA ACC AAC AAT GAC TAT GAA ACA GCT GAC GGC GGC TAC ATG ACT CTG AAC CCC AGG GCA CCT ACT GAC GAT AAA AAC ATC TAC CTG ACT CTT CCT CCC AAC GAC CAT GTC AAC AGT AAT AAC TAA. 34. A rεcεptor for the Fc portion of i mu n o g α b u 1 i n having the amino acid sequence set out in any onε of the accompanying drawings or so much thereof being a receptor for the Fc portion αf immuπogobulin . 35. A receptor for the Fc portion of i m m u n α g o b u 1 i π as claimed in any one of claims 1 -18 and 34 and modifisd by deletion, addition or substitution of amino acids or by chemical or irradiation modification not substantially deletεriously affecting thε ability tα bind to the Fc portion of immunαgobuliπ . 36. A nucleotide sequεncε, gene, cDNA clone, or a vector for same having the nucleotide sequence set out in any one of thε accompanying drawings or so much thereof being adapted to encode for a receptor for the Fc portion of immunogobuliπ or a fragment thereof. 37. A nucleotidε sequence, gene, cDNA clone, or a vector for same as claimed in any one of claims 19-33 and 36 and modified by deletion, addition or substitution of nucleotides or by chemical or irradiation modification not substantially deleteriously affecting the ability to code for a recεptor for thε Fc portion of i mu π o g ob u 1 i n or a fragment thereof. 38. A receptor for the Fc portion of immun o g ob u 1 in and bεing derived from pFc24, pFc113, Hc3.0, Hc3.1 or Hc3.47. 39. A material being pFc24, pFc113, Hc3.0, Hc3.1 or Hc3.47. 40. A cDNA clone, gene or nucleotide sequence being incorporated in pFc24, pFc113, Hc3.0, Hc3.1 or Hc3.47 and capable αf encoding for a recεptor for the Fc portion of immunogobuliπ or a fragment thereof.

Inventors: BONADONNA LISA, HIBBS MARGARET L, HOGARTH MARK P, MCKENZIE IAN F C, SCOTT BERNADETTE M, BONADONNA, LISA, HIBBS, MARGARET, L., HOGARTH, MARK, P., MCKENZIE, IAN, F., C., SCOTT, BERNADETTE, M.
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1987
Main classification: C07K13
IPC codes: A61K38, A61P37, C07H21, C07K14, C12N15, C12P21