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IL-6 Receptor Masahiko Hibi* and Toshio Hirano Division of Molecular Oncology, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine(C7), 2-2, Yamada-oka, Suita, Osaka, 565, Japan *corresponding author tel: 81-6-879-3880, fax: 81-6-879-3889, e-mail: [email protected] DOI: 10.1006/rwcy.2000.17001. SUMMARY binding receptors expressed on the surface of cells which can respond to IL-6 (Taga et al., 1987). The human cDNA for IL-6R(cid:11) (originally IL-6R) was Interleukin 6 (IL-6) is a pleiotropic cytokine that cloned by an expression-cloning strategy (Yamasaki regulates immune reaction, hematopoiesis, and dif- et al., 1988). Later, IL-6R(cid:11) turned out to be a ferentiation of the nervous system. The receptor for member of cytokine receptor family (type I cytokine IL-6 (IL-6R) consists of two chains, namely IL-6R(cid:11) receptorsuperfamily,hematopoietinreceptorfamily), and gp130. Both IL-6R(cid:11) and gp130 belong to the which includes receptors for many interleukins, type I cytokine receptor superfamily. IL-6R(cid:11) is the colony-stimulating factors, and hormones (e.g. pro- binding component specific to IL-6. In contrast, lactin, growth hormone, and leptin) (Bazan, 1990). gp130 transmits signals not only of IL-6 but also of Expression of IL-6R(cid:11) in cells constitutes the low- IL-6-related cytokines such as leukemia-inhibitory affinity binding sites, but IL-6R(cid:11) alone does not factor (LIF), ciliary neurotropic factor (CNTF), create either the high-affinity binding sites or signals oncostatin M (OSM), IL-11, cardiotropin 1 (CT-1), that elicit biological functions of IL-6, suggesting the and possibly neurotrophin-1/B cell-stimulating factor existenceofanothercomponentoftheIL-6Rcomplex 3 (NNT-1/BSF-3). The ligand’s binding to the (Taga et al., 1989) in a manner which is called receptor leads to homo- and heterodimerization of receptorconversion.ItwasshownthatinalltheIL-6- gp130, resulting in the activation of gp130-associated responsive cells, IL-6R(cid:11) associates with a transmem- JAKs (JAK1, JAK2, and TYK2) and subsequently brane glycoprotein, molecular weight 130kDa in the tyrosine phosphorylation of gp130. The tyrosine- human myeloma cell line U266, in response to IL-6 phosphorylated gp130 further transmits signals by (Taga et al., 1989). Thus it was named gp130. recruiting SH2 domain-containing signaling mole- Furthermore, a complex of IL-6 and soluble IL-6R(cid:11) cules such as the protein tyrosine phosphatase SHP-2 was shown to bind the extracellular domain of gp130 and STAT1 and STAT3 (signal transducers and acti- and transmit signals (Taga et al., 1989). Molecular vatorsoftranscription).Wedescribetherolesofthese cloning of gp130 further revealed that it also belongs signal transduction pathways in the biological to the type I cytokine receptor superfamily. responses of the IL-6 family cytokines. Expression of gp130 was reported to generate the high-affinity IL-6 binding sites, and render a certain cell line to receive the signal of IL-6 and soluble BACKGROUND IL-6R (Hibi et al., 1990). Thereafter it was found that gp130 transmits sig- Discovery nals not only of IL-6 but also of IL-6-related cyto- kines such as LIF, CNTF, OSM, IL-11, and CT-1, IL-6 is a pleiotropic cytokine that acts on various and possibly NNT-1/BSF-3 (Senaldi et al., 1999) cells.SignalsofIL-6aretransmittedintotheinsideof which share the biological function with IL-6. gp130 cellsthroughtheIL-6Rcomplexthatexistsonthecell forms a complex with (cid:11) subunits of IL-6R, IL-11R, surface.Initially,ligand-bindingassaysusingisotope- CNTFreceptor,andpossiblyCT-1receptor,andLIF labeled IL-6 revealed the existence of high-affinity receptor (cid:12) or OSM receptor (cid:12) to act as receptors for 1762 Masahiko Hibi and Toshio Hirano the IL-6-related cytokines (Hirano et al., 1997; Figure 1 Structure of the IL-6 receptor system. Heinrich et al., 1998). IL-6R(cid:11)andgp130belongtothecytokinereceptor typesuperfamily.Theextracellulardomainsofboth IL-6R(cid:11) and gp130 contain an immunoglobulin- Alternative names like (Ig) domain and a cytokine-binding module (CBM). In addition, gp130 has three repeats of IL-6 receptor (cid:11): B cell stimulatory factor-2, BSF-2 fibronectin type III (FNIII) domains in the extra- receptor, IL-6 receptor, CD126. cellulardomain.gp130hasregions(box1andbox2) gp130: IL-6 signal transducer, IL-6 receptor (cid:12) chain, conserved with the family and six tyrosines in its CD130. cytoplasmicdomain(numbersindicatelocationsof tyrosine in human gp130). Box 1 and box 2 are involvedintheassociationandactivationofJAKs Structure (JAK1,JAK2,andTYK2).Phosphorylationofthe Y759andthefourtyrosines(Y767,Y814,Y904,and TheIL-6receptorsystemiscomposedofIL-6R(cid:11)and Y915) in the C-terminal are involved in the phos- gp130 (Figure 1). A reconstitution experiment of the phorylation and activation of SHP-2 and STAT3. ThephosphorylationofY904andY915wasshown solubleIL-6RsystemsuggestedthattheIL-6receptor to be involved in the phosphorylation and activa- consistsoftwomoleculesofIL-6,IL-6R(cid:11),andgp130 tion of STAT1. (Paonessaetal.,1995).Alternatively,itwasproposed thattheIL-6receptorconsistsofonemoleculeofIL-6 andIL-6R(cid:11),andtwomoleculesofgp130(Grotzinger et al., 1997). The molecular weight of IL-6R(cid:11) is 80kDa (the molecular weight predicted from the amino acid sequence is 49.9kDa) (Yamasaki et al., 1988; Hirata et al., 1989; Taga et al., 1989). IL-6R(cid:11) is a type I transmembrane protein. The precursor of human IL- 6R(cid:11)contains468aminoacidsanditsputativemature formconsistsof449aminoacids.Itiscomposedofan extracellular region of 339 amino acids, a membrane- spanningregionof28aminoacids,andacytoplasmic region of 82 amino acids. It has five potential N- glycosylation sites. IL-6R(cid:11) belongs to the type I cytokine receptor superfamily. It has one cytokine- binding module (CBM) composed of two fibronectin typeIII-like(FNIII)domains,inwhichtheN-terminal domain contains a set of four conserved cysteine residues and the C-terminal one contains a WSXWS motif,conservedinthetypeIcytokinereceptorsuper- family. It was reported that the C-terminal FNIII domain in the CBM is sufficient for IL-6 binding but not for the association with gp130 (Ozbek et al., 1998). In addition to the CRM, IL-6R(cid:11) contains an immunoglobulin-like domain at the N-terminus. The structure and functions of the cytoplasmic domain of IL-6R(cid:11) remain to be clarified. The molecular weight of gp130 is 130–160kDa (Taga et al., 1989; Hibi et al., 1990), the variation in of 597 amino acids, a membrane-spanning region of the molecular weight probably being due to the gly- 22aminoacids,andacytoplasmicregionof277amino cosylation. Deglycosylated gp130 displays 100kDa, acids. gp130 is also a member of the type I cytokine whichisconsistentwiththepredictedmolecularweight receptor superfamily. It has a immunoglobulin-like from the amino acid sequence (101kDa) (Taga et al., domain at the N-terminus, which is followed by a 1989). gp130 is also a type I transmembrane protein. CBMcontainingtwoFNIIIdomains.gp130hasthree The precursor of human gp130 contains 918 amino additional FNIII domains following the CBM, and acids and its putative mature form consists of 896 thus contains five FNIII domains in the extracellular aminoacids.Itiscomposedofanextracellularregion region. IL-6 Receptor 1763 The three-dimensional structure of the CBM of Figure2 NucleotidesequencesforhumanIL-6receptor(cid:11) gp130 has been analyzed (Bravo et al., 1998). and gp130. Structurally, it is closely related to the CBMs of growth hormone receptor (de Vos et al., 1992) and Human IL-6 receptor a erythropoietin receptor. The cytoplasmic region of GGCGGTCCCCTGTTCTCCCCGCTCAGGTGCGGCGCTGTGGCAGGAAGCCA gp130 has several distinct domains. The membrane- CCGCCAGGGGACAAGAGGGGCGAGTCCACGCCGCGACACCGTCCTTCGGT proximal region of gp130 has a similar sequence to CCCCCTCGGTCGGCCGGTGCGCGGGGCTGTTGCGCCATCCGCTCCGGCTT those of all members of the type I cytokine receptor GGGGGAGCCAGCCGGCCACGCGCCCCGACAACGCGGTAGGCGAGGCCGAA superfamily. The conserved region was separated by TCGTAACCGCACCCTGGGACGGCCCAGAGACGCTCCAGCGCGAGTTCCTC AGCATTGGCGTGGGACCCTGCCGGGTCTCTGCGAGGTCGCGCTCAAGGAG a short nonconserved amino acid stretch and thus AAATGTTTTCCTGCGTTGCCAGGACCGTCCGCCGCTCTGAGTCATGTGCG these domains are named box 1 and box 2 domains TTTACAAAAGGACGCAACGGTCCTGGCAGGCGGCGAGACTCAGTACACGC (Murakami et al., 1991). The box 1 domain is a AGTGGGAAGTCGCACTGACACTGAGCCGGGCCAGAGGGAGAGGAGCCGAG proline-rich amino acid stretch and box 2 contains a TCACCCTTCAGCGTGACTGTGACTCGGCCCGGTCTCCCTCTCCTCGGCTC clusterofhydrophobicaminoacidsfollowedbyacidic CGCGGCGCGGGGCCGAGGGACTCGCAGTGTGTGTAGAGAGCCGGGCTCCT GCGCCGCGCCCCGGCTCCCTGAGCGTCACACACATCTCTCGGCCCGAGGA amino acid residues. The box 1 and box 2 regions GCGGATGGGGGCTGCCCCCGGGGCCTGAGCCCGCCTGCCCGCCCACCGCC constitute a docking site for Janus tyrosine kinases CGCCTACCCCCGACGGGGGCCCCGGACTCGGGCGGACGGGCGGGTGGCGG (JAK1,JAK2,andTYK2).gp130hasaregionwhich CCGCCCCGCCCCTGCCACCCCTGCCGCCCGGTTCCCATTAGCCTGTCCGC ishomologoustoG-CSFreceptorinthemiddleofthe GGCGGGGCGGGGACGGTGGGGACGGCGGGCCAAGGGTAATCGGACAGGCG cytoplasmic domain (amino acids 772–787) and this CTCTGCGGGACCATGGAGTGGTAGCCGAGGAGGAAGCATGCTGGCCGTCG region is termed box 3 (Fukunaga et al., 1991). In GAGACGCCCTGGTACCTCACCATCGGCTCCTCCTTCGTACGACCGGCAGC GCTGCGCGCTGCTGGCTGCCCTGCTGGCCGCGCCGGGAGCGGCGCTGGCC addition to box 1, box 2, and box 3, gp130 contains CGACGCGCGACGACCGACGGGACGACCGGCGCGGCCCTCGCCGCGACCGG six tyrosines, some of which have been shown to be CCAAGGCGCTGCCCTGCGCAGGAGGTGGCAAGAGGCGTGCTGACCAGTCT important for signal transduction. GGTTCCGCGACGGGACGCGTCCTCCACCGTTCTCCGCACGACTGGTCAGA GCCAGGAGACAGCGTGACTCTGACCTGCCCGGGGGTAGAGCCGGAAGACA CGGTCCTCTGTCGCACTGAGACTGGACGGGCCCCCATCTCGGCCTTCTGT Main activities and ATGCCACTGTTCACTGGGTGCTCAGGAAGCCGGCTGCAGGCTCCCACCCC TACGGTGACAAGTGACCCACGAGTCCTTCGGCCGACGTCCGAGGGTGGGG pathophysiological roles AGCAGATGGGCTGGCATGGGAAGGAGGCTGCTGCTGAGGTCGGTGCAGCT TCGTCTACCCGACCGTACCCTTCCTCCGACGACGACTCCAGCCACGTCGA CCACGACTCTGGAAACTATTCATGCTACCGGGCCGGCCGCCCAGCTGGGA IL-6R(cid:11) and gp130 transmit all the biological GGTGCTGAGACCTTTGATAAGTACGATGGCCCGGCCGGCGGGTCGACCCT functions of IL-6. gp130 is also required for the CTGTGCACTTGCTGGTGGATGTTCCCCCCGAGGAGCCCCAGCTCTCCTGC biological functions of the IL-6-related cytokines: GACACGTGAACGACCACCTACAAGGGGGGCTCCTCGGGGTCGAGAGGACG TTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGTGGGGTCCTCGGAG LIF, CNTF, IL-11, OSM, and CT-1. AAGGCCTTCTCGGGGGAGTCGTTACAACAAACACTCACCCCAGGAGCCTC CACCCCATCCCTGACGACAAAGGCTGTGCTCTTGGTGAGGAAGTTTCAGA GTGGGGTAGGGACTGCTGTTTCCGACACGAGAACCACTCCTTCAAAGTCT ACAGTCCGGCCGAAGACTTCCAGGAGCCGTGCCAGTATTCCCAGGAGTCC GENE TGTCAGGCCGGCTTCTGAAGGTCCTCGGCACGGTCATAAGGGTCCTCAGG CAGAAGTTCTCCTGCCAGTTAGCAGTCCCGGAGGGAGACAGCTCTTTCTA Accession numbers GTCTTCAAGAGGACGGTCAATCGTCAGGGCCTCCCTCTGTCGAGAAAGAT CATAGTGTCCATGTGCGTCGCCAGTAGTGTCGGGAGCAAGTTCAGCAAAA GTATCACAGGTACACGCAGCGGTCATCACAGCCCTCGTTCAAGTCGTTTT Human IL-6R(cid:11): X12830, M20566 CTCAAACCTTTCAGGGTTGTGGAATCTTGCAGCCTGATCCGCCTGCCAAC GAGTTTGGAAAGTCCCAACACCTTAGAACGTCGGACTAGGCGGACGGTTG Mouse IL-6R(cid:11): X51975, X51976 ATCACAGTCACTGCCGTGGCCAGAAACCCCCGCTGGCTCAGTGTCACCTG Human gp130: M57230 TAGTGTCAGTGACGGCACCGGTCTTTGGGGGCGACCGAGTCACAGTGGAC Mouse gp130: X62646, M83336 GCAAGACCCCCACTCCTGGAACTCATCTTTCTACAGACTACGGTTTGAGC Rat gp130: M92340 CGTTCTGGGGGTGAGGACCTTGAGTAGAAAGATGTCTGATGCCAAACTCG Chick gp130: AJ011688, GGA011688 TCAGATATCGGGCTGAACGGTCAAAGACATTCACAACATGGATGGTCAAG AGTCTATAGCCCGACTTGCCAGTTTCTGTAAGTGTTGTACCTACCAGTTC Genome: GACCTCCAGCATCACTGTGTCATCCACGACGCCTGGAGCGGCCTGAGGCA IL-6R(cid:11): chromosomal location not determined. CTGGAGGTCGTAGTGACACAGTAGGTGCTGCGGACCTCGCCGGACTCCGT Humangp130:chromosome5(apseudogeneexistsin CGTGGTGCAGCTTCGTGCCCAGGAGGAGTTCGGGCAAGGCGAGTGGAGCG chromosome 17). GCACCACGTCGAAGCACGGGTCCTCCTCAAGCCCGTTCCGCTCACCTCGC AGTGGAGCCCGGAGGCCATGGGCACGCCTTGGACAGAATCCAGGAGTCCT TCACCTCGGGCCTCCGGTACCCGTGCGGAACCTGTCTTAGGTCCTCAGGA Sequence CCAGCTGAGAACGAGGTGTCCACCCCCATGCAGGCACTTACTACTAATAA GGTCGACTCTTGCTCCACAGGTGGGGGTACGTCCGTGAATGATGATTATT AGACGATGATAATATTCTCTTCAGAGATTCTGCAAATGCGACAAGCCTCC See Figure 2. 1764 Masahiko Hibi and Toshio Hirano Figure 2 (Continued) Figure 2 (Continued) TCTGCTACTATTATAAGAGAAGTCTCTAAGACGTTTACGCTGTTCGGAGG Human gp130 CAGTGCAAGATTCTTCTTCAGTACCACTGCCCACATTCCTGGTTGCTGGA GTCACGTTCTAAGAAGAAGTCATGGTGACGGGTGTAAGGACCAACGACCT GAGCAGCCAAAAGGCCCGCGGAGTCGCGCTGGGCCGCCCCGGCGCAGCTG GGGAGCCTGGCCTTCGGAACGCTCCTCTGCATTGCCATTGTTCTGAGGTT CTCGTCGGTTTTCCGGGCGCCTCAGCGCGACCCGGCGGGGCCGCGTCGAC CCCTCGGACCGGAAGCCTTGCGAGGAGACGTAACGGTAACAAGACTCCAA AACCGGGGGCCGCGCCTGCCAGGCCGACGGGTCTGGCCCAGCCTGGCGCC CAAGAAGACGTGGAAGCTGCGGGCTCTGAAGGAAGGCAAGACAAGCATGC TTGGCCCCCGGCGCGGACGGTCCGGCTGCCCAGACCGGGTCGGACCGCGG GTTCTTCTGCACCTTCGACGCCCGAGACTTCCTTCCGTTCTGTTCGTACG AAGGGGTTCGTGCGCTGTGGAGACGCGGAGGGTCGAGGCGGCGCGGCCTG ATCCGCCGTACTCTTTGGGGCAGCTGGTCCCGGAGAGGCCTCGACCCACC TTCCCCAAGCACGCGACACCTCTGCGCCTCCCAGCTCCGCCGCGCCGGAC TAGGCGGCATGAGAAACCCCGTCGACCAGGGCCTCTCCGGAGCTGGGTGG AGTGAAACCCAATGGAAAAAGCATGACATTTAGAAGTAGAAGACTTAGCT CCAGTGCTTGTTCCTCTCATCTCCCCACCGGTGTCCCCCAGCAGCCTGGG GGTCACGAACAAGGAGAGTAGAGGGGTGGCCACAGGGGGTCGTCGGACCC TCACTTTGGGTTACCTTTTTCGTACTGTAAATCTTCATCTTCTGAATCGA GTCTGACAATACCTCGAGCCACAACCGACCAGATGCCAGGGACCCACGGA TCAAATCCCTACTCCTTCACTTACTAATTTTGTGATTTGGAAATATCCGC CAGACTGTTATGGAGCTCGGTGTTGGCTGGTCTACGGTCCCTGGGTGCCT AGTTTAGGGATGAGGAAGTGAATGATTAAAACACTAAACCTTTATAGGCG GCCCTTATGACATCAGCAATACAGACTACTTCTTCCCCAGATAGCTGGCT GCAAGATGTTGACGTTGCAGACTTGGGTAGTGCAAGCCTTGTTTATTTTC CGGGAATACTGTAGTCGTTATGTCTGATGAAGAAGGGGTCTATCGACCGA CGTTCTACAACTGCAACGTCTGAACCCATCACGTTCGGAACAAATAAAAG GGGTGGCACCAGCAGCCTGGACCCTGTGGATGACAAAACACAAACGGGCT CTCACCACTGAATCTACAGGTGAACTTCTAGATCCATGTGGTTATATCAG CCCACCGTGGTCGTCGGACCTGGGACACCTACTGTTTTGTGTTTGCCCGA GAGTGGTGACTTAGATGTCCACTTGAAGATCTAGGTACACCAATATAGTC CAGCAAAAGATGCTTCTCACTGCCATGCCAGCTTATCTCAGGGGTGTGCG TCCTGAATCTCCAGTTGTACAACTTCATTCTAATTTCACTGCAGTTTGTG GTCGTTTTCTACGAAGAGTGACGGTACGGTCGAATAGAGTCCCCACACGC GCCTTTGGCTTCACGGAAGAGCCTTGCGGAAGGTTCTACGCCAGGGGAAA AGGACTTAGAGGTCAACATGTTGAAGTAAGATTAAAGTGACGTCAAACAC CGGAAACCGAAGTGCCTTCTCGGAACGCCTTCCAAGATGCGGTCCCCTTT TGCTAAAGGAAAAATGTATGGATTATTTTCATGTAAATGCTAATTACATT ATCAGCCTGCTCCAGCTGTTCAGCTGGTTGAGGTTTCAAACCTCCCTTTC ACGATTTCCTTTTTACATACCTAATAAAAGTACATTTACGATTAATGTAA TAGTCGGACGAGGTCGACAAGTCGACCAACTCCAAAGTTTGGAGGGAAAG GTCTGGAAAACAAACCATTTTACTATTCCTAAGGAGCAATATACTATCAT CAAATGCCCAGCTTAAAGGGGTTAGAGTGAACTTGGGCCACTGTGAAGAG CAGACCTTTTGTTTGGTAAAATGATAAGGATTCCTCGTTATATGATAGTA GTTTACGGGTCGAATTTCCCCAATCTCACTTGAACCCGGTGACACTTCTC AAACAGAACAGCATCCAGTGTCACCTTTACAGATATAGCTTCATTAAATA AACCATATCAAGACTCTTTGGACACTCACACGGACACTCAAAAGCTGGGC TTTGTCTTGTCGTAGGTCACAGTGGAAATGTCTATATCGAAGTAATTTAT TTGGTATAGTTCTGAGAAACCTGTGAGTGTGCCTGTGAGTTTTCGACCCG TTCAGCTCACTTGCAACATTCTTACATTCGGACAGCTTGAACAGAATGTT AGGTTGGTGGGGGCCTCGGTGTGGAGAAGCGGCTGGCAGCCCACCCCTCA AAGTCGAGTGAACGTTGTAAGAATGTAAGCCTGTCGAACTTGTCTTACAA TCCAACCACCCCCGGAGCCACACCTCTTCGCCGACCGTCGGGTGGGGAGT ACACCTCTGCACAAGCTGCACCCTCAGGCAGGTGGGATGGATTTCCAGCC TATGGAATCACAATAATTTCAGGCTTGCCTCCAGAAAAACCTAAAAATTT TGTGGAGACGTGTTCGACGTGGGAGTCCGTCCACCCTACCTAAAGGTCGG ATACCTTAGTGTTATTAAAGTCCGAACGGAGGTCTTTTTGGATTTTTAAA AAAGCCTCCTCCAGCCGCCATGCTCCTGGCCCACTGCATCGTTTCATCTT GAGTTGCATTGTGAACGAGGGGAAGAAAATGAGGTGTGAGTGGGATGGTG TTTCGGAGGAGGTCGGCGGTACGAGGACCGGGTGACGTAGCAAAGTAGAA CTCAACGTAACACTTGCTCCCCTTCTTTTACTCCACACTCACCCTACCAC CCAACTCAAACTCTTAAAACCCAAGTGCCCTTAGCAAATTCTGTTTTTCT GAAGGGAAACACACTTGGAGACAAACTTCACTTTAAAATCTGAATGGGCA GGTTGAGTTTGAGAATTTTGGGTTCACGGGAATCGTTTAAGACAAAAAGA CTTCCCTTTGTGTGAACCTCTGTTTGAAGTGAAATTTTAGACTTACCCGT AGGCCTGGGGACGGCTTTTACTTAAACGCCAAGGCCTGGGGGAAGAAGCT ACACACAAGTTTGCTGATTGCAAAGCAAAACGTGACACCCCCACCTCATG TCCGGACCCCTGCCGAAAATGAATTTGCGGTTCCGGACCCCCTTCTTCGA TGTGTGTTCAAACGACTAACGTTTCGTTTTGCACTGTGGGGGTGGAGTAC CTCTCCTCCCTTTCTTCCCTACAGTTCAAAAACAGCTGAGGGTGAGTGGG GAGAGGAGGGAAAGAAGGGATGTCAAGTTTTTGTCGACTCCCACTCACCC CACTGTTGATTATTCTACTGTGTATTTTGTCAACATTGAAGTCTGGGTAG TGAATAATACAGTATGTCAGGGCCTGGTCGTTTTCAACAGAATTATAATT GTGACAACTAATAAGATGACACATAAAACAGTTGTAACTTCAGACCCATC ACTTATTATGTCATACAGTCCCGGACCAGCAAAAGTTGTCTTAATATTAA AAGCAGAGAATGCCCTTGGGAAGGTTACATCAGATCATATCAATTTTGAT AGTTCCTCATTAGCAGTTTTGCCTAAATGTGAATGATGATCCTAGGCATT TTCGTCTCTTACGGGAACCCTTCCAATGTAGTCTAGTATAGTTAAAACTA TCAAGGAGTAATCGTCAAAACGGATTTACACTTACTACTAGGATCCGTAA CCTGTATATAAAGTGAAGCCCAATCCGCCACATAATTTATCAGTGATCAA TGCTGAATACAGAGGCAACTGCATTGGCTTTGGGTTGCAGGACCTCAGGT GGACATATATTTCACTTCGGGTTAGGCGGTGTATTAAATAGTCACTAGTT ACGACTTATGTCTCCGTTGACGTAACCGAAACCCAACGTCCTGGAGTCCA CTCAGAGGAACTGTCTAGTATCTTAAAATTGACATGGACCAACCCAAGTA GAGAAGCAGAGGAAGGAGAGGAGAGGGGCACAGGGTCTCTACCATCCCCT GAGTCTCCTTGACAGATCATAGAATTTTAACTGTACCTGGTTGGGTTCAT CTCTTCGTCTCCTTCCTCTCCTCTCCCCGTGTCCCAGAGATGGTAGGGGA TTAAGAGTGTTATAATACTAAAATATAACATTCAATATAGGACCAAAGAT GTAGAGTGGGAGCTGAGTGGGGGATCACAGCCTCTGAAAACCAATGTTCT CATCTCACCCTCGACTCACCCCCTAGTGTCGGAGACTTTTGGTTACAAGA AATTCTCACAATATTATGATTTTATATTGTAAGTTATATCCTGGTTTCTA CTCTTCTCCACCTCCCACAAAGGAGAGCTAGCAGCAGGGAGGGCTTCTGC GCCTCAACTTGGAGCCAGATTCCTCCTGAAGACACAGCATCCACCCGATC GAGAAGAGGTGGAGGGTGTTTCCTCTCGATCGTCGTCCCTCCCGAAGACG CGGAGTTGAACCTCGGTCTAAGGAGGACTTCTGTGTCGTAGGTGGGCTAG CATTTCTGAGATCAAAACGGTTTTACTGCAGCTTTGTTTGTTGTCAGCTG TTCATTCACTGTCCAAGACCTTAAACCTTTTACAGAATATGTGTTTAGGA GTAAAGACTCTAGTTTTGCCAAAATGACGTCGAAACAAACAACAGTCGAC AAGTAAGTGACAGGTTCTGGAATTTGGAAAATGTCTTATACACAAATCCT AACCTGGGTAACTAGGGAAGATAATATTAAGGAAGACAATGTGAAAAGAA TTCGCTGTATGAAGGAAGATGGTAAGGGATACTGGAGTGACTGGAGTGAA TTGGACCCATTGATCCCTTCTATTATAATTCCTTCTGTTACACTTTTCTT AAGCGACATACTTCCTTCTACCATTCCCTATGACCTCACTGACCTCACTT AAATGAGCCTGGCAAGAATGCGTTTAAACTTGGTTTTTAAAAAACTGCTG GAAGCAAGTGGGATCACCTATGAAGATAGACCATCTAAAGCACCAAGTTT TTTACTCGGACCGTTCTTACGCAAATTTGAACCAAAAATTTTTTGACGAC ACTGTTTTCTCTTGAGAGGGTGGAATATCCAATATTCGCTGTGTCAGCAT CTTCGTTCACCCTAGTGGATACTTCTATCTGGTAGATTTCGTGGTTCAAA TGACAAAAGAGAACTCTCCCACCTTATAGGTTATAAGCGACACAGTCGTA CTGGTATAAAATAGATCCATCCCATACTCAAGGCTACAGAACTGTACAAC AGAAGTAACTTACTTAGGTGTGGGGGAAGCACCATAACTTTGTTTAGCCC GACCATATTTTATCTAGGTAGGGTATGAGTTCCGATGTCTTGACATGTTG TCTTCATTGAATGAATCCACACCCCCTTCGTGGTATTGAAACAAATCGGG TCGTGTGGAAGACATTGCCTCCTTTTGAAGCCAATGGAAAAATCTTGGAT AAAACCAAGTCAAGTGAAAAAGGAGGAAGAGAAAAAATATTTTCCTGCCA AGCACACCTTCTGTAACGGAGGAAAACTTCGGTTACCTTTTTAGAACCTA TTTTGGTTCAGTTCACTTTTTCCTCCTTCTCTTTTTTATAAAAGGACGGT TATGAAGTGACTCTCACAAGATGGAAATCACATTTACAAAATTACACAGT GGCATGGAGGCCCACGCACTTCGGGAGGTCGAGGCAGGAGGATCACTTGA ATACTTCACTGAGAGTGTTCTACCTTTAGTGTAAATGTTTTAATGTGTCA CCGTACCTCCGGGTGCGTGAAGCCCTCCAGCTCCGTCCTCCTAGTGAACT TAATGCCACAAAACTGACAGTAAATCTCACAAATGATCGCTATCTAGCAA GTCCAGAAGTTTGAGATCAGCCTGGGCAATGTGATAAAACCCCATCTCTA ATTACGGTGTTTTGACTGTCATTTAGAGTGTTTACTAGCGATAGATCGTT CAGGTCTTCAAACTCTAGTCGGACCCGTTACACTATTTTGGGGTAGAGAT CAAAAAGCATAAAAATTAGCCAAGTGTGGTAGAGTGTGCCTGAAGTCCCA CCCTAACAGTAAGAAATCTTGTTGGCAAATCAGATGCAGCTGTTTTAACT GTTTTTCGTATTTTTAATCGGTTCACACCATCTCACACGGACTTCAGGGT GGGATTGTCATTCTTTAGAACAACCGTTTAGTCTACGTCGACAAAATTGA GATACTTGGGGGGCTGAGGTGGGAGGATCTCTTGAGCCTGGGAGGTCAAG ATCCCTGCCTGTGACTTTCAAGCTACTCACCCTGTAATGGATCTTAAAGC CTATGAACCCCCCGACTCCACCCTCCTAGAGAACTCGGACCCTCCAGTTC TAGGGACGGACACTGAAAGTTCGATGAGTGGGACATTACCTAGAATTTCG GCTGCAGTGAGCCGAGATTGCACCACTGCACTCCAGCCTGGGGTGACAGA ATTCCCCAAAGATAACATGCTTTGGGTGGAATGGACTACTCCAAGGGAAT CGACGTCACTCGGCTCTAACGTGGTGACGTGAGGTCGGACCCCACTGTCT GCAAGTGAGACCCTGTCTCCGTTCACTCTGGGACAGAG IL-6 Receptor 1765 Figure 2 (Continued) PROTEIN TAAGGGGTTTCTATTGTACGAAACCCACCTTACCTGATGAGGTTCCCTTA Accession numbers CTGTAAAGAAATATATACTTGAGTGGTGTGTGTTATCAGATAAAGCACCC GACATTTCTTTATATATGAACTCACCACACACAATAGTCTATTTCGTGGG TGTATCACAGACTGGCAACAAGAAGATGGTACCGTGCATCGCACCTATTT Human IL-6R(cid:11): 49726, 33846 ACATAGTGTCTGACCGTTGTTCTTCTACCATGGCACGTAGCGTGGATAAA Mouse IL-6R(cid:11): 52693 AAGAGGGAACTTAGCAGAGAGCAAATGCTATTTGATAACAGTTACTCCAG Rat IL-6R(cid:11): 111882 TTCTCCCTTGAATCGTCTCTCGTTTACGATAAACTATTGTCAATGAGGTC Human gp130: 729833, 106982, 186354 TATATGCTGATGGACCAGGAAGCCCTGAATCCATAAAGGCATACCTTAAA ATATACGACTACCTGGTCCTTCGGGACTTAGGTATTTCCGTATGGAATTT Mouse gp130: 729834, 2137360, 840817, 193592 CAAGCTCCACCTTCCAAAGGACCTACTGTTCGGACAAAAAAAGTAGGGAA Rat gp130: 729835, 348455 GTTCGAGGTGGAAGGTTTCCTGGATGACAAGCCTGTTTTTTTCATCCCTT AAACGAAGCTGTCTTAGAGTGGGACCAACTTCCTGTTGATGTTCAGAATG TTTGCTTCGACAGAATCTCACCCTGGTTGAAGGACAACTACAAGTCTTAC Sequence GATTTATCAGAAATTATACTATATTTTATAGAACCATCATTGGAAATGAA CTAAATAGTCTTTAATATGATATAAAATATCTTGGTAGTAACCTTTACTT ACTGCTGTGAATGTGGATTCTTCCCACACAGAATATACATTGTCCTCTTT See Figure 3. TGACGACACTTACACCTAAGAAGGGTGTGTCTTATATGTAACAGGAGAAA GACTAGTGACACATTGTACATGGTACGAATGGCAGCATACACAGATGAAG Description of protein CTGATCACTGTGTAACATGTACCATGCTTACCGTCGTATGTGTCTACTTC GTGGGAAGGATGGTCCAGAATTCACTTTTACTACCCCAAAGTTTGCTCAA CACCCTTCCTACCAGGTCTTAAGTGAAAATGATGGGGTTTCAAACGAGTT See Table 1. GGAGAAATTGAAGCCATAGTCGTGCCTGTTTGCTTAGCATTCCTATTGAC CCTCTTTAACTTCGGTATCAGCACGGACAAACGAATCGTAAGGATAACTG AACTCTTCTGGGAGTGCTGTTCTGCTTTAATAAGCGAGACCTAATTAAAA Relevant homologies and species TTGAGAAGACCCTCACGACAAGACGAAATTATTCGCTCTGGATTAATTTT AACACATCTGGCCTAATGTTCCAGATCCTTCAAAGAGTCATATTGCCCAG differences TTGTGTAGACCGGATTACAAGGTCTAGGAAGTTTCTCAGTATAACGGGTC TGGTCACCTCACACTCCTCCAAGGCACAATTTTAATTCAAAAGATCAAAT ACCAGTGGAGTGTGAGGAGGTTCCGTGTTAAAATTAAGTTTTCTAGTTTA TheoverallhomologybetweenhumanandmouseIL- GTATTCAGATGGCAATTTCACTGATGTAAGTGTTGTGGAAATAGAAGCAA 6R(cid:11) is 69% and 54% at the DNA and protein level, CATAAGTCTACCGTTAAAGTGACTACATTCACAACACCTTTATCTTCGTT ATGACAAAAAGCCTTTTCCAGAAGATCTGAAATCATTGGACCTGTTCAAA respectively(Yamasakietal.,1988;Sugitaetal.,1990). TACTGTTTTTCGGAAAAGGTCTTCTAGACTTTAGTAACCTGGACAAGTTT The overall homology between human and mouse AAGGAAAAAATTAATACTGAAGGACACAGCAGTGGTATTGGGGGGTCTTC gp130 is 76.6% and 76.8% at the DNA and protein TTCCTTTTTTAATTATGACTTCCTGTGTCGTCACCATAACCCCCCAGAAG level,respectively(Hibietal.,1990;Saitoetal.,1992). ATGCATGTCATCTTCTAGGCCAAGCATTTCTAGCAGTGATGAAAATGAAT TACGTACAGTAGAAGATCCGGTTCGTAAAGATCGTCACTACTTTTACTTA The homologies in the extracellular and cytoplasmic CTTCACAAAACACTTCGAGCACTGTCCAGTATTCTACCGTGGTACACAGT regions between human and mouse gp130 are 72% GAAGTGTTTTGTGAAGCTCGTGACAGGTCATAAGATGGCACCATGTGTCA and 85%, respectively. The transmembrane domains GGCTACAGACACCAAGTTCCGTCAGTCCAAGTCTTCTCAAGATCCGAGTC ofhumanandmousegp130areidentical.Thehomol- CCGATGTCTGTGGTTCAAGGCAGTCAGGTTCAGAAGAGTTCTAGGCTCAG TACCCAGCCCTTGTTAGATTCAGAGGAGCGGCCAGAAGATCTACAATTAG ogy between human and chick gp130 is 74% at the ATGGGTCGGGAACAATCTAAGTCTCCTCGCCGGTCTTCTAGATGTTAATC amino acid level (Geissen et al., 1998). TAGATCATGTAGATGGCGGTGATGGTATTTTGCCCAGGCAACAGTACTTC ATCTAGTACATCTACCGCCACTACCATAAAACGGGTCCGTTGTCATGAAG AAACAGAACTGCAGTCAGCATGAATCCAGTCCAGATATTTCACATTTTGA Affinity for ligand(s) TTTGTCTTGACGTCAGTCGTACTTAGGTCAGGTCTATAAAGTGTAAAACT AAGGTCAAAGCAAGTTTCATCAGTCAATGAGGAAGATTTTGTTAGACTTA TTCCAGTTTCGTTCAAAGTAGTCAGTTACTCCTTCTAAAACAATCTGAAT Low-affinity binding sites for IL-6: K (cid:136)1–6nM. d AACAGCAGATTTCAGATCATATTTCACAATCCTGTGGATCTGGGCAAATG High-affinity binding sites for IL-6: K (cid:136)40–70pM. TTGTCGTCTAAAGTCTAGTATAAAGTGTTAGGACACCTAGACCCGTTTAC d The low-affinity binding site for IL-6 is composed AAAATGTTTCAGGAAGTTTCTGCAGCAGATGCTTTTGGTCCAGGTACTGA of IL-6R(cid:11) alone (Hibi et al., 1990). The high-affinity TTTTACAAAGTCCTTCAAAGACGTCGTCTACGAAAACCAGGTCCATGACT GGGACAAGTAGAAAGATTTGAAACAGTTGGCATGGAGGCTGCGACTGATG bindingsiteforIL-6iscomposedofbothIL-6R(cid:11)and CCCTGTTCATCTTTCTAAACTTTGTCAACCGTACCTCCGACGCTGACTAC gp130,althoughgp130doesnothavetheabilitytobind AAGGCATGCCTAAAAGTTACTTACCACAGACTGTACGGCAAGGCGGCTAC IL-6 by itself (Taga et al., 1989; Hibi et al., 1990). TTCCGTACGGATTTTCAATGAATGGTGTCTGACATGCCGTTCCGCCGATG ATGCCTCAGTGAAGGACTAGTAGTTCCTGCTACAACTTCAGCAGTACCTA TACGGAGTCACTTCCTGATCATCAAGGACGATGTTGAAGTCGTCATGGAT Cell types and tissues expressing TAAAGTAAAGCTAAAATGATTTTATCTGTGAATTC ATTTCATTTCGATTTTACTAAAATAGACACTTAAG the receptor IL-6R(cid:11) is expressed in all the cell lines that respond to IL-6. These include T cell lymphoma and B cell 1766 Masahiko Hibi and Toshio Hirano Figure3 AminoacidsequencesforhumanandmouseIL-6receptor (cid:11) and human and mouse gp130. Signal peptides are underlined; transmembrane regions are in bold. Human IL-6 receptor a MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVT LTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRP AGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAED FQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDP PANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHH CVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQAL TTNKDDDNILFRDSANATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTW KLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDA RDPRSPYDISNTDYFFPR Mouse IL-6 receptor a MLTVGCTLLVALLAAPAVALVLGSCRALEVANGTVTSLPGATVT LICPGKEAAGNVTIHWVYSGSQNREWTTTGNTLVLRDVQLSDTGDYLCSLNDHLVGTV PLLVDVPPEEPKLSCFRKNPLVNAICEWRPSSTPSPTTKAVLFAKKINTTNGKSDFQV PCQYSQQLKSFSCQVEILEGDKVYHIVSLCVANSVGSKSSHNEAFHSLKMVQPDPPAN LVVSAIPGRPRWLKVSWQHPETWDPSYYLLQFQLRYRPVWSKEFTVLLLPVAQYQCVI HDALRGVKHVVQVRGKEELDLGQWSEWSPEVTGTPWIAEPRTTPAGILWNPTQVSVED SANHEDQYESSTEATSVLAPVQESSSMSLPTFLVAGGSLAFGLLLCVFIILRLKQKWK SEAEKESKTTSPPPPPYSLGPLKPTFLLVPLLTPHSSGSDNTVNHSCLGVRDAQSPYD NSNRDYLFPR Human gp130 MLTLQTWVVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNF TAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLT CNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTL KSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVY KVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPED TASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWY KIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLT NDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESV KKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPE SIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNET AVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVP VCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPS ISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHI SQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGG YMPQ Mouse gp130 MSAPRIWLAQALLFFLTTESIGQLLEPCGYIYPEFPVVQRGSNF TAICVLKEACLQHYYVNASYIVWKTNHAAVPREQVTVINRTTSSVTFTDVVLPSVQLT CNILSFGQIEQNVYGVTMLSGFPPDKPTNLTCIVNEGKNMLCQWDPGRETYLETNYTL KSEWATEKFPDCQSKHGTSCMVSYMPTYYVNIEVWVEAENALGKVSSESINFDPVDKV KPTPPYNLSVTNSEELSSILKLSWVSSGLGGLLDLKSDIQYRTKDASTWIQVPLEDTM SPRTSFTVQDLKPFTEYVFRIRSIKDSGKGYWSDWSEEASGTTYEDRPSRPPSFWYKT NPSHGQEYRSVRLIWKALPLSEANGKILDYEVILTQSKSVSQTYTVTGTELTVNLTND RYVASLAARNKVGKSAAAVLTIPSPHVTAAYSVVNLKAFPKDNLLWVEWTPPPKPVSK YILEWCVLSENAPCVEDWQQEDATVNRTHLRGRLLESKCYQITVTPVFATGPGGSESL KAYLKQAAPARGPTVRTKKVGKNEAVLAWDQIPVDDQNGFIRNYSISYRTSVGKEMVV HVDSSHTEYTLSSLSSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVC LAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYS DGNFTDVSVVEIEANNKKPCPDDLKSVDLFKKEKVSTEGHSSGIGGSSCMSSSRPSIS SNEENESAQSTASTVEYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD SVDGGDEILPRQPYFKQNCSQPEACPEISHFERSNQVLSGNEEDFVRLKQQQVSDHIS QPYGSEQRRLFQEGSTADALGTGADGQMERFESVGMETTIDEEIPKSYLPQTVRQGGY MPQ IL-6 Receptor 1767 Table 1 Composition of human IL-6 receptor (cid:11) and IL-6 and soluble IL-6R(cid:11) can act on cells expressing gp130 only gp130 and such a mechanism, which is called receptor conversion, may generate functional diver- Human Human gp130 sity of cytokines in vivo (Hirano et al., 1997). In fact, IL-6R(cid:11) double transgenic mice expressing human IL-6 and soluble IL-6R(cid:11) showed myocardial hypertrophy Composition (amino acids) (Hirota et al., 1995) and extramedullary expansion Precursor 468 918 of hematopoietic progenitors (Peters et al., 1997; Mature form 449 896 Schirmacher et al., 1998). Furthermore, the fusion Extracellular domain 339 597 proteinconsistingofIL-6andtheextracellulardomain of IL-6R(cid:11) linked together by a flexible peptide chain Transmembrane domain 28 22 was shown to stimulate hematopoietic progenitor Cytoplasmic domain 82 277 cells effectively (Fischer et al., 1997). However, it is Molecular weight (kDa) not yet clear whether soluble IL-6R(cid:11) acts positively Predicted 49.9 101 on gp130 signaling under physiological conditions. Soluble gp130 was also found in human serum at a Observed 80 130–160 concentration of 300ng/mL (Narazaki et al., 1993). Soluble gp130 was reported to be generated by alternative splicing (Diamant et al., 1997). lymphoma,multiplemyeloma(plasmacytoma),hepa- toma, hepatocellular carcinoma, and glioma cell lines (Taga et al., 1987; Sugita et al., 1990). IL-6R(cid:11) is SIGNAL TRANSDUCTION expressed in spleen, liver, lung, and thymus strongly in mice (Sugita et al., 1990). The size of mRNA for Associated or intrinsic kinases humanandmouseIL-6R(cid:11)isabout5.5kb(Sugitaetal., 1990).InamouseplasmacytomalineP3U1,thecyto- Januskinases,JAK1,JAK2,andTYK2constitutively plasmic domain of IL-6R(cid:11) was replaced by a part of associatewithgp130(Luttickenetal.,1994;Narazaki LTR intracisternal A particle gene and the mRNA et al., 1994;Stahlet al.,1994).Thestructure ofJanus size of the aberrant receptor is 1.8kb (Sugita et al., kinases is shown in Figure 4. Janus kinases contain a 1990). tyrosine kinase domain in their C-termini. Adjacent gp130 is ubiquitously expressed (Hibi et al., 1990; tothekinasedomain,theyhaveakinase-likedomain, Saito et al., 1992). Two mRNAs for gp130 are which is similar to the authentic kinase domain but detected by northern blotting. The size of gp130 lacksseveralaminoacidsconservedamongthekinase mRNAsare 7kband 10kb.Therelative ratioof7kb domains (Ihle, 1995). Upon stimulation of IL-6- and10kbgp130mRNAvariesamongtissues:inheart, related cytokines, these Janus kinases are tyrosine spleen,andlung;expressionof10kbmRNAwasmuch phosphorylated and become activated, resulting in lower than 7kb mRNA (Saito et al., 1992). tyrosine phosphorylation of the cytoplasmic domain of gp130. The box 1/box 2 domain in the membrane- proximal regions of the cytoplasmic domain is Release of soluble receptors involved in the interaction between gp130 and the JAKs. Mutations of the box 1 region reduces the Both IL-6R(cid:11) and gp130 have soluble forms: 30– interaction between gp130 and JAKs (Tanner et al., 70ng/mLofsolubleIL-6R(cid:11)isfoundinhumanserum 1995), and abrogates ligand-dependent activation of (Honda et al., 1992; Muller-Newen et al., 1996). JAK1 and JAK2 (Narazaki et al., 1994) and SolubleIL-6R(cid:11)wasreportedtobeelevatedinserum downstream signaling (Murakami et al., 1991; Lai of multiple myeloma (Gaillard et al., 1993), juvenile et al., 1995a). Truncations of the box 2 region also chronicarthritis(Keuletal.,1998),HIV(Hondaetal., reduced the interaction between gp130 and JAK2, 1992), and Graves’ disease (Salvi et al., 1996). and abrogated the biological functions mediated by Although soluble IL-6R(cid:11) was generated by shedding gp130 (Murakami et al., 1991; Lai et al., 1995a). in vitro (Mullberg et al., 1993), it is possible that it is However, JAKs were reported to interact with gp130 generated by alternative splicing (Lust et al., 1992; lacking the box 2region when theyare overexpressed Muller-Newen et al., 1996). It was reported that a (Lai et al., 1995a; Tanner et al., 1995). These indicate complex of soluble IL-6R(cid:11) and IL-6 has agonistic thatthebox1regionisrequiredandsufficientforthe effects on gp130 (Taga et al., 1989). The complex of JAK binding to some extent, and the box 2 region 1768 Masahiko Hibi and Toshio Hirano Figure 4 Structure of JAK and STAT. A JAK contains seven domains (JAK homology: JH1–7) conserved among JAKs. The JH1 and JH2 do- mains correspond to the tyrosine kinase and kinase-like (pseudokinase) domains,respectively.ASTATcontainsanSH2domain,anSH3-likedomain (functionalsignificanceunknown).Y(tyrosine)indicatesaphosphoacceptor site for JAKs. S indicates a serine residue, the phosphorylation of which is involved in the activation of STATs. may support the interaction or should be necessary gp130 (Matsuda et al., 1995a). It was shown that for the activation of the JAKs. Although JAK1, erbB2interactswithgp130andtheactivityoferbB2is JAK2, and TYK2 interact with and are activated by necessaryforERK/MAPkinaseactivationinprostate gp130, it is not clear which JAKs are involved in carcinoma cells (Qiu et al., 1998). The biological downstreamsignalingandbiologicalfunctionsandto significance of these tyrosine kinases remains to be what extent. elucidated. It was demonstrated that tyrosine phosphorylation of gp130 and STAT3 induced by soluble IL-6R(cid:11) and IL-6 was significantly reduced in a JAK1-deficient Cytoplasmic signaling cascades fibrosarcomacelllinebutnotinaJAK2-deficientcell line(Guschinetal.,1995).Thisisalsoconsistentwith data from knockout experiments. Several types of The current model of gp130-mediated signal trans- cells derived from JAK1-deficient mice did not duction is illustrated in Figure 5 (also see review by respond to the IL-6-related cytokines (Rodig et al., Hirano et al., 1997). Once the ligand is bound to the 1998), but JAK2-deficient fibroblasts and embryonic receptor complex, gp130 becomes a homodimer, and stem (ES) cells did respond to IL-6 and LIF, gp130-associated JAKs come close and transphos- respectively (Neubauer et al., 1998; Parganas et al., phorylate each other. It is believed that transphos- 1998). Furthermore, the embryonic lethal phenotype phorylation of JAKs activates its kinase activity. In of JAK1-deficient mice resembled that of gp130- the case of JAK2 and Tyk2, phosphorylation of the deficient mice (Yoshida et al., 1996; Rodig et al., twotyrosinesintheactivationloopofJAKswasshown 1998). Further analysis will be required to reveal to be required for tyrosine kinase activity (Gauzzi which JAK is involved in individual biological et al., 1996; Feng et al., 1997). The gp130-associated responses elicited by gp130. JAKs, once activated by the transphosphorylation, InadditiontoJanuskinase,severaltyrosinekinases phosphorylatetyrosinesinthecytoplasmicdomainof wereshowntointeractwithgp130directlyorthrough gp130.gp130containssixtyrosinesinthecytoplasmic the JAKs in certain cells. Hck, a Src-family tyrosine region (Y683, Y759, Y767, Y814, Y905, and Y915 in kinase,wasreportedtoassociatewithgp130,andwas humangp130;the numbersareindicatedaminoacids activated in response to LIF in ES cells (Ernst et al., from the translational initiation site). It is not known 1994).TecandBtk,whicharedistantmembersofthe which tyrosines are preferentially phosphorylated on Src-family tyrosine kinases and contain a pleckstrin stimulation. However, phosphorylated tyrosines on homology (PH) domain, were reported to associate gp130 are known to recruit signal transducing with gp130 through the JAKs and tyrosine phos- molecules such as SHP-2 and STATs (described phorylated upon the stimulation of gp130 (Matsuda below), resulting in the tyrosine phosphorylation of etal.,1995b;Takahashi-Tezukaetal.,1997).Feswas these molecules by the gp130-associated JAKs. also reported to interact with and was activated by Experiments of truncation and point mutations in IL-6 Receptor 1769 Figure 5 Model of gp130-mediated signal transduction. gp130 revealed the roles of individual tyrosine for Figure 6 Structure of SHP-2, Gab1, and Gab2. SHP-2 downstreamsignaling.Y683isnotsignificantlyphos- containstwoSH2andaphosphatasedomainsinitsN-and phorylated since a truncated mutant gp130, which C-terminal, respectively. Y (tyrosine) indicates possible possesses the membrane-proximal 68 amino acid phosphorylation sites. Both Gab1 and Gab2 have a pleckstrin homology (PH) domain, a c-Met-binding do- containing only Y683, could not be tyrosyl phos- main(MBD),andtyrosine-basedmotifsforSHP-2andp85 phorylatedinspiteofthetyrosinephosphorylationof PI-3 kinase. The region around the MBD contains the JAKs(Fukadaetal.,1996).PhosphorylationofY759 proline-rich PXXP motifs, which are known to bind the was shown to be required for tyrosine phosphoryla- SH3 domains. The white bars indicate predicted tyrosine- tion of SHP-2, a protein tyrosine phosphatase (Stahl phosphorylated sites, the surrounding sequences of which et al., 1995; Fukada et al., 1996; Yamanaka et al., fit with the consensus-binding sequences for Grb2, Crk, 1996). The four tyrosines (Y767, Y814, Y905, and and PLC(cid:13). Y915)in the C-terminushave a glutamineat position +3 of tyrosines (YXXQ) and phosphorylation of any one of these tyrosines has been shown to be required for tyrosine phosphorylation and activation of STAT3 (Stahl et al., 1995; Fukada et al., 1996; Gerhartz et al., 1996; Yamanaka et al., 1996). The two tyrosines in the carboxy-end (Y905 and Y915) among these four tyrosines have a proline at position +2 of tyrosines (YXPQ) and their phos- phorylation was further reported to be necessary for tyrosine phosphorylation of STAT1 (Gerhartz et al., 1996). In addition to SHP-2, STATs, and tyrosine kinases,thereareseveralproteinsthatarereportedto be tyrosyl phosphorylated on stimulation of gp130. TheseincludeShc(Boultonetal.,1994;Kumaretal., 1994),IRS-1(Burfootetal.,1997),Gab1(Takahashi- Tezuka et al., 1998), and Gab2 (Nishida et al., 1999). Tyrosine phosphorylation of STAT5, Gab1, and SHP-2 is a protein tyrosine phosphatase bearing Gab2 does not require tyrosine phosphorylation two SH2 domains in the N-terminal region and a of gp130 (Lai et al., 1995b; Fujitani et al., 1997; phosphatase domain in the C-terminal region Takahashi-Tezuka et al., 1998; Nishida et al., (Figure 6) (Adachi et al., 1996). In comparison to 1999). SHP-1,whichisanegativeregulatorforsignalingofa 1770 Masahiko Hibi and Toshio Hirano cytokinereceptor(Klingmulleretal.,1995)andBcell The mutation of Y759 disrupted the interaction antigen receptor (Blery et al., 1998; Maeda et al., between SHP-2 and Gab1, and Gab1 and PI-3 1998),SHP-2 is apositive regulatorof signaling from kinases, suggesting that Gab1-mediating signals are receptor tyrosine kinases and cytokine receptors dependent on Y759 of gp130. Gab1-dependent ERK (Tonks and Neel, 1996; Neel and Tonks, 1997). activation was shown to be inhibited by a dominant SHP-2 is tyrosyl phosphorylated on stimulation of negative p85, wortmannin, an inhibitor for PI-3 gp130 (Stahl et al., 1995; Fukada et al., 1996).SHP-2 kinases, or a dominant negative Ras (Takahashi- contains three to four YXNX (depending on splicing Tezuka et al., 1998). These results suggest that Gab variants) motifs, which is the consensus sequence for family proteins mediate signals from SHP-2 to PI-3 Grb2 binding, in its C-terminal region (Feng et al., kinases to activate ERK/MAP kinases. Further 1993; Vogel et al., 1993). Upon stimulation, tyrosine analysis will be required to clarify this point. phosphorylated SHP-2 was reported to interact with A negative role of SHP-2 in gp130 signaling has Grb2 (Bennett et al., 1994; Li et al., 1994; Fukada alsobeenreported.ThemutationofY759wasshown et al., 1996). Since Grb2 constitutively interacts with to prolong the activation of a STAT3-mediated Sos, the GDP–GTP exchange factor for Ras, SHP-2 pathway (Kim et al., 1998). Since gp130 and JAK1 may act as an adapter molecule mediating gp130 butnotSTAT3couldbesubstratesforSHP-2invitro signaling to the Ras pathway. The mutation of Y759 (Nishida et al., 1999), SHP-2 may distinguish gp130- to a phenylalanine abrogated tyrosine phosphoryla- mediated signals as a feedback mechanism. tion of SHP-2, the interaction between SHP-2 and STATsaretranscriptionfactorscontaininganSH2 Grb2, and activation of an ERK/MAP kinase, a domain(Figure4)(Darnelletal.,1994;IhleandKerr, major target of the Ras pathway, suggest a role for 1995). Upon stimulation, STAT1 and STAT3 are SHP-2 in the Ras pathway (Fukada et al., 1996). In recruitedongp130andtyrosylphosphorylatedbythe addition to the possible adapter function of SHP-2, gp130-associated JAKs (Akira et al., 1994; Zhong the catalytic activity of SHP-2 was shown to be et al., 1994; Guschin et al., 1995). Tyrosine 701 of necessary for transmitting signals to the ERK/MAP STAT1 and tyrosine 705 of STAT3 are phospho- kinasesinthecaseofreceptortyrosinekinasessuchas acceptor sites by JAKs. Phosphorylated STAT1 and receptorsforinsulin,EGF, andFGF(Noguchiet al., STAT3 form homodimers (STAT1/STAT1, STAT3/ 1994;Tangetal.,1995;Bennettetal.,1996).Ingp130 STAT3) or a heterodimer (STAT1/STAT3) through signaling, SHP-2 wasshown to interact with Gab1 or theinteractionbetweenthe phosphotyrosinesandthe Gab2 upon stimulation (Takahashi-Tezuka et al., SH2 domains. It was also reported that STAT5 1998; Nishida et al., 1999). Tyrosyl phosphorylated (STAT5a and STAT5b) is tyrosine phosphorylated Gab1 and Gab2 were shown to be dephosphorylated independently of tyrosine phosphorylation of gp130 by SHP-2 in vitro (Nishida et al., 1999). Gab1 and (Lai et al., 1995a; Fujitani et al., 1997). Tyrosine Gab2areadapterproteinswhichdisplayhomologyto phosphorylationofSTAT5byJAKswasshowntobe Drosophila Dos (daughter of sevenless) (Figure 6) mediated in part by a direct interaction between (Herbst et al., 1996; Raabe et al., 1996), a substrate STAT5 and the JH2 domain of JAKs (Fujitani et al., for corkscrew, a Drosophila homolog of SHP-2 1997). In addition to tyrosine phosphorylation of (Perkins et al., 1992). They contain a PH domain, STATs, serine phosphorylation of STAT1 and and proline-rich sequences including c-Met-binding STAT3 was required for full transcriptional activity domain (MBD), and tyrosine-based motifs that bind ofthemolecules(Boultonetal.,1995;Nakajimaetal., to SH2 domains when they are phosphorylated 1995; Wen et al., 1995). Serine 727 of both STAT1 (Holgado-Madruga et al., 1996; Weidner et al., and STAT3 is responsible for the phosphorylation 1996). site and their mutations were shown to reduce Tyrosine phosphorylation of Gab1 and Gab2 was STATs-dependent transcription (Wen and Darnell, independent of tyrosine phosphorylation of gp130 1997). Serine 727 is located prior to proline and fits a (Takahashi-Tezuka et al., 1998; Nishida et al., 1999). part of the consensus sequence of phosphoacceptor They are likely phosphorylated by the gp130-asso- sites by ERK/MAP kinase. In fact, serine 727 could ciated JAKs, since overexpression of JAKs induced be phosphorylated by ERK/MAP kinase in vitro their phosphorylation. Gab1 and Gab2 interacted (Wen et al., 1995). Upon growth hormone stimula- with not only SHP-2 but also the p85 PI-3 kinase tion, STAT3 is phosphorylated dependent on ERK/ on stimulation. Overexpression of Gab1 or Gab2 MAP kinases, whereas serine phosphorylation of was reported to enhance the gp130-dependent acti- STAT3inducedbyIL-6isindependentofERK/MAP vation of ERK/MAP kinases, suggesting that Gab kinase (Chung et al., 1997; Ng and Cantrell, 1997). family proteins act upstream of ERK/MAP kinases Several groups reported that the gp130-mediated (Takahashi-Tezuka et al., 1998; Nishida et al., 1999). serine phosphorylation of STAT3 was inhibited by a

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IL-6 Receptor PDF

18 Pages·0.29 MB·English
by  Hibi M., Hirano T.
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IL-6 Receptor Masahiko Hibi* and Toshio Hirano Division of Molecular Oncology, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine(C7), 2-2, Yamada-oka, Suita, Osaka, 565, Japan *corresponding author tel: 81-6-879-3880, fax: 81-6-879-3889, e-mail: [email protected] DOI: 10.1006/rwcy.2000.17001. SUMMARY binding receptors expressed on the surface of cells which can respond to IL-6 (Taga et al., 1987). The human cDNA for IL-6R(cid:11) (originally IL-6R) was Interleukin 6 (IL-6) is a pleiotropic cytokine that cloned by an expression-cloning strategy (Yamasaki regulates immune reaction, hematopoiesis, and dif- et al., 1988). Later, IL-6R(cid:11) turned out to be a ferentiation of the nervous system. The receptor for member of cytokine receptor family (type I cytokine IL-6 (IL-6R) consists of two chains, namely IL-6R(cid:11) receptorsuperfamily,hematopoietinreceptorfamily), and gp130. Both IL-6R(cid:11) and gp130 belong to the which includes receptors for many interleukins, type I cytokine receptor superfamily. IL-6R(cid:11) is the colony-stimulating factors, and hormones (e.g. pro- binding component specific to IL-6. In contrast, lactin, growth hormone, and leptin) (Bazan, 1990). gp130 transmits signals not only of IL-6 but also of Expression of IL-6R(cid:11) in cells constitutes the low- IL-6-related cytokines such as leukemia-inhibitory affinity binding sites, but IL-6R(cid:11) alone does not factor (LIF), ciliary neurotropic factor (CNTF), create either the high-affinity binding sites or signals oncostatin M (OSM), IL-11, cardiotropin 1 (CT-1), that elicit biological functions of IL-6, suggesting the and possibly neurotrophin-1/B cell-stimulating factor existenceofanothercomponentoftheIL-6Rcomplex 3 (NNT-1/BSF-3). The ligand’s binding to the (Taga et al., 1989) in a manner which is called receptor leads to homo- and heterodimerization of receptorconversion.ItwasshownthatinalltheIL-6- gp130, resulting in the activation of gp130-associated responsive cells, IL-6R(cid:11) associates with a transmem- JAKs (JAK1, JAK2, and TYK2) and subsequently brane glycoprotein, molecular weight 130kDa in the tyrosine phosphorylation of gp130. The tyrosine- human myeloma cell line U266, in response to IL-6 phosphorylated gp130 further transmits signals by (Taga et al., 1989). Thus it was named gp130. recruiting SH2 domain-containing signaling mole- Furthermore, a complex of IL-6 and soluble IL-6R(cid:11) cules such as the protein tyrosine phosphatase SHP-2 was shown to bind the extracellular domain of gp130 and STAT1 and STAT3 (signal transducers and acti- and transmit signals (Taga et al., 1989). Molecular vatorsoftranscription).Wedescribetherolesofthese cloning of gp130 further revealed that it also belongs signal transduction pathways in the biological to the type I cytokine receptor superfamily. responses of the IL-6 family cytokines. Expression of gp130 was reported to generate the high-affinity IL-6 binding sites, and render a certain cell line to receive the signal of IL-6 and soluble BACKGROUND IL-6R (Hibi et al., 1990). Thereafter it was found that gp130 transmits sig- Discovery nals not only of IL-6 but also of IL-6-related cyto- kines such as LIF, CNTF, OSM, IL-11, and CT-1, IL-6 is a pleiotropic cytokine that acts on various and possibly NNT-1/BSF-3 (Senaldi et al., 1999) cells.SignalsofIL-6aretransmittedintotheinsideof which share the biological function with IL-6. gp130 cellsthroughtheIL-6Rcomplexthatexistsonthecell forms a complex with (cid:11) subunits of IL-6R, IL-11R, surface.Initially,ligand-bindingassaysusingisotope- CNTFreceptor,andpossiblyCT-1receptor,andLIF labeled IL-6 revealed the existence of high-affinity receptor (cid:12) or OSM receptor (cid:12) to act as receptors for 1762 Masahiko Hibi and Toshio Hirano the IL-6-related cytokines (Hirano et al., 1997; Figure 1 Structure of the IL-6 receptor system. Heinrich et al., 1998). IL-6R(cid:11)andgp130belongtothecytokinereceptor typesuperfamily.Theextracellulardomainsofboth IL-6R(cid:11) and gp130 contain an immunoglobulin- Alternative names like (Ig) domain and a cytokine-binding module (CBM). In addition, gp130 has three repeats of IL-6 receptor (cid:11): B cell stimulatory factor-2, BSF-2 fibronectin type III (FNIII) domains in the extra- receptor, IL-6 receptor, CD126. cellulardomain.gp130hasregions(box1andbox2) gp130: IL-6 signal transducer, IL-6 receptor (cid:12) chain, conserved with the family and six tyrosines in its CD130. cytoplasmicdomain(numbersindicatelocationsof tyrosine in human gp130). Box 1 and box 2 are involvedintheassociationandactivationofJAKs Structure (JAK1,JAK2,andTYK2).Phosphorylationofthe Y759andthefourtyrosines(Y767,Y814,Y904,and TheIL-6receptorsystemiscomposedofIL-6R(cid:11)and Y915) in the C-terminal are involved in the phos- gp130 (Figure 1). A reconstitution experiment of the phorylation and activation of SHP-2 and STAT3. ThephosphorylationofY904andY915wasshown solubleIL-6RsystemsuggestedthattheIL-6receptor to be involved in the phosphorylation and activa- consistsoftwomoleculesofIL-6,IL-6R(cid:11),andgp130 tion of STAT1. (Paonessaetal.,1995).Alternatively,itwasproposed thattheIL-6receptorconsistsofonemoleculeofIL-6 andIL-6R(cid:11),andtwomoleculesofgp130(Grotzinger et al., 1997). The molecular weight of IL-6R(cid:11) is 80kDa (the molecular weight predicted from the amino acid sequence is 49.9kDa) (Yamasaki et al., 1988; Hirata et al., 1989; Taga et al., 1989). IL-6R(cid:11) is a type I transmembrane protein. The precursor of human IL- 6R(cid:11)contains468aminoacidsanditsputativemature formconsistsof449aminoacids.Itiscomposedofan extracellular region of 339 amino acids, a membrane- spanningregionof28aminoacids,andacytoplasmic region of 82 amino acids. It has five potential N- glycosylation sites. IL-6R(cid:11) belongs to the type I cytokine receptor superfamily. It has one cytokine- binding module (CBM) composed of two fibronectin typeIII-like(FNIII)domains,inwhichtheN-terminal domain contains a set of four conserved cysteine residues and the C-terminal one contains a WSXWS motif,conservedinthetypeIcytokinereceptorsuper- family. It was reported that the C-terminal FNIII domain in the CBM is sufficient for IL-6 binding but not for the association with gp130 (Ozbek et al., 1998). In addition to the CRM, IL-6R(cid:11) contains an immunoglobulin-like domain at the N-terminus. The structure and functions of the cytoplasmic domain of IL-6R(cid:11) remain to be clarified. The molecular weight of gp130 is 130–160kDa (Taga et al., 1989; Hibi et al., 1990), the variation in of 597 amino acids, a membrane-spanning region of the molecular weight probably being due to the gly- 22aminoacids,andacytoplasmicregionof277amino cosylation. Deglycosylated gp130 displays 100kDa, acids. gp130 is also a member of the type I cytokine whichisconsistentwiththepredictedmolecularweight receptor superfamily. It has a immunoglobulin-like from the amino acid sequence (101kDa) (Taga et al., domain at the N-terminus, which is followed by a 1989). gp130 is also a type I transmembrane protein. CBMcontainingtwoFNIIIdomains.gp130hasthree The precursor of human gp130 contains 918 amino additional FNIII domains following the CBM, and acids and its putative mature form consists of 896 thus contains five FNIII domains in the extracellular aminoacids.Itiscomposedofanextracellularregion region. IL-6 Receptor 1763 The three-dimensional structure of the CBM of Figure2 NucleotidesequencesforhumanIL-6receptor(cid:11) gp130 has been analyzed (Bravo et al., 1998). and gp130. Structurally, it is closely related to the CBMs of growth hormone receptor (de Vos et al., 1992) and Human IL-6 receptor a erythropoietin receptor. The cytoplasmic region of GGCGGTCCCCTGTTCTCCCCGCTCAGGTGCGGCGCTGTGGCAGGAAGCCA gp130 has several distinct domains. The membrane- CCGCCAGGGGACAAGAGGGGCGAGTCCACGCCGCGACACCGTCCTTCGGT proximal region of gp130 has a similar sequence to CCCCCTCGGTCGGCCGGTGCGCGGGGCTGTTGCGCCATCCGCTCCGGCTT those of all members of the type I cytokine receptor GGGGGAGCCAGCCGGCCACGCGCCCCGACAACGCGGTAGGCGAGGCCGAA superfamily. The conserved region was separated by TCGTAACCGCACCCTGGGACGGCCCAGAGACGCTCCAGCGCGAGTTCCTC AGCATTGGCGTGGGACCCTGCCGGGTCTCTGCGAGGTCGCGCTCAAGGAG a short nonconserved amino acid stretch and thus AAATGTTTTCCTGCGTTGCCAGGACCGTCCGCCGCTCTGAGTCATGTGCG these domains are named box 1 and box 2 domains TTTACAAAAGGACGCAACGGTCCTGGCAGGCGGCGAGACTCAGTACACGC (Murakami et al., 1991). The box 1 domain is a AGTGGGAAGTCGCACTGACACTGAGCCGGGCCAGAGGGAGAGGAGCCGAG proline-rich amino acid stretch and box 2 contains a TCACCCTTCAGCGTGACTGTGACTCGGCCCGGTCTCCCTCTCCTCGGCTC clusterofhydrophobicaminoacidsfollowedbyacidic CGCGGCGCGGGGCCGAGGGACTCGCAGTGTGTGTAGAGAGCCGGGCTCCT GCGCCGCGCCCCGGCTCCCTGAGCGTCACACACATCTCTCGGCCCGAGGA amino acid residues. The box 1 and box 2 regions GCGGATGGGGGCTGCCCCCGGGGCCTGAGCCCGCCTGCCCGCCCACCGCC constitute a docking site for Janus tyrosine kinases CGCCTACCCCCGACGGGGGCCCCGGACTCGGGCGGACGGGCGGGTGGCGG (JAK1,JAK2,andTYK2).gp130hasaregionwhich CCGCCCCGCCCCTGCCACCCCTGCCGCCCGGTTCCCATTAGCCTGTCCGC ishomologoustoG-CSFreceptorinthemiddleofthe GGCGGGGCGGGGACGGTGGGGACGGCGGGCCAAGGGTAATCGGACAGGCG cytoplasmic domain (amino acids 772–787) and this CTCTGCGGGACCATGGAGTGGTAGCCGAGGAGGAAGCATGCTGGCCGTCG region is termed box 3 (Fukunaga et al., 1991). In GAGACGCCCTGGTACCTCACCATCGGCTCCTCCTTCGTACGACCGGCAGC GCTGCGCGCTGCTGGCTGCCCTGCTGGCCGCGCCGGGAGCGGCGCTGGCC addition to box 1, box 2, and box 3, gp130 contains CGACGCGCGACGACCGACGGGACGACCGGCGCGGCCCTCGCCGCGACCGG six tyrosines, some of which have been shown to be CCAAGGCGCTGCCCTGCGCAGGAGGTGGCAAGAGGCGTGCTGACCAGTCT important for signal transduction. GGTTCCGCGACGGGACGCGTCCTCCACCGTTCTCCGCACGACTGGTCAGA GCCAGGAGACAGCGTGACTCTGACCTGCCCGGGGGTAGAGCCGGAAGACA CGGTCCTCTGTCGCACTGAGACTGGACGGGCCCCCATCTCGGCCTTCTGT Main activities and ATGCCACTGTTCACTGGGTGCTCAGGAAGCCGGCTGCAGGCTCCCACCCC TACGGTGACAAGTGACCCACGAGTCCTTCGGCCGACGTCCGAGGGTGGGG pathophysiological roles AGCAGATGGGCTGGCATGGGAAGGAGGCTGCTGCTGAGGTCGGTGCAGCT TCGTCTACCCGACCGTACCCTTCCTCCGACGACGACTCCAGCCACGTCGA CCACGACTCTGGAAACTATTCATGCTACCGGGCCGGCCGCCCAGCTGGGA IL-6R(cid:11) and gp130 transmit all the biological GGTGCTGAGACCTTTGATAAGTACGATGGCCCGGCCGGCGGGTCGACCCT functions of IL-6. gp130 is also required for the CTGTGCACTTGCTGGTGGATGTTCCCCCCGAGGAGCCCCAGCTCTCCTGC biological functions of the IL-6-related cytokines: GACACGTGAACGACCACCTACAAGGGGGGCTCCTCGGGGTCGAGAGGACG TTCCGGAAGAGCCCCCTCAGCAATGTTGTTTGTGAGTGGGGTCCTCGGAG LIF, CNTF, IL-11, OSM, and CT-1. AAGGCCTTCTCGGGGGAGTCGTTACAACAAACACTCACCCCAGGAGCCTC CACCCCATCCCTGACGACAAAGGCTGTGCTCTTGGTGAGGAAGTTTCAGA GTGGGGTAGGGACTGCTGTTTCCGACACGAGAACCACTCCTTCAAAGTCT ACAGTCCGGCCGAAGACTTCCAGGAGCCGTGCCAGTATTCCCAGGAGTCC GENE TGTCAGGCCGGCTTCTGAAGGTCCTCGGCACGGTCATAAGGGTCCTCAGG CAGAAGTTCTCCTGCCAGTTAGCAGTCCCGGAGGGAGACAGCTCTTTCTA Accession numbers GTCTTCAAGAGGACGGTCAATCGTCAGGGCCTCCCTCTGTCGAGAAAGAT CATAGTGTCCATGTGCGTCGCCAGTAGTGTCGGGAGCAAGTTCAGCAAAA GTATCACAGGTACACGCAGCGGTCATCACAGCCCTCGTTCAAGTCGTTTT Human IL-6R(cid:11): X12830, M20566 CTCAAACCTTTCAGGGTTGTGGAATCTTGCAGCCTGATCCGCCTGCCAAC GAGTTTGGAAAGTCCCAACACCTTAGAACGTCGGACTAGGCGGACGGTTG Mouse IL-6R(cid:11): X51975, X51976 ATCACAGTCACTGCCGTGGCCAGAAACCCCCGCTGGCTCAGTGTCACCTG Human gp130: M57230 TAGTGTCAGTGACGGCACCGGTCTTTGGGGGCGACCGAGTCACAGTGGAC Mouse gp130: X62646, M83336 GCAAGACCCCCACTCCTGGAACTCATCTTTCTACAGACTACGGTTTGAGC Rat gp130: M92340 CGTTCTGGGGGTGAGGACCTTGAGTAGAAAGATGTCTGATGCCAAACTCG Chick gp130: AJ011688, GGA011688 TCAGATATCGGGCTGAACGGTCAAAGACATTCACAACATGGATGGTCAAG AGTCTATAGCCCGACTTGCCAGTTTCTGTAAGTGTTGTACCTACCAGTTC Genome: GACCTCCAGCATCACTGTGTCATCCACGACGCCTGGAGCGGCCTGAGGCA IL-6R(cid:11): chromosomal location not determined. CTGGAGGTCGTAGTGACACAGTAGGTGCTGCGGACCTCGCCGGACTCCGT Humangp130:chromosome5(apseudogeneexistsin CGTGGTGCAGCTTCGTGCCCAGGAGGAGTTCGGGCAAGGCGAGTGGAGCG chromosome 17). GCACCACGTCGAAGCACGGGTCCTCCTCAAGCCCGTTCCGCTCACCTCGC AGTGGAGCCCGGAGGCCATGGGCACGCCTTGGACAGAATCCAGGAGTCCT TCACCTCGGGCCTCCGGTACCCGTGCGGAACCTGTCTTAGGTCCTCAGGA Sequence CCAGCTGAGAACGAGGTGTCCACCCCCATGCAGGCACTTACTACTAATAA GGTCGACTCTTGCTCCACAGGTGGGGGTACGTCCGTGAATGATGATTATT AGACGATGATAATATTCTCTTCAGAGATTCTGCAAATGCGACAAGCCTCC See Figure 2. 1764 Masahiko Hibi and Toshio Hirano Figure 2 (Continued) Figure 2 (Continued) TCTGCTACTATTATAAGAGAAGTCTCTAAGACGTTTACGCTGTTCGGAGG Human gp130 CAGTGCAAGATTCTTCTTCAGTACCACTGCCCACATTCCTGGTTGCTGGA GTCACGTTCTAAGAAGAAGTCATGGTGACGGGTGTAAGGACCAACGACCT GAGCAGCCAAAAGGCCCGCGGAGTCGCGCTGGGCCGCCCCGGCGCAGCTG GGGAGCCTGGCCTTCGGAACGCTCCTCTGCATTGCCATTGTTCTGAGGTT CTCGTCGGTTTTCCGGGCGCCTCAGCGCGACCCGGCGGGGCCGCGTCGAC CCCTCGGACCGGAAGCCTTGCGAGGAGACGTAACGGTAACAAGACTCCAA AACCGGGGGCCGCGCCTGCCAGGCCGACGGGTCTGGCCCAGCCTGGCGCC CAAGAAGACGTGGAAGCTGCGGGCTCTGAAGGAAGGCAAGACAAGCATGC TTGGCCCCCGGCGCGGACGGTCCGGCTGCCCAGACCGGGTCGGACCGCGG GTTCTTCTGCACCTTCGACGCCCGAGACTTCCTTCCGTTCTGTTCGTACG AAGGGGTTCGTGCGCTGTGGAGACGCGGAGGGTCGAGGCGGCGCGGCCTG ATCCGCCGTACTCTTTGGGGCAGCTGGTCCCGGAGAGGCCTCGACCCACC TTCCCCAAGCACGCGACACCTCTGCGCCTCCCAGCTCCGCCGCGCCGGAC TAGGCGGCATGAGAAACCCCGTCGACCAGGGCCTCTCCGGAGCTGGGTGG AGTGAAACCCAATGGAAAAAGCATGACATTTAGAAGTAGAAGACTTAGCT CCAGTGCTTGTTCCTCTCATCTCCCCACCGGTGTCCCCCAGCAGCCTGGG GGTCACGAACAAGGAGAGTAGAGGGGTGGCCACAGGGGGTCGTCGGACCC TCACTTTGGGTTACCTTTTTCGTACTGTAAATCTTCATCTTCTGAATCGA GTCTGACAATACCTCGAGCCACAACCGACCAGATGCCAGGGACCCACGGA TCAAATCCCTACTCCTTCACTTACTAATTTTGTGATTTGGAAATATCCGC CAGACTGTTATGGAGCTCGGTGTTGGCTGGTCTACGGTCCCTGGGTGCCT AGTTTAGGGATGAGGAAGTGAATGATTAAAACACTAAACCTTTATAGGCG GCCCTTATGACATCAGCAATACAGACTACTTCTTCCCCAGATAGCTGGCT GCAAGATGTTGACGTTGCAGACTTGGGTAGTGCAAGCCTTGTTTATTTTC CGGGAATACTGTAGTCGTTATGTCTGATGAAGAAGGGGTCTATCGACCGA CGTTCTACAACTGCAACGTCTGAACCCATCACGTTCGGAACAAATAAAAG GGGTGGCACCAGCAGCCTGGACCCTGTGGATGACAAAACACAAACGGGCT CTCACCACTGAATCTACAGGTGAACTTCTAGATCCATGTGGTTATATCAG CCCACCGTGGTCGTCGGACCTGGGACACCTACTGTTTTGTGTTTGCCCGA GAGTGGTGACTTAGATGTCCACTTGAAGATCTAGGTACACCAATATAGTC CAGCAAAAGATGCTTCTCACTGCCATGCCAGCTTATCTCAGGGGTGTGCG TCCTGAATCTCCAGTTGTACAACTTCATTCTAATTTCACTGCAGTTTGTG GTCGTTTTCTACGAAGAGTGACGGTACGGTCGAATAGAGTCCCCACACGC GCCTTTGGCTTCACGGAAGAGCCTTGCGGAAGGTTCTACGCCAGGGGAAA AGGACTTAGAGGTCAACATGTTGAAGTAAGATTAAAGTGACGTCAAACAC CGGAAACCGAAGTGCCTTCTCGGAACGCCTTCCAAGATGCGGTCCCCTTT TGCTAAAGGAAAAATGTATGGATTATTTTCATGTAAATGCTAATTACATT ATCAGCCTGCTCCAGCTGTTCAGCTGGTTGAGGTTTCAAACCTCCCTTTC ACGATTTCCTTTTTACATACCTAATAAAAGTACATTTACGATTAATGTAA TAGTCGGACGAGGTCGACAAGTCGACCAACTCCAAAGTTTGGAGGGAAAG GTCTGGAAAACAAACCATTTTACTATTCCTAAGGAGCAATATACTATCAT CAAATGCCCAGCTTAAAGGGGTTAGAGTGAACTTGGGCCACTGTGAAGAG CAGACCTTTTGTTTGGTAAAATGATAAGGATTCCTCGTTATATGATAGTA GTTTACGGGTCGAATTTCCCCAATCTCACTTGAACCCGGTGACACTTCTC AAACAGAACAGCATCCAGTGTCACCTTTACAGATATAGCTTCATTAAATA AACCATATCAAGACTCTTTGGACACTCACACGGACACTCAAAAGCTGGGC TTTGTCTTGTCGTAGGTCACAGTGGAAATGTCTATATCGAAGTAATTTAT TTGGTATAGTTCTGAGAAACCTGTGAGTGTGCCTGTGAGTTTTCGACCCG TTCAGCTCACTTGCAACATTCTTACATTCGGACAGCTTGAACAGAATGTT AGGTTGGTGGGGGCCTCGGTGTGGAGAAGCGGCTGGCAGCCCACCCCTCA AAGTCGAGTGAACGTTGTAAGAATGTAAGCCTGTCGAACTTGTCTTACAA TCCAACCACCCCCGGAGCCACACCTCTTCGCCGACCGTCGGGTGGGGAGT ACACCTCTGCACAAGCTGCACCCTCAGGCAGGTGGGATGGATTTCCAGCC TATGGAATCACAATAATTTCAGGCTTGCCTCCAGAAAAACCTAAAAATTT TGTGGAGACGTGTTCGACGTGGGAGTCCGTCCACCCTACCTAAAGGTCGG ATACCTTAGTGTTATTAAAGTCCGAACGGAGGTCTTTTTGGATTTTTAAA AAAGCCTCCTCCAGCCGCCATGCTCCTGGCCCACTGCATCGTTTCATCTT GAGTTGCATTGTGAACGAGGGGAAGAAAATGAGGTGTGAGTGGGATGGTG TTTCGGAGGAGGTCGGCGGTACGAGGACCGGGTGACGTAGCAAAGTAGAA CTCAACGTAACACTTGCTCCCCTTCTTTTACTCCACACTCACCCTACCAC CCAACTCAAACTCTTAAAACCCAAGTGCCCTTAGCAAATTCTGTTTTTCT GAAGGGAAACACACTTGGAGACAAACTTCACTTTAAAATCTGAATGGGCA GGTTGAGTTTGAGAATTTTGGGTTCACGGGAATCGTTTAAGACAAAAAGA CTTCCCTTTGTGTGAACCTCTGTTTGAAGTGAAATTTTAGACTTACCCGT AGGCCTGGGGACGGCTTTTACTTAAACGCCAAGGCCTGGGGGAAGAAGCT ACACACAAGTTTGCTGATTGCAAAGCAAAACGTGACACCCCCACCTCATG TCCGGACCCCTGCCGAAAATGAATTTGCGGTTCCGGACCCCCTTCTTCGA TGTGTGTTCAAACGACTAACGTTTCGTTTTGCACTGTGGGGGTGGAGTAC CTCTCCTCCCTTTCTTCCCTACAGTTCAAAAACAGCTGAGGGTGAGTGGG GAGAGGAGGGAAAGAAGGGATGTCAAGTTTTTGTCGACTCCCACTCACCC CACTGTTGATTATTCTACTGTGTATTTTGTCAACATTGAAGTCTGGGTAG TGAATAATACAGTATGTCAGGGCCTGGTCGTTTTCAACAGAATTATAATT GTGACAACTAATAAGATGACACATAAAACAGTTGTAACTTCAGACCCATC ACTTATTATGTCATACAGTCCCGGACCAGCAAAAGTTGTCTTAATATTAA AAGCAGAGAATGCCCTTGGGAAGGTTACATCAGATCATATCAATTTTGAT AGTTCCTCATTAGCAGTTTTGCCTAAATGTGAATGATGATCCTAGGCATT TTCGTCTCTTACGGGAACCCTTCCAATGTAGTCTAGTATAGTTAAAACTA TCAAGGAGTAATCGTCAAAACGGATTTACACTTACTACTAGGATCCGTAA CCTGTATATAAAGTGAAGCCCAATCCGCCACATAATTTATCAGTGATCAA TGCTGAATACAGAGGCAACTGCATTGGCTTTGGGTTGCAGGACCTCAGGT GGACATATATTTCACTTCGGGTTAGGCGGTGTATTAAATAGTCACTAGTT ACGACTTATGTCTCCGTTGACGTAACCGAAACCCAACGTCCTGGAGTCCA CTCAGAGGAACTGTCTAGTATCTTAAAATTGACATGGACCAACCCAAGTA GAGAAGCAGAGGAAGGAGAGGAGAGGGGCACAGGGTCTCTACCATCCCCT GAGTCTCCTTGACAGATCATAGAATTTTAACTGTACCTGGTTGGGTTCAT CTCTTCGTCTCCTTCCTCTCCTCTCCCCGTGTCCCAGAGATGGTAGGGGA TTAAGAGTGTTATAATACTAAAATATAACATTCAATATAGGACCAAAGAT GTAGAGTGGGAGCTGAGTGGGGGATCACAGCCTCTGAAAACCAATGTTCT CATCTCACCCTCGACTCACCCCCTAGTGTCGGAGACTTTTGGTTACAAGA AATTCTCACAATATTATGATTTTATATTGTAAGTTATATCCTGGTTTCTA CTCTTCTCCACCTCCCACAAAGGAGAGCTAGCAGCAGGGAGGGCTTCTGC GCCTCAACTTGGAGCCAGATTCCTCCTGAAGACACAGCATCCACCCGATC GAGAAGAGGTGGAGGGTGTTTCCTCTCGATCGTCGTCCCTCCCGAAGACG CGGAGTTGAACCTCGGTCTAAGGAGGACTTCTGTGTCGTAGGTGGGCTAG CATTTCTGAGATCAAAACGGTTTTACTGCAGCTTTGTTTGTTGTCAGCTG TTCATTCACTGTCCAAGACCTTAAACCTTTTACAGAATATGTGTTTAGGA GTAAAGACTCTAGTTTTGCCAAAATGACGTCGAAACAAACAACAGTCGAC AAGTAAGTGACAGGTTCTGGAATTTGGAAAATGTCTTATACACAAATCCT AACCTGGGTAACTAGGGAAGATAATATTAAGGAAGACAATGTGAAAAGAA TTCGCTGTATGAAGGAAGATGGTAAGGGATACTGGAGTGACTGGAGTGAA TTGGACCCATTGATCCCTTCTATTATAATTCCTTCTGTTACACTTTTCTT AAGCGACATACTTCCTTCTACCATTCCCTATGACCTCACTGACCTCACTT AAATGAGCCTGGCAAGAATGCGTTTAAACTTGGTTTTTAAAAAACTGCTG GAAGCAAGTGGGATCACCTATGAAGATAGACCATCTAAAGCACCAAGTTT TTTACTCGGACCGTTCTTACGCAAATTTGAACCAAAAATTTTTTGACGAC ACTGTTTTCTCTTGAGAGGGTGGAATATCCAATATTCGCTGTGTCAGCAT CTTCGTTCACCCTAGTGGATACTTCTATCTGGTAGATTTCGTGGTTCAAA TGACAAAAGAGAACTCTCCCACCTTATAGGTTATAAGCGACACAGTCGTA CTGGTATAAAATAGATCCATCCCATACTCAAGGCTACAGAACTGTACAAC AGAAGTAACTTACTTAGGTGTGGGGGAAGCACCATAACTTTGTTTAGCCC GACCATATTTTATCTAGGTAGGGTATGAGTTCCGATGTCTTGACATGTTG TCTTCATTGAATGAATCCACACCCCCTTCGTGGTATTGAAACAAATCGGG TCGTGTGGAAGACATTGCCTCCTTTTGAAGCCAATGGAAAAATCTTGGAT AAAACCAAGTCAAGTGAAAAAGGAGGAAGAGAAAAAATATTTTCCTGCCA AGCACACCTTCTGTAACGGAGGAAAACTTCGGTTACCTTTTTAGAACCTA TTTTGGTTCAGTTCACTTTTTCCTCCTTCTCTTTTTTATAAAAGGACGGT TATGAAGTGACTCTCACAAGATGGAAATCACATTTACAAAATTACACAGT GGCATGGAGGCCCACGCACTTCGGGAGGTCGAGGCAGGAGGATCACTTGA ATACTTCACTGAGAGTGTTCTACCTTTAGTGTAAATGTTTTAATGTGTCA CCGTACCTCCGGGTGCGTGAAGCCCTCCAGCTCCGTCCTCCTAGTGAACT TAATGCCACAAAACTGACAGTAAATCTCACAAATGATCGCTATCTAGCAA GTCCAGAAGTTTGAGATCAGCCTGGGCAATGTGATAAAACCCCATCTCTA ATTACGGTGTTTTGACTGTCATTTAGAGTGTTTACTAGCGATAGATCGTT CAGGTCTTCAAACTCTAGTCGGACCCGTTACACTATTTTGGGGTAGAGAT CAAAAAGCATAAAAATTAGCCAAGTGTGGTAGAGTGTGCCTGAAGTCCCA CCCTAACAGTAAGAAATCTTGTTGGCAAATCAGATGCAGCTGTTTTAACT GTTTTTCGTATTTTTAATCGGTTCACACCATCTCACACGGACTTCAGGGT GGGATTGTCATTCTTTAGAACAACCGTTTAGTCTACGTCGACAAAATTGA GATACTTGGGGGGCTGAGGTGGGAGGATCTCTTGAGCCTGGGAGGTCAAG ATCCCTGCCTGTGACTTTCAAGCTACTCACCCTGTAATGGATCTTAAAGC CTATGAACCCCCCGACTCCACCCTCCTAGAGAACTCGGACCCTCCAGTTC TAGGGACGGACACTGAAAGTTCGATGAGTGGGACATTACCTAGAATTTCG GCTGCAGTGAGCCGAGATTGCACCACTGCACTCCAGCCTGGGGTGACAGA ATTCCCCAAAGATAACATGCTTTGGGTGGAATGGACTACTCCAAGGGAAT CGACGTCACTCGGCTCTAACGTGGTGACGTGAGGTCGGACCCCACTGTCT GCAAGTGAGACCCTGTCTCCGTTCACTCTGGGACAGAG IL-6 Receptor 1765 Figure 2 (Continued) PROTEIN TAAGGGGTTTCTATTGTACGAAACCCACCTTACCTGATGAGGTTCCCTTA Accession numbers CTGTAAAGAAATATATACTTGAGTGGTGTGTGTTATCAGATAAAGCACCC GACATTTCTTTATATATGAACTCACCACACACAATAGTCTATTTCGTGGG TGTATCACAGACTGGCAACAAGAAGATGGTACCGTGCATCGCACCTATTT Human IL-6R(cid:11): 49726, 33846 ACATAGTGTCTGACCGTTGTTCTTCTACCATGGCACGTAGCGTGGATAAA Mouse IL-6R(cid:11): 52693 AAGAGGGAACTTAGCAGAGAGCAAATGCTATTTGATAACAGTTACTCCAG Rat IL-6R(cid:11): 111882 TTCTCCCTTGAATCGTCTCTCGTTTACGATAAACTATTGTCAATGAGGTC Human gp130: 729833, 106982, 186354 TATATGCTGATGGACCAGGAAGCCCTGAATCCATAAAGGCATACCTTAAA ATATACGACTACCTGGTCCTTCGGGACTTAGGTATTTCCGTATGGAATTT Mouse gp130: 729834, 2137360, 840817, 193592 CAAGCTCCACCTTCCAAAGGACCTACTGTTCGGACAAAAAAAGTAGGGAA Rat gp130: 729835, 348455 GTTCGAGGTGGAAGGTTTCCTGGATGACAAGCCTGTTTTTTTCATCCCTT AAACGAAGCTGTCTTAGAGTGGGACCAACTTCCTGTTGATGTTCAGAATG TTTGCTTCGACAGAATCTCACCCTGGTTGAAGGACAACTACAAGTCTTAC Sequence GATTTATCAGAAATTATACTATATTTTATAGAACCATCATTGGAAATGAA CTAAATAGTCTTTAATATGATATAAAATATCTTGGTAGTAACCTTTACTT ACTGCTGTGAATGTGGATTCTTCCCACACAGAATATACATTGTCCTCTTT See Figure 3. TGACGACACTTACACCTAAGAAGGGTGTGTCTTATATGTAACAGGAGAAA GACTAGTGACACATTGTACATGGTACGAATGGCAGCATACACAGATGAAG Description of protein CTGATCACTGTGTAACATGTACCATGCTTACCGTCGTATGTGTCTACTTC GTGGGAAGGATGGTCCAGAATTCACTTTTACTACCCCAAAGTTTGCTCAA CACCCTTCCTACCAGGTCTTAAGTGAAAATGATGGGGTTTCAAACGAGTT See Table 1. GGAGAAATTGAAGCCATAGTCGTGCCTGTTTGCTTAGCATTCCTATTGAC CCTCTTTAACTTCGGTATCAGCACGGACAAACGAATCGTAAGGATAACTG AACTCTTCTGGGAGTGCTGTTCTGCTTTAATAAGCGAGACCTAATTAAAA Relevant homologies and species TTGAGAAGACCCTCACGACAAGACGAAATTATTCGCTCTGGATTAATTTT AACACATCTGGCCTAATGTTCCAGATCCTTCAAAGAGTCATATTGCCCAG differences TTGTGTAGACCGGATTACAAGGTCTAGGAAGTTTCTCAGTATAACGGGTC TGGTCACCTCACACTCCTCCAAGGCACAATTTTAATTCAAAAGATCAAAT ACCAGTGGAGTGTGAGGAGGTTCCGTGTTAAAATTAAGTTTTCTAGTTTA TheoverallhomologybetweenhumanandmouseIL- GTATTCAGATGGCAATTTCACTGATGTAAGTGTTGTGGAAATAGAAGCAA 6R(cid:11) is 69% and 54% at the DNA and protein level, CATAAGTCTACCGTTAAAGTGACTACATTCACAACACCTTTATCTTCGTT ATGACAAAAAGCCTTTTCCAGAAGATCTGAAATCATTGGACCTGTTCAAA respectively(Yamasakietal.,1988;Sugitaetal.,1990). TACTGTTTTTCGGAAAAGGTCTTCTAGACTTTAGTAACCTGGACAAGTTT The overall homology between human and mouse AAGGAAAAAATTAATACTGAAGGACACAGCAGTGGTATTGGGGGGTCTTC gp130 is 76.6% and 76.8% at the DNA and protein TTCCTTTTTTAATTATGACTTCCTGTGTCGTCACCATAACCCCCCAGAAG level,respectively(Hibietal.,1990;Saitoetal.,1992). ATGCATGTCATCTTCTAGGCCAAGCATTTCTAGCAGTGATGAAAATGAAT TACGTACAGTAGAAGATCCGGTTCGTAAAGATCGTCACTACTTTTACTTA The homologies in the extracellular and cytoplasmic CTTCACAAAACACTTCGAGCACTGTCCAGTATTCTACCGTGGTACACAGT regions between human and mouse gp130 are 72% GAAGTGTTTTGTGAAGCTCGTGACAGGTCATAAGATGGCACCATGTGTCA and 85%, respectively. The transmembrane domains GGCTACAGACACCAAGTTCCGTCAGTCCAAGTCTTCTCAAGATCCGAGTC ofhumanandmousegp130areidentical.Thehomol- CCGATGTCTGTGGTTCAAGGCAGTCAGGTTCAGAAGAGTTCTAGGCTCAG TACCCAGCCCTTGTTAGATTCAGAGGAGCGGCCAGAAGATCTACAATTAG ogy between human and chick gp130 is 74% at the ATGGGTCGGGAACAATCTAAGTCTCCTCGCCGGTCTTCTAGATGTTAATC amino acid level (Geissen et al., 1998). TAGATCATGTAGATGGCGGTGATGGTATTTTGCCCAGGCAACAGTACTTC ATCTAGTACATCTACCGCCACTACCATAAAACGGGTCCGTTGTCATGAAG AAACAGAACTGCAGTCAGCATGAATCCAGTCCAGATATTTCACATTTTGA Affinity for ligand(s) TTTGTCTTGACGTCAGTCGTACTTAGGTCAGGTCTATAAAGTGTAAAACT AAGGTCAAAGCAAGTTTCATCAGTCAATGAGGAAGATTTTGTTAGACTTA TTCCAGTTTCGTTCAAAGTAGTCAGTTACTCCTTCTAAAACAATCTGAAT Low-affinity binding sites for IL-6: K (cid:136)1–6nM. d AACAGCAGATTTCAGATCATATTTCACAATCCTGTGGATCTGGGCAAATG High-affinity binding sites for IL-6: K (cid:136)40–70pM. TTGTCGTCTAAAGTCTAGTATAAAGTGTTAGGACACCTAGACCCGTTTAC d The low-affinity binding site for IL-6 is composed AAAATGTTTCAGGAAGTTTCTGCAGCAGATGCTTTTGGTCCAGGTACTGA of IL-6R(cid:11) alone (Hibi et al., 1990). The high-affinity TTTTACAAAGTCCTTCAAAGACGTCGTCTACGAAAACCAGGTCCATGACT GGGACAAGTAGAAAGATTTGAAACAGTTGGCATGGAGGCTGCGACTGATG bindingsiteforIL-6iscomposedofbothIL-6R(cid:11)and CCCTGTTCATCTTTCTAAACTTTGTCAACCGTACCTCCGACGCTGACTAC gp130,althoughgp130doesnothavetheabilitytobind AAGGCATGCCTAAAAGTTACTTACCACAGACTGTACGGCAAGGCGGCTAC IL-6 by itself (Taga et al., 1989; Hibi et al., 1990). TTCCGTACGGATTTTCAATGAATGGTGTCTGACATGCCGTTCCGCCGATG ATGCCTCAGTGAAGGACTAGTAGTTCCTGCTACAACTTCAGCAGTACCTA TACGGAGTCACTTCCTGATCATCAAGGACGATGTTGAAGTCGTCATGGAT Cell types and tissues expressing TAAAGTAAAGCTAAAATGATTTTATCTGTGAATTC ATTTCATTTCGATTTTACTAAAATAGACACTTAAG the receptor IL-6R(cid:11) is expressed in all the cell lines that respond to IL-6. These include T cell lymphoma and B cell 1766 Masahiko Hibi and Toshio Hirano Figure3 AminoacidsequencesforhumanandmouseIL-6receptor (cid:11) and human and mouse gp130. Signal peptides are underlined; transmembrane regions are in bold. Human IL-6 receptor a MLAVGCALLAALLAAPGAALAPRRCPAQEVARGVLTSLPGDSVT LTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRP AGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAED FQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDP PANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHH CVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQAL TTNKDDDNILFRDSANATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTW KLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDA RDPRSPYDISNTDYFFPR Mouse IL-6 receptor a MLTVGCTLLVALLAAPAVALVLGSCRALEVANGTVTSLPGATVT LICPGKEAAGNVTIHWVYSGSQNREWTTTGNTLVLRDVQLSDTGDYLCSLNDHLVGTV PLLVDVPPEEPKLSCFRKNPLVNAICEWRPSSTPSPTTKAVLFAKKINTTNGKSDFQV PCQYSQQLKSFSCQVEILEGDKVYHIVSLCVANSVGSKSSHNEAFHSLKMVQPDPPAN LVVSAIPGRPRWLKVSWQHPETWDPSYYLLQFQLRYRPVWSKEFTVLLLPVAQYQCVI HDALRGVKHVVQVRGKEELDLGQWSEWSPEVTGTPWIAEPRTTPAGILWNPTQVSVED SANHEDQYESSTEATSVLAPVQESSSMSLPTFLVAGGSLAFGLLLCVFIILRLKQKWK SEAEKESKTTSPPPPPYSLGPLKPTFLLVPLLTPHSSGSDNTVNHSCLGVRDAQSPYD NSNRDYLFPR Human gp130 MLTLQTWVVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNF TAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLT CNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTL KSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVY KVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPED TASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWY KIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLT NDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESV KKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPE SIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNET AVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVP VCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQM YSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPS ISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQL VDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHI SQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGG YMPQ Mouse gp130 MSAPRIWLAQALLFFLTTESIGQLLEPCGYIYPEFPVVQRGSNF TAICVLKEACLQHYYVNASYIVWKTNHAAVPREQVTVINRTTSSVTFTDVVLPSVQLT CNILSFGQIEQNVYGVTMLSGFPPDKPTNLTCIVNEGKNMLCQWDPGRETYLETNYTL KSEWATEKFPDCQSKHGTSCMVSYMPTYYVNIEVWVEAENALGKVSSESINFDPVDKV KPTPPYNLSVTNSEELSSILKLSWVSSGLGGLLDLKSDIQYRTKDASTWIQVPLEDTM SPRTSFTVQDLKPFTEYVFRIRSIKDSGKGYWSDWSEEASGTTYEDRPSRPPSFWYKT NPSHGQEYRSVRLIWKALPLSEANGKILDYEVILTQSKSVSQTYTVTGTELTVNLTND RYVASLAARNKVGKSAAAVLTIPSPHVTAAYSVVNLKAFPKDNLLWVEWTPPPKPVSK YILEWCVLSENAPCVEDWQQEDATVNRTHLRGRLLESKCYQITVTPVFATGPGGSESL KAYLKQAAPARGPTVRTKKVGKNEAVLAWDQIPVDDQNGFIRNYSISYRTSVGKEMVV HVDSSHTEYTLSSLSSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVC LAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYS DGNFTDVSVVEIEANNKKPCPDDLKSVDLFKKEKVSTEGHSSGIGGSSCMSSSRPSIS SNEENESAQSTASTVEYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVD SVDGGDEILPRQPYFKQNCSQPEACPEISHFERSNQVLSGNEEDFVRLKQQQVSDHIS QPYGSEQRRLFQEGSTADALGTGADGQMERFESVGMETTIDEEIPKSYLPQTVRQGGY MPQ IL-6 Receptor 1767 Table 1 Composition of human IL-6 receptor (cid:11) and IL-6 and soluble IL-6R(cid:11) can act on cells expressing gp130 only gp130 and such a mechanism, which is called receptor conversion, may generate functional diver- Human Human gp130 sity of cytokines in vivo (Hirano et al., 1997). In fact, IL-6R(cid:11) double transgenic mice expressing human IL-6 and soluble IL-6R(cid:11) showed myocardial hypertrophy Composition (amino acids) (Hirota et al., 1995) and extramedullary expansion Precursor 468 918 of hematopoietic progenitors (Peters et al., 1997; Mature form 449 896 Schirmacher et al., 1998). Furthermore, the fusion Extracellular domain 339 597 proteinconsistingofIL-6andtheextracellulardomain of IL-6R(cid:11) linked together by a flexible peptide chain Transmembrane domain 28 22 was shown to stimulate hematopoietic progenitor Cytoplasmic domain 82 277 cells effectively (Fischer et al., 1997). However, it is Molecular weight (kDa) not yet clear whether soluble IL-6R(cid:11) acts positively Predicted 49.9 101 on gp130 signaling under physiological conditions. Soluble gp130 was also found in human serum at a Observed 80 130–160 concentration of 300ng/mL (Narazaki et al., 1993). Soluble gp130 was reported to be generated by alternative splicing (Diamant et al., 1997). lymphoma,multiplemyeloma(plasmacytoma),hepa- toma, hepatocellular carcinoma, and glioma cell lines (Taga et al., 1987; Sugita et al., 1990). IL-6R(cid:11) is SIGNAL TRANSDUCTION expressed in spleen, liver, lung, and thymus strongly in mice (Sugita et al., 1990). The size of mRNA for Associated or intrinsic kinases humanandmouseIL-6R(cid:11)isabout5.5kb(Sugitaetal., 1990).InamouseplasmacytomalineP3U1,thecyto- Januskinases,JAK1,JAK2,andTYK2constitutively plasmic domain of IL-6R(cid:11) was replaced by a part of associatewithgp130(Luttickenetal.,1994;Narazaki LTR intracisternal A particle gene and the mRNA et al., 1994;Stahlet al.,1994).Thestructure ofJanus size of the aberrant receptor is 1.8kb (Sugita et al., kinases is shown in Figure 4. Janus kinases contain a 1990). tyrosine kinase domain in their C-termini. Adjacent gp130 is ubiquitously expressed (Hibi et al., 1990; tothekinasedomain,theyhaveakinase-likedomain, Saito et al., 1992). Two mRNAs for gp130 are which is similar to the authentic kinase domain but detected by northern blotting. The size of gp130 lacksseveralaminoacidsconservedamongthekinase mRNAsare 7kband 10kb.Therelative ratioof7kb domains (Ihle, 1995). Upon stimulation of IL-6- and10kbgp130mRNAvariesamongtissues:inheart, related cytokines, these Janus kinases are tyrosine spleen,andlung;expressionof10kbmRNAwasmuch phosphorylated and become activated, resulting in lower than 7kb mRNA (Saito et al., 1992). tyrosine phosphorylation of the cytoplasmic domain of gp130. The box 1/box 2 domain in the membrane- proximal regions of the cytoplasmic domain is Release of soluble receptors involved in the interaction between gp130 and the JAKs. Mutations of the box 1 region reduces the Both IL-6R(cid:11) and gp130 have soluble forms: 30– interaction between gp130 and JAKs (Tanner et al., 70ng/mLofsolubleIL-6R(cid:11)isfoundinhumanserum 1995), and abrogates ligand-dependent activation of (Honda et al., 1992; Muller-Newen et al., 1996). JAK1 and JAK2 (Narazaki et al., 1994) and SolubleIL-6R(cid:11)wasreportedtobeelevatedinserum downstream signaling (Murakami et al., 1991; Lai of multiple myeloma (Gaillard et al., 1993), juvenile et al., 1995a). Truncations of the box 2 region also chronicarthritis(Keuletal.,1998),HIV(Hondaetal., reduced the interaction between gp130 and JAK2, 1992), and Graves’ disease (Salvi et al., 1996). and abrogated the biological functions mediated by Although soluble IL-6R(cid:11) was generated by shedding gp130 (Murakami et al., 1991; Lai et al., 1995a). in vitro (Mullberg et al., 1993), it is possible that it is However, JAKs were reported to interact with gp130 generated by alternative splicing (Lust et al., 1992; lacking the box 2region when theyare overexpressed Muller-Newen et al., 1996). It was reported that a (Lai et al., 1995a; Tanner et al., 1995). These indicate complex of soluble IL-6R(cid:11) and IL-6 has agonistic thatthebox1regionisrequiredandsufficientforthe effects on gp130 (Taga et al., 1989). The complex of JAK binding to some extent, and the box 2 region 1768 Masahiko Hibi and Toshio Hirano Figure 4 Structure of JAK and STAT. A JAK contains seven domains (JAK homology: JH1–7) conserved among JAKs. The JH1 and JH2 do- mains correspond to the tyrosine kinase and kinase-like (pseudokinase) domains,respectively.ASTATcontainsanSH2domain,anSH3-likedomain (functionalsignificanceunknown).Y(tyrosine)indicatesaphosphoacceptor site for JAKs. S indicates a serine residue, the phosphorylation of which is involved in the activation of STATs. may support the interaction or should be necessary gp130 (Matsuda et al., 1995a). It was shown that for the activation of the JAKs. Although JAK1, erbB2interactswithgp130andtheactivityoferbB2is JAK2, and TYK2 interact with and are activated by necessaryforERK/MAPkinaseactivationinprostate gp130, it is not clear which JAKs are involved in carcinoma cells (Qiu et al., 1998). The biological downstreamsignalingandbiologicalfunctionsandto significance of these tyrosine kinases remains to be what extent. elucidated. It was demonstrated that tyrosine phosphorylation of gp130 and STAT3 induced by soluble IL-6R(cid:11) and IL-6 was significantly reduced in a JAK1-deficient Cytoplasmic signaling cascades fibrosarcomacelllinebutnotinaJAK2-deficientcell line(Guschinetal.,1995).Thisisalsoconsistentwith data from knockout experiments. Several types of The current model of gp130-mediated signal trans- cells derived from JAK1-deficient mice did not duction is illustrated in Figure 5 (also see review by respond to the IL-6-related cytokines (Rodig et al., Hirano et al., 1997). Once the ligand is bound to the 1998), but JAK2-deficient fibroblasts and embryonic receptor complex, gp130 becomes a homodimer, and stem (ES) cells did respond to IL-6 and LIF, gp130-associated JAKs come close and transphos- respectively (Neubauer et al., 1998; Parganas et al., phorylate each other. It is believed that transphos- 1998). Furthermore, the embryonic lethal phenotype phorylation of JAKs activates its kinase activity. In of JAK1-deficient mice resembled that of gp130- the case of JAK2 and Tyk2, phosphorylation of the deficient mice (Yoshida et al., 1996; Rodig et al., twotyrosinesintheactivationloopofJAKswasshown 1998). Further analysis will be required to reveal to be required for tyrosine kinase activity (Gauzzi which JAK is involved in individual biological et al., 1996; Feng et al., 1997). The gp130-associated responses elicited by gp130. JAKs, once activated by the transphosphorylation, InadditiontoJanuskinase,severaltyrosinekinases phosphorylatetyrosinesinthecytoplasmicdomainof wereshowntointeractwithgp130directlyorthrough gp130.gp130containssixtyrosinesinthecytoplasmic the JAKs in certain cells. Hck, a Src-family tyrosine region (Y683, Y759, Y767, Y814, Y905, and Y915 in kinase,wasreportedtoassociatewithgp130,andwas humangp130;the numbersareindicatedaminoacids activated in response to LIF in ES cells (Ernst et al., from the translational initiation site). It is not known 1994).TecandBtk,whicharedistantmembersofthe which tyrosines are preferentially phosphorylated on Src-family tyrosine kinases and contain a pleckstrin stimulation. However, phosphorylated tyrosines on homology (PH) domain, were reported to associate gp130 are known to recruit signal transducing with gp130 through the JAKs and tyrosine phos- molecules such as SHP-2 and STATs (described phorylated upon the stimulation of gp130 (Matsuda below), resulting in the tyrosine phosphorylation of etal.,1995b;Takahashi-Tezukaetal.,1997).Feswas these molecules by the gp130-associated JAKs. also reported to interact with and was activated by Experiments of truncation and point mutations in IL-6 Receptor 1769 Figure 5 Model of gp130-mediated signal transduction. gp130 revealed the roles of individual tyrosine for Figure 6 Structure of SHP-2, Gab1, and Gab2. SHP-2 downstreamsignaling.Y683isnotsignificantlyphos- containstwoSH2andaphosphatasedomainsinitsN-and phorylated since a truncated mutant gp130, which C-terminal, respectively. Y (tyrosine) indicates possible possesses the membrane-proximal 68 amino acid phosphorylation sites. Both Gab1 and Gab2 have a pleckstrin homology (PH) domain, a c-Met-binding do- containing only Y683, could not be tyrosyl phos- main(MBD),andtyrosine-basedmotifsforSHP-2andp85 phorylatedinspiteofthetyrosinephosphorylationof PI-3 kinase. The region around the MBD contains the JAKs(Fukadaetal.,1996).PhosphorylationofY759 proline-rich PXXP motifs, which are known to bind the was shown to be required for tyrosine phosphoryla- SH3 domains. The white bars indicate predicted tyrosine- tion of SHP-2, a protein tyrosine phosphatase (Stahl phosphorylated sites, the surrounding sequences of which et al., 1995; Fukada et al., 1996; Yamanaka et al., fit with the consensus-binding sequences for Grb2, Crk, 1996). The four tyrosines (Y767, Y814, Y905, and and PLC(cid:13). Y915)in the C-terminushave a glutamineat position +3 of tyrosines (YXXQ) and phosphorylation of any one of these tyrosines has been shown to be required for tyrosine phosphorylation and activation of STAT3 (Stahl et al., 1995; Fukada et al., 1996; Gerhartz et al., 1996; Yamanaka et al., 1996). The two tyrosines in the carboxy-end (Y905 and Y915) among these four tyrosines have a proline at position +2 of tyrosines (YXPQ) and their phos- phorylation was further reported to be necessary for tyrosine phosphorylation of STAT1 (Gerhartz et al., 1996). In addition to SHP-2, STATs, and tyrosine kinases,thereareseveralproteinsthatarereportedto be tyrosyl phosphorylated on stimulation of gp130. TheseincludeShc(Boultonetal.,1994;Kumaretal., 1994),IRS-1(Burfootetal.,1997),Gab1(Takahashi- Tezuka et al., 1998), and Gab2 (Nishida et al., 1999). Tyrosine phosphorylation of STAT5, Gab1, and SHP-2 is a protein tyrosine phosphatase bearing Gab2 does not require tyrosine phosphorylation two SH2 domains in the N-terminal region and a of gp130 (Lai et al., 1995b; Fujitani et al., 1997; phosphatase domain in the C-terminal region Takahashi-Tezuka et al., 1998; Nishida et al., (Figure 6) (Adachi et al., 1996). In comparison to 1999). SHP-1,whichisanegativeregulatorforsignalingofa 1770 Masahiko Hibi and Toshio Hirano cytokinereceptor(Klingmulleretal.,1995)andBcell The mutation of Y759 disrupted the interaction antigen receptor (Blery et al., 1998; Maeda et al., between SHP-2 and Gab1, and Gab1 and PI-3 1998),SHP-2 is apositive regulatorof signaling from kinases, suggesting that Gab1-mediating signals are receptor tyrosine kinases and cytokine receptors dependent on Y759 of gp130. Gab1-dependent ERK (Tonks and Neel, 1996; Neel and Tonks, 1997). activation was shown to be inhibited by a dominant SHP-2 is tyrosyl phosphorylated on stimulation of negative p85, wortmannin, an inhibitor for PI-3 gp130 (Stahl et al., 1995; Fukada et al., 1996).SHP-2 kinases, or a dominant negative Ras (Takahashi- contains three to four YXNX (depending on splicing Tezuka et al., 1998). These results suggest that Gab variants) motifs, which is the consensus sequence for family proteins mediate signals from SHP-2 to PI-3 Grb2 binding, in its C-terminal region (Feng et al., kinases to activate ERK/MAP kinases. Further 1993; Vogel et al., 1993). Upon stimulation, tyrosine analysis will be required to clarify this point. phosphorylated SHP-2 was reported to interact with A negative role of SHP-2 in gp130 signaling has Grb2 (Bennett et al., 1994; Li et al., 1994; Fukada alsobeenreported.ThemutationofY759wasshown et al., 1996). Since Grb2 constitutively interacts with to prolong the activation of a STAT3-mediated Sos, the GDP–GTP exchange factor for Ras, SHP-2 pathway (Kim et al., 1998). Since gp130 and JAK1 may act as an adapter molecule mediating gp130 butnotSTAT3couldbesubstratesforSHP-2invitro signaling to the Ras pathway. The mutation of Y759 (Nishida et al., 1999), SHP-2 may distinguish gp130- to a phenylalanine abrogated tyrosine phosphoryla- mediated signals as a feedback mechanism. tion of SHP-2, the interaction between SHP-2 and STATsaretranscriptionfactorscontaininganSH2 Grb2, and activation of an ERK/MAP kinase, a domain(Figure4)(Darnelletal.,1994;IhleandKerr, major target of the Ras pathway, suggest a role for 1995). Upon stimulation, STAT1 and STAT3 are SHP-2 in the Ras pathway (Fukada et al., 1996). In recruitedongp130andtyrosylphosphorylatedbythe addition to the possible adapter function of SHP-2, gp130-associated JAKs (Akira et al., 1994; Zhong the catalytic activity of SHP-2 was shown to be et al., 1994; Guschin et al., 1995). Tyrosine 701 of necessary for transmitting signals to the ERK/MAP STAT1 and tyrosine 705 of STAT3 are phospho- kinasesinthecaseofreceptortyrosinekinasessuchas acceptor sites by JAKs. Phosphorylated STAT1 and receptorsforinsulin,EGF, andFGF(Noguchiet al., STAT3 form homodimers (STAT1/STAT1, STAT3/ 1994;Tangetal.,1995;Bennettetal.,1996).Ingp130 STAT3) or a heterodimer (STAT1/STAT3) through signaling, SHP-2 wasshown to interact with Gab1 or theinteractionbetweenthe phosphotyrosinesandthe Gab2 upon stimulation (Takahashi-Tezuka et al., SH2 domains. It was also reported that STAT5 1998; Nishida et al., 1999). Tyrosyl phosphorylated (STAT5a and STAT5b) is tyrosine phosphorylated Gab1 and Gab2 were shown to be dephosphorylated independently of tyrosine phosphorylation of gp130 by SHP-2 in vitro (Nishida et al., 1999). Gab1 and (Lai et al., 1995a; Fujitani et al., 1997). Tyrosine Gab2areadapterproteinswhichdisplayhomologyto phosphorylationofSTAT5byJAKswasshowntobe Drosophila Dos (daughter of sevenless) (Figure 6) mediated in part by a direct interaction between (Herbst et al., 1996; Raabe et al., 1996), a substrate STAT5 and the JH2 domain of JAKs (Fujitani et al., for corkscrew, a Drosophila homolog of SHP-2 1997). In addition to tyrosine phosphorylation of (Perkins et al., 1992). They contain a PH domain, STATs, serine phosphorylation of STAT1 and and proline-rich sequences including c-Met-binding STAT3 was required for full transcriptional activity domain (MBD), and tyrosine-based motifs that bind ofthemolecules(Boultonetal.,1995;Nakajimaetal., to SH2 domains when they are phosphorylated 1995; Wen et al., 1995). Serine 727 of both STAT1 (Holgado-Madruga et al., 1996; Weidner et al., and STAT3 is responsible for the phosphorylation 1996). site and their mutations were shown to reduce Tyrosine phosphorylation of Gab1 and Gab2 was STATs-dependent transcription (Wen and Darnell, independent of tyrosine phosphorylation of gp130 1997). Serine 727 is located prior to proline and fits a (Takahashi-Tezuka et al., 1998; Nishida et al., 1999). part of the consensus sequence of phosphoacceptor They are likely phosphorylated by the gp130-asso- sites by ERK/MAP kinase. In fact, serine 727 could ciated JAKs, since overexpression of JAKs induced be phosphorylated by ERK/MAP kinase in vitro their phosphorylation. Gab1 and Gab2 interacted (Wen et al., 1995). Upon growth hormone stimula- with not only SHP-2 but also the p85 PI-3 kinase tion, STAT3 is phosphorylated dependent on ERK/ on stimulation. Overexpression of Gab1 or Gab2 MAP kinases, whereas serine phosphorylation of was reported to enhance the gp130-dependent acti- STAT3inducedbyIL-6isindependentofERK/MAP vation of ERK/MAP kinases, suggesting that Gab kinase (Chung et al., 1997; Ng and Cantrell, 1997). family proteins act upstream of ERK/MAP kinases Several groups reported that the gp130-mediated (Takahashi-Tezuka et al., 1998; Nishida et al., 1999). serine phosphorylation of STAT3 was inhibited by a

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