Articulo 4

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Articulo 4

  1. 1. <br />Vijay Shankaran and Robert D. Schreiber*<br />Center for immunology and Department of Pathology. Washington University School of Medicine, 660 South Euclid Avenue, Mailstop 8118, St. Louis, MO 63108, USA<br />* corresponding author Tel: 314-362-8747, fax: 314-747-4888. e-mail: schreiber@immunology.wustl.edu DOI: 10.1006/rwcy.2000.18001.<br />SUMMARY<br />The IFNy receptor consists of two sub-units, IFNGR1 and IFNGR2, and is expressed nearly ubiquitously on a!3 cell surfaces. The 90kDa IFNGR1 glyco-protein is predominantly responsible for mediating high-affinity, species-specific ligand binding, ligand trafficking, and signal transduction. The 62kDa IFNGR2 glycoprotein plays a minor role in ligand binding but is required for signaling. Activated IFNy receptors utilize the JAK./STAT signaling pathway and specifically JAK1, JAK2, and STAT1 for mediating many IFNy-dependent effects on cells. A large number of immediate-early genes have been identified that are regulated through an [FNy- and STAT1-dependent mechanism. These genes contain common promoter elements known as GAS sites which function as target sites for activated STAT1 homodirners. Interference with the IFNy-dependent JAK/STAT signaling pathway (i.e. through the use of blocking antibodies or gene deletion) renders the host exquisitely sensitive to infection by a variety of microbial pathogens and certain viruses and also increases host susceptibility to tumors. Recently, patients suffering from rare mycobacteria! infections have been identified who have mutations in either IFNGR1 or IFNGR2. Thus the physiologic relevance of signaling through the IFNy receptor has been unequivocally established.<br />BACKGROUND<br />Discovery<br />The IFNy receptor was initially characterized in the early 1980s in radioligand-binding studies conducted<br />in several laboratories on a variety of different cell types (Anderson el al., 1982; Ceiada et a/., 1984, 1985; reviewed in Farrar and Schreiber, 1993), These experiments showed that most primary end cultured cells expressed a moderate level (250-25,000 sites/ceil) of high-affinity (ATa= 109-1010M" ') binding sites for IFNy. The interaction of IFNy with its receptor was not inhibited by other intcrferon classes, which explained the basis for the biologic specificity of IFNy. In addition, human and murine IFNy bound to their respective receptors in a strictly species-specific manner and thereby induced biologic responses only in species-matched cells. The latter observation proved to be critical in defining the sub-units of the functionally active IFNy receptor and in determining the structure-function relationships operative within each subunit.<br />A major step forward in defining the subunit composition of IFNy receptors came from key genetic experiments conducted in 1987 (Jung et al., 1987). These studies employed a family of stable rmirine-human somatic cell hybrids that contained the full complement of murine chromosomes and a random assortment of human chromosomes. Ail hybrids that contained human chromosome 6 bound human IFNy with high affinity - an observation that was later explained by the presence of the human IFNy receptor a chain gene on this chromosome (Pfizenmaier et al., 1988). However, biologic responsiveness to human IFNy was found only in hybrids that contained both human chromosomes 6 and 21. These observations, together with similar studies using hamster-murine somatic cell hybrids, led to the hypothesis that functionally active human or murine IFNy receptors consist of two (or more) species-matched subunits (Jung et a!., 1987; Hibino et al., 1991). The first is the receptor subunit responsible for binding ligand in a species-specific manner. The 1820 Vijay Shankaran and Robert D. Schreiber<br /> <br />second is a species-matched subunit that is required for induction of biologic responses.<br />This concept., was further refined-.,by independent reports-.in ;l_9;87ry8£( o;f. the,purification..of .the ligand-bindiijgscompqjieuti,p(','1Uie!..;huma.n,;I,FNY- receptor iAgu£t^d,^er;k^ .1^^^<br />ei a/., ,1988) 'and_4he;Subsequent-doning of its gene (Aguet et al., 1988). This event was followed one year later, by the .isolation of the gene encoding the murine homolog (Gray et al., 1989; Hemmi et-ai, 1985; JCumar ,et.tal,, .;19.89-;.....Ivjunro,and Manialis, 1989; Cofano et al., 1990). When the ligand-binding chains of the human or murine IFNy receptor were expressed al high levels in murine or human cells, respectively, the transfected cells bound human or murine ligand in a manner that was identical to endogenous receptors expressed on homologous cells. However, treatment of the transfected cells with heterologous ligand failed to effect induction of cellular responses. In contrast, when the human IFNy-binding protein was expressed in murine cells that also contained human-chromosome 21. these cells not only bound' the human ligand but also responded to it (Fischer et al., 1990a; Jung et al., 1990; Farrar et al., 1991). -These observations thus added significant support to the concept that functionally active IFNy receptors require a second, species-specific subunit. Definitive proof of this concept came in 1994 when the second subunit of both the human and murine IFNy receptors were simultaneously identified by two independent laboratories using complementation cloning approaches (Hemmi et ai, 1994; Soh et ai., 1994). Thus, functionally active IFNy receptors are now known to consist of two species-matched chains: a 90kDa pofypeplide responsible for mediating high-affinity Hgand binding, ligand trafficking through the cell and signal transduction, and a distinct 62kDa subunit which is responsible for signaling (Figure 1).<br />Alternative names<br />The nomenclature for the IFNy receptor subunits was recently established by the investigators in the field. Currently, the ligand-binding component of the IFNy receptor (originally denoted the IFNy receptor a-chain or IFNyRl) is now known as IFNGR1 or CDwll9. The second subunit, originally designated the IFNy receptor f3 chain, accessory factor-1 (AF-1) or IFNyR2, is now known as IFNGR2.<br />Structure<br />Functionally active IFNy receptors are comprised of two species-matched poiypeptide chains (Figure 1 and<br />Table 1). IFNGR1 is a 90kDa glycoprotein that plays important roles in [mediating ligand binding, ligand trafficking through, the cell, and signal transduction. IFNGR2 is a 62kDa glycoprotein that plays only a minor role in ligand binding but which is required in an obligatory manner for signaling. In-unstimulated cells the two receptor subunits are not tightly preassociated. with one another. Association is induced upon exposure of the cell to ligand and this association initiates the signal transduction process.<br />Siail binding<br />site when K phosphoryfaied P<br />472 Ser<br />IFNGR2<br />Figure 1 Poiypeptide chain structure of the human IFNy receptor. The IFNy receptor consists of two species-matched poiypeptide subunits. IFNGR1 is a 90kDa glycoprotein required for ligand binding, trafficking of ligand through the cell, and signal transduction. IFNGR2 is a 62kDa glycoprotein required predominantly for signaling. The intracellular domain of IFNGR1 contains two functionally critical sequences: first, an LPKS sequence that participates in the binding of JAK1 to IFNGR1, and second, a YDKPH sequence that, when phosphoryiated. forms the docking site on the activated receptor for STAT1. The intracelluiar domain of IFNGR2 contains a 12 amino acid sequence required for JAK2 association.<br />iFNGRJJFNGR2PropertyHuman MurineHumanMurinePrimary sequence^— ——————— — ————— — — ^ ———————— ^ _ _ . ———— _ ., „ —————— . — __^_5:gnaJ peptidei7aa 26aa.21 aaiSaaMature form472aa 451 aa3i6aa314aaHomology52%58%Chromosomal localization6 102116Domain structureExtracellular228 aa 228 aa226 aa224 aaTransmembrane23 aa 23 aa24 aa24 aaLntracellular221aa 200 aa66 aa66 aaPotential A- Jinked gJycosylatiolt Sites5 • $ -; •66Predicted Mt (JiDa) .••••.. '" " $&£• ; . :-., ' " ;%g. ,, ; ,34.8•; -.. 35.6Mature protein Mr (kDa)- ii : ' • gg •6J-67• . 60-65Conserved iniraceliulaf lyrosines53<br />Main activities and pafhophysioJogical roles<br />Analysis of IFNy receptor md IFNa//? receptor signaling led to the discovery pf a nove/ signaling pathway known as the JAK/S|" AT signaling pathway (reviewed in Darnel! et at., J994; IhJe et a/., J995; Schindler and Dameli, 1995; .t^onard and O'Shea, J998). This work has had far-reaching effects in understanding cyfokine receptor signaling in general because this signal transaction pathway was subsequently found to participate in the development of many biologic responses induced by a wide variety of different cytokines. The study of IFN receptor signaling led to the, identification of two classes of signaling proteins that comprised this pathway. One was a family of latent cytosolic transcription factors that eventually became known as STAT proteins (abbreviation, of signal transducers and activators of Iranscription) (Fu et al., 1992; Schindler et a!., 1992). The other was a family of structurally distinct proiein tyrosiiie fcfnases known as Janus family Jdnases or JAKs (Darnel! et al., 1994). Th'c unique feature of the JAK/'STAT signaling pathway was that receptor Jigation resulted in the activation of specific stibsels of Janus family protein tyrosine kinases (i.e. the JAKs) that subsequently tyrosine phosphorylated and activated cytosolic STAT proteins. The phos-phorvlated STAT proteins then dimerized and<br />translocated directly from the membrane to the nucleus and effected transcriptional activation of specific target genes. The events linking receptor Jigation with signal transduction were ultimately defined when IFNy was found to effect the tyrosine phosphorylation of the IFNy receptor a chain, thereby forming a specific docking site on the acii-vated receptor for a particular STAT family member, namely STATI (Greenhmd et al., 1994). Subsequent work generalized this finding by showing that other STAT family members were recruited to their distinct cytokine receptors by a similar mechanism.<br />Accession numbers<br />Human IFNGRI: J03143<br />Mouse IFNGRI: M2671I, M28233, M25764,<br />M28995<br />Human IFNGR2: U05877<br />Mouse IFNGR2: S69336<br />Sequence<br />See Figure 2.<br />J822 Vijay Shankaran and Robert D. Schreiber<br />Figure 2 Nucfeoiide sequences for human and mouse IFNGRI.<br />Human II-XGR3<br />GA AITCCGCA GGCGCTCGCGGTTGGAGCCAGCGA CCGTCGGTAGCAGCA TGGCTCTCCTCTTTCTCCTA CCCCTTGTCA TGCAGGGTGTGAGCAGGGCTG AGATGGGCA CCGCGGATCTGGGGCCGTCCTCAGTGCCTA CACCAACTA A TGTTA CAATTGAATCCTATA A CATGAACCCTATCGTA TA TTGGG AGTA CC AGATCATGCCACAGGTCCCTGrmTACCGTAGAGGTAAAGAACTATGGT GTTAA GA A'lTCAGA ATGGATTGA TGCCTCCATCA ATA TITCTCATCATTAT TGTA ATAT7TCTG ATCA TGTTGGTGA TCCA TCA AA TTCTCTTTGGGTCAGA GTTAAAGCCAGGG7TGGACAAAAAGAATCTGCCTATGCAAAGTCAGAAG A ATTTGCTGTATGCCGAGA TGGAAA AATTGGACCACCTA A ACTGGA TA TC AGAAA GGAGGAGA AGCA A ATCATGATTGACA TATTTCA CCC'TTCA GTTTT TGTAA A TGGAGA CG AGCAGGA AGTCGATTATGATCCCGA A ACTACCTGT TA CA TTA GGGTGTACA A1GTGTA TGTG AGAA TGAACGGA AGTGAGATCC AGTATA A A A TACTCA CGCA GA AGGA AGA'fGAH'GTGA CGAGA 'JTCAGTC CCAGTTAGCGA TTCCAGTA TCCTCA CTG A A1TCTCAGTACTGTGTTTCACC<br />AGAAGGAGTCTTACATGTGTGGGGTGTTAGAAC7GAAAAGTCAAAAGA; GTTTGTATI'ACCATTTTCAATAGCAGTATAAAAGGTTCTCTrTGGATTCCA GlTGTTGCTGCTTTACTACTCTTTCTAGTGCTTAGCCrGGTAlTCATCTGIT TTTA TA TTA AGA A A A TTAA TCCA TTG A AGGAA A AAAGCATA A TA TTA <br />CCC AAGTCCTTGATCTCTGrGGTAAGAAGTGCrACTTTAGAGACAAAACCTGA A TCA AAATATGTATCACTCATCACGTCATA CCAGCCATrTTCCTTAGA AA AGGAGGTGGTCTGTGAAGAGCCGTTGTCTCCAGCAACAGTTCCAGGCAT .GCATACCGAAGACAATCCAGGAAAAGTGGAACATACAGAAGAACTTTCT AGTATAACAGAAGTGGTGACTACTGAAGAAAATATTCCTGACGrGGrCC CGGGCAGCCATCTGACTCCAATAGAGAGAGAGAGTTCTTCACCTTTAAGT AGTAA CCAGTCTGA A CCTGGCAGCA TCGCTTTA A ACTCGTATCACTCCAG AAATTGTTCTGAGAGTG ATCA CTCCAGA AA TGGTTTTGA TACTGATTCCA GCTGTCTGGAATCACATAGCTCCTTATCTGACTCAGAATTTCCCCCAAAT AATAAAGGTGAAATAAAAACAGAAGGACAAGAGCTCATAACCGTAATA AAAGCCCCCACCTCCTTTGGTTATGATAAACCACATGTGCTAGTGGATCT ACTTGTGGATGATAGCGGTAAAGAGTCCTTGATTGGTTATAGACCAACAG A AGA TTCCA A AGA ATTTTCA TGAGA TCAGCTAAGTTGCACCA A CTTTGAA GTCTGATTTTCCTGGACAGmTCTGCnTAAnTCATGAAAAGATTATGA TCTGAGAAATTGTATCTTAGTTGGTATCAACCAAATGGAGTGACTTAGTG TACATGA AAGCGTAA AGAGGA TGTGTGGCA TTTTCACTTrrGGCTTGIA A AGTACAGACTTTrrTTITrnTTAAACAAAAAAAGCATTG7'AACTTATGAA CCTTTA CATCCAGATAGGTTACCAGTAACGGAA CATA TCCAGTACTCCTG G7TCCTAGGTGAGCAGGTGA TGCCCCAGGGACCnTGTAGCCACTTCACT TTTTrrCTnTCTCTGCCrTGGTA TA GCA TA TGTGTT7TGTA AGTTTA TGCA T ACAGTA ATTTTAAGTA A TTTCAGAAGA A ATTCTCGA AGCTTTTCA AA A TT<br />IFNy Receptor 1823<br /> <br />Figure 2 (Continued}<br /> <br />GGA CTTA A A ATCTA ATTCAAACTAATAGAATTA ATGGA ATA TGTA A A TA CA A A CGTGTATATTTTTTA TGA A ACATTACAGITAGAGATTTTTA A A TAA AGA A 7T7TA A A A CTC<br />Mouse IFNGK1<br />CGGCAGGCCGCTTGCGGACTTGGCGACTAGTCTGCGGCGGACGTGACGC CAAGGCCAGGCCACGGGCAGCGCGGGTCCCCTGTCAGAGGTGTCCCTGG CGCAGGAATGGGCCCGCAGGCGGCAGCTGGCAGGATGATTCTGCTGGTG GTCCTGATCCTGTCTGCGA A GGTCGGGAGTGGAGCTTTGACGAGCA CTGA GGATCCTGAGCCTCCCTCGGTGCCTGTA COGACGA ATGTTCTA A TTA ACT CTTAIAA CTTGA ACCCTGTCGTA TGCTGGGA ATACCAGAA CA TGTCA CAG ACTCCTA TTnTACTGTACAGGTA A AGGTGTA7TCGGGT TCCTGGACTGA T TCCTGCACGAA CATITCTGATCATTGTTCTA ATATCTATGA A CA A ATTATG TA TCCTGA TGTATCTGCCTGGGCCAGAGTTA AAGCTAAGGTTGGA CAA A A AGAATCTGACTArGCACGGTCAAAAGAGTTCCTTATG7GCCTAAAGGGA AAGGTCGGGCCCCCTGGCCTGGAGATCAGGAGGAAGAAGGAAGAACAG CrCTCCGTCCTCGTATTrCACCCTGAAGTCGTrGTGAATGGAGAGACCCA GGGAACCA TGTTTGGrGACGGGAGCA CCTGTTACACATTGGA CTATA CTG TGTATGTGGAGCATAACCGGAGTGGGGAGArCCTACATACGAAACATAC GGTCGA AA AAGAAGAGTGTAA TGAGA CrCTGTGTGAGTTAAACATCTCA GTATCCA CACTGG A rfCCAG ATA TTGTATTTCAGTAG ACGGA ATCTCATCT TrCTGGCA AGTTAGA A CAGA AA A ATCGA A AG A CGTCTGTATCCCTCC11T -CCATGATGA CAG A AAGGATTCA ATTTGGA TTC1GGTGGITGCTCCTCTTA C CGTCrrrACAGTAGTTATCCTGGTA TTTGCGTA1TGGTATA CTAAG AAGA A TTCATTCA AGAGAA AAAGCATA A TGTTA CCTAAGTCCTTGCTCTCTGTGG<br />Chromosome location and linkages<br />Human IFNGRI is encoded by a 30kb gene located on the long arm of chromosome 6 (Table I) (Pfizenmaier et al., 1988). The murine homolog is a 22kb gene present on chromosome 10 .(Mariano et al., J987). Both genes consist of seven exons. Exons 1-5 encode the receptor extracellular domain; exon 6 encodes a small portion of the membrane-proximal region of the extracellular domain and the transmembrane domain; and exon 7 encodes the entire intracelluJar domain. Transcription of the human and murine IFNGRI genes gives rise to mRNA transcripts of 2.3kb fFarrar and Schreiber, 1993).<br />The human JFNGR2 gene has been localized to chromosome 2lq22.l (Cook el al, 1994;<br />Soh ei al., 1994). The murine homolog resides on chromosome 16 (Table 1) (Hibino et al,, J99J). These syntenic chromosomal regions also contain the genes of several other IFN receptor family members, including the subunifs of the IFNa/B receptor (IFNAR1 and IFNAR2) and the nonligand-binding subunit of the IL-IQ receptor, originally denoted CRF2-4 (Lutfa.'ia et al., 1993; Cook et al., 1994).<br />At the present time, structural data are only available for the mouse JFNGR2 gene. This 17kb gene appears to consist of .seven exons. Transcriptional activation of the IFNGR2 gene results in the generation of an mRNA transcript of 1.8kb in human cells or 2kb in mouse ceils (Hemmi et a/., 1994; Soh eta/., 1994),<br />!?2- Viiav Shankaran and Robert D. Schreibcr<br />Figure 2 (Continued)<br />TAAAAAGTGCCACGTTAGAGACAAAACCTGAATCGAAGTATTCACTTGT<br />CACACCGCACCAGCCAGCTGTCCTAGAGAGTGAGACGGTGATCTGTGAA<br />GAGCCCCTGTCCACAGTGACAGCTCCAGACAGCCCCGAAGCAGCAGAAC<br />AGGAAGAACTITCAAAAGAAACAAAGGCTCTGGAGGCTGGAGGAAGCA<br />CGTCTGCCATGACCCCAGACAGCCCTCCAACTCCGACACAAAGACGCAG<br />CTTrTCCCTGTTAAGTAGTAACCAGTCAGGCCCTTGl'AGCCTCACCGCCTA<br />TCACTCCCGAAACGGCTCTGACAGTGGCCTCGTGGGATCGGGCAGCTCC<br />ATATCGGACITGGAATCTCTCCCAAACAACAACTCAGAAACAAAGATGG<br />CAGAGCACGACCCTCCACCCGTGAGAAAGGCCCCCATGGCCTCCGGTTA<br />TGACAAACCGCACATGTTGGTGGACGTGCITGTGGATGrrGGGGGGAAG<br />GAGTCTCTCATGGGGTATAGACTCACAGGAGAGGCCCAGGAGCTGTCCT<br />AAGGTCTCCCGAGGCCrGCTGGTGGTAAAGAAACTGACCTTTTAGGCAGT<br />'nTTCTGCArrGATTTCATGAAAGAAGCTATACAITAGCTAATACTAACCA<br />CATAGAATATCAGACTTAGATACGTGAATAAGGATCCTGTGGGCACTGCT<br />GGGTCCACTCTGCAAATGCCAAGACTATCAAAGGAACGTATTGTCGGTTC<br />TGGCTCCTTCCCAGGTGGGCTAGCATCTGTGAGTTTGCCTCGGCTAGCCTT<br />GCrrCCTACAGCCGCCACTGCTCCTCCACCCTGATCATCTCACAGGACAG<br />GGTGGACCGGGTTTlTrTTTTTTTCACACACCTTTGTATATGTAAGTTCATG<br />TATATAATATGTTTACATGTTTCACTTTGAACTGAAAGCTACTCAAAGCCA<br />GCCGTAAGTCTATGGTAGAATGTGATGGAACATGTTGGTGGAAGCTTGTA<br />CAATAGAACACATTGGTGGGAGCTTGTACATACTTTrTTATGGAGCATTA<br />CTTACGATTTfTTAAGTAAAATGTTrrGAAACCAAAAAAAAAA<br />PROTEIN<br />Accession numbers<br />Human IFNGRl: PI5260 Mouse IFNGRl: P15261 Human IFNGR2: P38484 Mouse IFNGR2: A49947<br />Sequence<br />See Figure 3.<br />Description of protein<br />The human and murine IFNGRl proteins are organized in a similar manner and are symmetrically<br />oriented around a single transmembrane domain (Figure 1 and Table 1) (Aguet et ai, 1988; Gray el at., 1989; Hemmi et al., 1989; Kumar et al., 1989; Munro and Maniatis, 1989; Cofano et al., 1990). The mature proteins consist of 472 and 451 arnino acids, respectively, and have predicted molecular masses of 52.5 and 49.8kDa. Both proteins are symmetrically oriented around single 23 amino acid transmembrane domains. Each possesses a 228 amino acid extracellular domain that contains 10 cysteine residues and five potential AMinked glycosylation sites. Based on biosynthetic labeling experiments, all five glycosyia-iion sites appear to be occupied (Hershey and Schreiber, 1989; Mao et a!,, 1989; Fischer et al., I990b) and AMinked oligosaccharides contribute approximately 25kDa to the apparent molecular mass of the fuiiy mature protein. The size of the fully mature IFNGRl derived from different cell types ranges from 85 to 105 kDa depending on the extent of<br />IFNy Receptor 1825<br /> <br /> <br />Figure 3 Amino acid sequences for human and mouse IFNGR1 and IFKGR2.<br />Human IFNGR1<br />MALLFLLPLVMQGVSRAEMCTADLGPSSVPTPTNYTIESYNMNPIYYWEYQ1<br />MPQVPVFTVEVKNYGVKNSEWIDACINISHHYCNISDHVGDPSNSLWVRVKA<br />RVGQKESAYAKSEEFAVCRDGKiGPPKLDIRKEEKQIMIDIFHPSVFVNGDEQE<br />VDYDPETTCYTRVYNVYVRMNGSEIQYKILTQKEDDCDEIQCQLAIPVSSLNSQ<br />YCVSAEGVLHVWGVTTEKSKEVCmFNSSIKG5LVvfIPVVAAL,LLFLVLSLVFlCF<br />YIKKINPLKEKSIILPKSLISVVRSATLETKPESKYVSUTSYQPFSLEKEVVCEEPLS<br />PATVPGMHTEDNPGKVEHTEELSSITEVVTTEENIPDVVPGSHLTPIERESSSPLS<br />SNQSEPGSIALN5YHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEI<br />KTEGQELlTVIKAPTSFGYDKPHVLVDLLVDDSGKESLiGYRPTEDSKEFS<br />Mouse IFNGR1<br />MGPQAAAGRMILLVVLMLSAKVGSGALTSTEDPEPPSVPVPTNVLIKSYNLNP<br />VVCWEYQNMSQTPIFrv'QYKVYSGSWTDSCTNISDHCCNIYGQlMYPDVSAW<br />ARVKAKVGQKESDYAR3KEFLMCLKGKVGPPGLEIRRKKEEQLSVLVFHPEVV<br />VNGESQGTMFGDGSTCYTFDYTVYVEKNRSGEILHTKHTVEKEECNETLCEL<br />NISVSTLDSRYCISVDGISSFWQVRTEKSKDVCIPPFHDDRKDSIW1LVVAPLTVF<br />TVVILVFAYWYTKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYSLVTPHQPA<br />VLESETVICEEPLSTVTAPDSPEAAEQEELSKETKALEAGGSTSAMTPDSPPTPT<br />cell-specific glycosylation (Hershey find Schreiber, 1989; Mao el a!., 1989; Fischer el a!., 1990b).<br />The human and murine IFNGR2 proteins are also structurally similar to one another (Figure 1 and Table 1) (Hemmi et al., 1994; Soh et al., 1994). The mature human protein consists of a 226 arnino acid extracellular domain, a 24 amino acid transmembrane domain, and a 66 amino acid intracellular domain. It has a predicted molecular mass of 34.8 kDa. The mature marine equivalent has an extracellular domain that is two amino acids shorter. The predicted molecular mass of murine 1FNGR2 is 35.6kDa. The human and murine proteins contain five and six N-linked glycosylation sites, respectively, arid both are heavily glycosylated. Glycosylation contributes to the significant size heterogeneity seen in the mature proteins, even when derived from the same cell. The fully mature human protein displays Mr values that range from 61 to 67 kDa, while mature forms of the mouse<br />equivalent display Mt values of 60-65 kDa (Bach etui., 1995).<br />Relevant homologies and species differences<br />Examination of the extracellular domains of both IFNGR1 and 1FNGR2 reveal that they belong to a small family of cytokine receptors known as the class 2 cytokine receptors. The other members of this protein family include the two subunits of the IFNa/,5 receptor (IFNAR1 and IFNAR2), the two subunits of the IL-10 receptor, and tissue factor (Bazan, 3990). The members of this protein family share a similarly organized 210 amino acid-binding domain which contains conserved cysteine pairs at both N- and C-termini. The class 2 receptor family appears to be<br />1826 Vijay Shankaran and Robert D. Schreiber<br />Figure 3 (Continued)<br />QKRSFSLLSSNQSGPCSLTAYHSRNGSDSGLVGSGSSISDLESLPNNNSETKMAE HDPPPVRKAPMA5GYDKPHMLVDVLVDVGGKESLMGYRLTGEAQELS<br />Human IFNGR2<br />MRPTLLWSLLLLLGVFAAAAAAPPDPLSQLPAPQHPKIRLYNAEQVLSWEPV<br />ALSNSTRPVVYRVQFKYTDSKWFTADIMS1GVNCTQ1TATECDFTAASPSAGFP<br />MDFNVTLRLRAHLGAULSAWVTMPWFQHYRNVTVGPPENIEVTPGEGSLIIR<br />FSSPFDIADTSTAFFCYYVHYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQV<br />QAQLLWNKSNIFRVGHLSNISCYETMADASTELQQVILISVGTFSLLSVLAGAC<br />FFLVLKYRGUKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSI<br />ISFPEKEQEDVLQTL<br />Mouse IFNGR2<br />MRPLPLWU'SLLLCGLGAAASSFDSFSQLAAPLNFRLHLYNDEQILTWEPSPSS<br />NDPRPVVYQVEYSFIDGSWHRLLEPNCTDITETKCDLTGGGRLKLFPHPFTVFL<br />RVRAKRGNLTSKWVGLEPFQHYEKVTVGPPKNISVTPGKGSLVIHFSPPFDVF<br />HGATFQYLVHYWEKSETQQEQVEGPFKSNS1VLGNLKPYRVYGLQTEAQLILK<br />NKKIRPHGLLSNVSCHETTANASARLQQVILIPLGIFALLLGLTGACFTLFLKY<br />QSRVKYWFQAPPNIPEQIEEYLKDPDQFILEVLDKDGSPKEDSWDSVSIISSPEK<br />ERDDVLQTP<br />only distantly related structurally to the much larger class S cytokine receptor famiiy.<br />IFNGR1 and IFNGR2 display a strict species-specificity in their ability to bind and respond to ligand, i.e. the human IFNy receptor binds and responds on!y to human and not lo murine IFNy while the murine IFN-y receptor binds and responds only to the murine ligand (reviewed in Farrar and Schreiber, 1993). The sequence identity between human and murine IFNGR1 is only 52% overall (50% identity between the extracellular domains and 55% identity between the intracelluiar domains). Human and murine IFNGR2 exhibit 58% identity overall but show greater identity (73%) within their intracelluiar domains (Hernmi et a!., 1994; Son et ai, 1994). The species-dependent sequence differences within the receptor subunits are the reason for the species-restricted interactions between the receptor<br />subunits with ligand and with one another. In contrast, the functionally critical regions within the intracellular domains of each subunit have been preserved between mice and humans and thus no species-specificity is observed in the interactions of the IFNy receptor subunits with the intracelluiar proteins required for signal transduction.<br />Affinity for ligand(s)<br />Tmmunochcmical and radioligand binding experiments on intact ceils indicate that the IFNy receptor binds ligand with a single high affinity (Ka) of 10y-lO^M'1 (Farrar and Schreiber, 1993). The majority of this affinity is derived from the interaction of<br />1826 Vijay Shankaran and Robert D. Schreiber<br />Figure 3 (Continued)<br />QKRSFSLLSSNQSGPCSLTAYHSRNGSDSGLVGSGSSISDLESLPNNNSETKMAE HDPPPVRKAPMA5GYDKPHMLVDVLVDVGGKESLMGYRLTGEAQELS<br />Human IFNGR2<br />MRPTLLWSLLLLLGVFAAAAAAPPDPLSQLPAPQHPKIRLYNAEQVLSWEPV<br />ALSNSTRPVVYRVQFKYTDSKWFTADIMS1GVNCTQ1TATECDFTAASPSAGFP<br />MDFNVTLRLRAHLGAULSAWVTMPWFQHYRNVTVGPPENIEVTPGEGSLIIR<br />FSSPFDIADTSTAFFCYYVHYWEKGGIQQVKGPFRSNSISLDNLKPSRVYCLQV<br />QAQLLWNKSNIFRVGHLSNISCYETMADASTELQQVILISVGTFSLLSVLAGAC<br />FFLVLKYRGUKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSI<br />ISFPEKEQEDVLQTL<br />Mouse IFNGR2<br />MRPLPLWU'SLLLCGLGAAASSFDSFSQLAAPLNFRLHLYNDEQILTWEPSPSS<br />NDPRPVVYQVEYSFIDGSWHRLLEPNCTDITETKCDLTGGGRLKLFPHPFTVFL<br />RVRAKRGNLTSKWVGLEPFQHYEKVTVGPPKNISVTPGKGSLVIHFSPPFDVF<br />HGATFQYLVHYWEKSETQQEQVEGPFKSNS1VLGNLKPYRVYGLQTEAQLILK<br />NKKIRPHGLLSNVSCHETTANASARLQQVILIPLGIFALLLGLTGACFTLFLKY<br />QSRVKYWFQAPPNIPEQIEEYLKDPDQFILEVLDKDGSPKEDSWDSVSIISSPEK<br />ERDDVLQTP<br />only distantly related structurally to the much larger class S cytokine receptor famiiy.<br />IFNGR1 and IFNGR2 display a strict species-specificity in their ability to bind and respond to ligand, i.e. the human IFNy receptor binds and responds on!y to human and not lo murine IFNy while the murine IFN-y receptor binds and responds only to the murine ligand (reviewed in Farrar and Schreiber, 1993). The sequence identity between human and murine IFNGR1 is only 52% overall (50% identity between the extracellular domains and 55% identity between the intracelluiar domains). Human and murine IFNGR2 exhibit 58% identity overall but show greater identity (73%) within their intracelluiar domains (Hernmi et a!., 1994; Son et ai, 1994). The species-dependent sequence differences within the receptor subunits are the reason for the species-restricted interactions between the receptor<br />subunits with ligand and with one another. In contrast, the functionally critical regions within the intracellular domains of each subunit have been preserved between mice and humans and thus no species-specificity is observed in the interactions of the IFNy receptor subunits with the intracelluiar proteins required for signal transduction.<br />Affinity for ligand(s)<br />Tmmunochcmical and radioligand binding experiments on intact ceils indicate that the IFNy receptor binds ligand with a single high affinity (Ka) of 10y-lO^M'1 (Farrar and Schreiber, 1993). The majority of this affinity is derived from the interaction of<br />iFNy Receptor 1827<br /> <br /> <br />IFNGR1 with the hgand which is characterized by a A'., of lOMo^M" 1. Using iigand-binding assays, sucrose density gradient ultracentrifugation, and HPLC ge! filtration chromatography, a soluble form of the 1FNGK1 cxiracellular domain (sECD) was found to form stable complexes in free solution with IFNy that consisted of 1 mole ligand and 2 rnole soluble receptor (Fountoulakis et a/., 1992; Greenlund et al.. 1993). Formation of the 2:1 (receptor-ligand) complex was also demonstrated on cell surfaces using either chemical crosslinking or irnmunochemical approaches. The crystal structure of the IFNy-lFNy receptor complex has been solved and confirms the structure predicted by the biochemical approaches (Walter er al., 1995).<br />Deletional mutagenesis analysis of the soluble IFNGR1 extracellular domain showed that the majority of the sECD (residues 6-227) was required for expression of Iigand-binding activity (Founloulakis el al., 1991). However, by exchanging corresponding regions between the human and murine IFNGR1 extracellular domains, several important internal sequences were identified throughout the extracellular domain that contributed to the species-specificity of the Iigand-binding process (Axelrod et al., 1994). Moreover, this study also revealed the presence of distinct regions within IFNGR1 that played an obligate role in biologic response induction, but not in ligand binding. One explanation for the latter observation is that the functionally important sequences may contribute to the interaction between the IFNGR1 and IFNGR2 subunits.<br />The contribution of IFNGR2 to the Iigand-binding process has also been established (Marsters et al., 1995). Using an experimental system where the two human IFNy receptor subunits were expressed either individually or together in murine fibrobiasts, no direct interaction was detected between human IFNy and human IFNG R2. However, when human IFNG R2 was expressed in murine cells that also expressed human IFNGR1, IFNy binding was increased 4-fold over that observed on cells that expressed the human IFNGR1 chain alone. Thus, one function of IFNGR2 is to stabilize the complex formed between ligand and the IFNGR1 subunit.<br />Cell types and tissues expressing the receptor<br />On the basis of immunochemical, radioligand binding, and molecular genetic analyses, IFNGR1 is ubiquitously expressed on nearly all cells (except erythrocytes)<br />(Farrar and Schreiber. 1993). Even pSatelets express IFNy receptors at a level of 300 receptors/cell. Considering the large number of platelets in the circulation (3 x 108/mL), it is possible that this cell plays an important role in transporting IFNy through the circulatory system. It is noteworthy that when receptor expression in different tissues is analyzed at either the mRNA or protein levels, the highest expression is observed in tissues which are not generally considered to have primary immunologic functions. Specifically, skin, nerve, and syncytial trophoblasts of the placenta express levels of IFNy receptor that are often 10-100 times that observed in spleen or on hematopoietic cells. Expression patterns of the IFNGR2 gene generally follow those of the IFNGR1 gene, except in T lymphocytes.<br />Regulation of receptor expression<br />In most cell types. IFNGR1 expression is constitutive and unregulated. The 5' flanking regions of the IFNGR1 gene contains a GC-rich region with no TATA box like that of promoters for noninducible housekeeping genes (Pfizenmaier el al., 1988; Merlin et «/., 1989). This observation suggests that expression of IFNGR1 is not regulated by external stimuli, a result that has been largely confirmed experimentally. Nevertheless, expression of the fully mature IFNGR1 protein at the plasma membrane varies widely between tissues (250-25,000 sites/cell). However there does not appear to be a direct correlation between the extent of receptor a chain expression and the magnitude of IFN-y-induced responses in cells (Farrar and Schreiber, 1993). Following IFNy receptor ligation, the IFNGR1-ligand complex is internalized and enters an acidified compartment. Within this compartment, the complex dissociates and free IFNy is trafficked to the lysosome where it is degraded. In many cells, such as fibrobiasts and macrophages, the uncoupled IFNGR1 enters a large intracellular pool of 1FNGR1 subunits and eventually recycles back to the cell surface. In most cells, the size of the intracellular pool is approximately 2-4 times that of the receptors expressed at the cell surface (Anderson et al., 1983; Celada and Schreiber, 1987; Finbloom, 1988; Hershey and Schreiber, 1989; Farrar et al., 1991).<br />Little is known about the intracellular trafficking of IFNGR2. However, in cenain cells (such as T lymphocytes), IFNGR2 expression is altered in a stimulus-linked manner. Several potential binding sites for a variety of externally regulated activated transcription factors have been identified within the 5 flanking region of the IFNGR2 gene (Ebensperger<br />1828 Vijay Shankaran and Robert D. Schreiber<br /> <br />el a!.. 1996). This observation suggested that transcription of the IFNGR2 gene may be tightly regulated. In fact, this hypothesis has been supported experimentally by the observation of differentia! expression of IFNGR2 on distinct murine helper T ceil subsets. Based on the observation that murine CD4+ T helper cell subsets differed in their ability to respond to IFNy (Gajewski and Fitch, 1988), two independent groups demonstrated in 1995 that the IFNy-unresponsive state seen exclusively in TH1 cells was due to a lack of cellular expression of IFNGR2 (Bach et a!., 1995; Pernis el al., 1995). Unrespon-siveness was shown to be a result of IFNy-dependent IFNGR2 downregulation and was not linked to T cell differentiation (Bach et a!., 1995). Thus, mouse TH1 cells, which produce IFNy, downregulate expression of IFNGR2, become IFNy-unresponsive, and thus circumvent the growth-inhibitory effects of the cylo-kine that they produce. In contrast, TH2 ceils, which do not produce IFNy, express IFNGR2 and remain IFNy-responsive. However, IFNGR2 downregulation could aiso be induced in murine TH2 cells, and also in human peripheral blood T cells, upon exposure to IFNy (Bach et a!., 1995; Sakatsume and Finbloom, 1996). Interestingly, ligand-induced IFNGR2 downregulation did not occur in certain fibroblast cell lines. Thus, IFNy appears to regulate expression of 1FNGR2 on certain cell types and thereby determines the ability of these cells to respond to subsequent exposure to IFNy. Recently, treatment of T cells with phorbol esters or with CD3 antibodies has been shown to effect induction of IFNGR2 mRNA (Sakatsume and FinbJoom, 1996). Taken together, these results demonstrate that IFNGR2 expression can be regulated either negatively or positively in a stimulus-specific manner.<br />SIGNAL TRANSDUCTION<br />Associated or intrinsic kinases<br />Like all members of the class 1 and class 2 cytokine receptor families, the intracellular domains of the IFNy receptor subunits do not express intrinsic kinase activity. However, distinct sequences within each intracellular domain function as the specific binding sites on the receptor for two members of the Janus family of protein tyrosine kinases. IFNGRE associates with JAKl and IFNGR2 associates with JAK2. The association of the IFNy receptor subunits with distinct Janus kinases was revealed by three lines of evidence. First, using-human fibrosareoma cells that<br />were mutated and selected for lack of responsiveness to both type I 1FN and IFNy, two of the Janus kinases - JAKl and JAK.2 - were found to be required for induction of IFNy-dependent biologic responses (Pellegrini el al., 1989; McKendry et al., 1991; Muiler et al., 1993; Walling et al., 1993). U1A cells that lack TYK2 did not respond to IFNa but responded normally to IFNy while ylA cells that lack JAK2 responded to IFNy but did not respond to IFNy. In contrast, U4A cells that lack JAKl responded to neither type I 1FN nor to IFNy. Responsiveness was restored in each cell Sine following transfection with plasmids encoding each of the missing JAKs. Thus IFNy signaling, at icast in this human fibrosareoma line, required the concomitant presence of JAKl and JAK2, while IFNa//? signaling required the concomitant presence of JAKl and TYK2.<br />Second, using a combination of deletion and substitution mutagenesis approaches, distinct sequences within the intracellular domains of IFNGR1 and IFNGR2 were identified that acted as specific binding sites on the receptor subunits for JAKl and JAK2 (reviewed in Farrar and Schreiber, 1993; Bach et al., 1997). In IFNGR1, a membrane proximal Leu-Pro-Lys-Ser (LPKS) sequence residing at positions 266-269 in the intracellular domain was found to function as the constitutive binding site for JAKl (Farrar et al., 1991; Greenlund et al., 1994). Replacement of this sequence with four alanine residues or substitution of alanine for proline at position 267 produced mutant IFNGR1 proteins that first, did not associate with JAKl; second, did not support ligand-induced activation of protein tyrosine kinase activity; and ihird, did not promote development of IFNy-dependent cellular responses (Kaplan et al., 1996). Coprecipitation studies coupled with biologic function analyses performed on ceils treated with either buffer or IFNy revealed that, whereas inactive JAKl constitutively associates with the IFNy receptor a chain, its activation is ligand-dependent. Similar studies conducted on the IFNGR2 subunit demonstrated that a 12 amino acid sequence (263-PPSIPLQIEEYL-274) located 13 amino acids away from the membrane in IFNGR2 functioned as the major site of attachment for JAK2 (Bach et al., 1996). Mutation within this region produced an IFNGR2 that was unable to interact with JAK2 and failed to support IFNy-dependent biologic response induction. Third, disruption of the genes encoding JAKl or JAK2 in mice led to IFNy unresponsiveness in primary cells derived from these animals (Neubauer et al., 1998; Paraganas et al., 1998; Rodig et al., 1998). In contrast, cells derived from JAK3-/-mice and cells lacking TYK2 responded normally to IFNy.<br />IFNy Receptor 1829<br /> <br /> <br />The role of STAT1 in IFNy signaling was also revealed by three experimental approaches. First, IFNy treatment of cells was shown to result in the selective activation of only one member of the STAT protein family, namely STAT1 (Fu et ai, 1992; Schindler et ai, 1992; Shuai et at., 1992). Following addition of IFNy to receptor-bearing cells, STATI was rapidly (30 seconds to 5 minutes) phosphorylated on a single tyrosine residue residing ai position 701, forming a homodimer that translocated to the nucleus where it associated with the promoter regions of IFNy-inducible genes and promoted gene transcription. IFNy-induced tyrosine phosphorylation of STAT1 required the presence in the cell of both JAK1 and JAK.2, a result that directly linked the JAKs to the STATs.<br />Second, structure-function analyses performed on IFNGR1 revealed the presence of a functionally critical five ami no acid region located &i positions 440-444 near the C-terminus that contains the residues Tyr-Asp-Lys-Pro-His (YDKPH) (Farrar et ai, 3992). The tyrosine residue at position 440 in human IFNGR1 (Tyr420 in murine IFNGRI) was found to be a major substrate site for the activated JAKs (Greenlund ei ai, 1994; Igarashi et ai, 1994). Once phosphorylated, the Y(PO4)DKPH sequence functioned as the attachment site on the activated receptor for STAT1. Mutationai analysis of these five residues demonstrated that only Tyr440, Asp441, and His444 were functionally important. IFNGRI harboring alanine substitutions at any of these three residues failed to activate STATi and failed to induce 1FNV dependent cellular responses. Moreover, STATI bound to peptides containing the Y(PO4)DKPH sequence (but not to unphosphorylated forms of the peptides) in a specific and high-affinity manner and phosphopeptides containing this specific sequence were able to inhibit IFNy-dependent STATI activation in a broken celi experimental system (Greenlund et ai. 1995). Using antibodies specific for different regions of the STATI protein and STAT1/STAT2 chimeric proteins, the SH2 domain of STATI was shown to be responsible for mediating association with the phosphorylated receptor sequence (Greenlund et ai, 1995; Heim et ai, 19'95).<br />Third, cells from mice lacking the STATI gene failed to respond to IFN-y (and also failed to respond to IFNa/,3) and the mice themselves were highly susceptible to infection with a wide variety of microbial pathogens and viruses (Durbin et ai, 1996; Meraz et ai, 1996). In addition, STATI-deficient mice were more sensitive than their wild-type counterparts to tumor formation induced by chemical carcinogens (Kaplan et ai, 1998). However, STATI-deficient mice showed no other defects that could be attributed to<br />signaling through other cytokine receptors and mice lacking other STAT proteins (i.e. STAT2, 3, 4, 5a, 5b, and 6) responded normally to IFNy. Thus, under physiologic conditions STATI plays a dedicated role in promoting IFN signaling and most IFN-dependent biologic responses require the participation of STATI.<br />Cytoplasmic signaling cascades<br />Based on the results discussed above, it has been possible to formulate a relatively complete model of the IFNy receptor signaling process (Figure 4) (Bach et ai, 1997). In unstimulated cells, the IFNy receptor subunits are not preassociated with each other but rather associate through their intracellular domains with inactive forms of specific Janus family kinases. JAK1 associates with the IFNGRI subunit by binding to the 266-LPKS-269 sequence and JAK2 constitutively associates with the 263-PPS1PLQIE-EYL-274 sequence in IFNGR2. Addition of IFNy, a homodimeric ligand, to the cells induces the rapid dimerization of 1FNGR1 subunits, thereby forming a site that is recognized, in a species-specific manner, by the extracellular domains of IFNGR2 subunits. The Hgand-induced assembly of the complete receptor complex containing two IFNGRI and two 1FNGR2 subunits brings into close juxtaposition the intracel-Sular domains of these proteins together with the inactive JAK enzymes that they carry. In this complex, JAK1 and JAK2 transactivate one another and then phosphorylate the functionally critical Tyr440 residue on IFNGRI, thereby forming a paired set of STATI docking sites on the activated receptor. Two STATI molecules then associate with the paired docking sites, are brought into close proximity with receptor-associated, activated JAK enzymes, and are activated by phosphorylation of the STATI TyrVOl residue. Tyrosme-phosphorylated STATI molecules dissociate from their receptor tether and form homodimeric complexes. The activated STATI complex is then phosphorylaled on a specific C-terminal serine residue (Ser723) (Wen et ai, 1995). Recent reports suggest that the serine phosphorylation is mediated by an as yet undefined enzyme with MAP kinase-like specificity (David ef ai, 1995; Wen et ai, 1995). Activated STATI translocates to the nucleus and, after binding to a specific sequence in the promoter region of immediate-early IFNy-inducible genes, effects gene transcription.<br />This signaling pathway is controlled by five distinct mechanisms. First, because the pathway is driven by<br />IS30 Viiav Shankaran and Robert D. Schreiber<br /> <br />Ubiquiyin-Proteasome<br />Degradation<br />PIAS<br />Stat! serine pliosptiorylalJon, nuclear translation and initiation of gene transcription<br />xxxxxxxx<br />Figure 4 Signaling through the IFNy receptor. The details of this process are described in the text. In unstimulated cells, inactive forms of JAKl and JAK2 associate in a constitutive manner with the IFNGR1 and IFNGR2 receptor subuniis, respectively. Upon ligand addition, the subunits ohgomerize, leading to the transactivation of JAKl and JAK2. The activated kinases then^phosphoryiate Y440 of IFNGR1, forming a paired set of docking sites on the activated receptor for STATI. Two STAT1 proteins bind to the receptor, become tyrosine and serine phos-phorylated and then dimerize, translocate to the nucleus, and initiate IFNy-dependem gene transcription, This process is regulated by phosphatases, kinase inhibitors, STAT inhibitors, and by degradation and/or transcriptional silencing of the receptor subunits and signaling proteins. The sites of regulation in the pathway are noted.<br />tyrosine phosphorylation, protein tyrosine phosphatases were considered obvious candidates capable of regulating IFNy receptor signaling. In fact, there is some evidence that suggests that both SHP-1 and SHP-2 may play a role in pathway regulation (You and Zhao, 1997; You el al., 1999). Second, a family of proteins was identified that regulate the activation of the janus kinases. This family is known as suppressors of cytokine signaling (SOCS), JAK-binding proteins (JABs), or STAT-induced STAT inhibitors (SSIs) (Endo el a!., 1997;Nakaefa/., 1997;Starr eta!., 1997). Theseproteins bind to activated JAKs and inhibit their catalytic activity. There are eight members of this family that participate in regulating the JAK/STAT signaling pathway and another 12 members whose function remains unknown (Hiltone/a/., S998).<br />Interestingly, mice lacking SOCS-1 die within 3 weeks of age of a massive inflammatory disease (Naka ei at., T998; Starr et al, 1998). This disease is prevented if the SOCS-1 deficiency is bred into an !FNy-deficient background (Alexander et a!.. 1999). Thus, SOCS-1 appears to be important in regulating signaling through the IFNy receptor pathway. The third family of proteins, known as proteins that inhibit activated STATs (PIAS), act downstream of the receptor and function by binding to activated STAT complexes and inhibiting their capacity to bind DNA (Chung et ai, 1997; Lm et al., 1998). Two family members have been characterized thus far. One of these, termed PIAS1, binds selectively to STATI homodimers and thus is likely to play an important role in controlling IFNy receptor signaling. The fourth proposed mechanism is one in which components of the IFNy signaling pathway are marked for degradation by the proteasome via ubiquination. Whereas one group showed that an inhibitor of the proteasome (MG132) stabilized phos-phorylated STATI in IFNy-treated HeLa ceils, another group showed that STATI molecules quantitatively cycle between the cytosol and nucleus as nonphosphorylated and phosphorylated proteins, respectively {^iaspe! et ai, 1996; Kim and Maniatis, 1996). Thus, it remains unclear how much this pathway contributes to signaling regulation under physiologic conditions. Finally, as discussed above, IFNy receptor signaling can be regulated in certain cells by mechanisms involving expression of the receptor IFNGR2 subunit (Bach et al, 1995; Pernis et a/,, 1995).<br />Thus, IFNy signaling is an ordered, affinity-driven, and highly regulated process that derives its specificity from first, the specific binding of a particular STAT protein, i.e. STATI, to a defined, ligand-induced but transiently expressed docking site on the activated receptor, and second, the ability of the<br />IFNy Receptor 1831<br /> <br /> <br />STAT1 homodimer specifically to activate IFN'y-induced genes.<br />DOWNSTREAM<br />ACTIVATION<br />Genes induced<br />The rapid signaling of the JAK/STAT pathway makes it an ideal system to regulate the activation of immediate-early genes and thus provides the host with a rapid mechanism to respond to an infectious agent. In fact, over the years it has been possible to identify a large number of IFNy-stimulated genes that are induced rapidly (within 15-30 minutes) after IFNy treatment of cells, and whose transcription does not depend on new protein synthesis (KLerr and Stark, 1991; Lewin el ai, 1991).<br />Examples of IFNy-induced immediate-early genes include interferon regulatory factor 1 (IRF-1), gua-nylate-binding protein 1 (GBP-1), and the type I Fey receptor (FcyRI), which encode proteins that participate in inflammatory and immune responses. Several IFNy-reguiated intermediate genes have also been identified. These genes are induced within 6-8 hours of stimulation and require additional protein synthesis for transcriptional activation to occur. Examples of ihese genes include those that encode class I and class II major histocornpatibility (MHC) proteins that play a centra! role in determining adaptive immune responses. Studies utilizing STATl" , JAK.1-, or JAK2-deficient mice have shown that these three signaling proteins play an obligate role in activating most IFNy-inducible genes (Durum et at., 1996; Meraz ei ai, 1996; Neubauer et ai, 1998; Paraganas et al., 1998). More than 100 IFNy regulated genes have been identified. This subject has recently been reviewed elsewhere and the reader is referred to several excellent reviews on the subject (Kerr and Stark, 1991; Boehm et a!., 1997; Der et ai, 1998).<br />Promoter regions involved<br />The promoter regions of IFNy-stimulated immediate-early genes contain GAS elements (IFNy-activated sequence) which act as the binding sites for activated STAT3 homodimers. When occupied, these exacting elements collaborate with basai transcriptional factors and promote gene transcription. The GAS site is a 9 nucleotide sequence with a consensus motif of<br />TTNCNNNAA. For STAT1 dimers, the most common sequence recognized is TTCC(G > C)GGAA.<br />Two major pathways account for induction of IFNy-induced intermediate genes. These pathways are driven by the transcription factors IRF-1 and CIITA (Tanaka and Taniguchi, 1992; Mach et ai, 1996). IRF-1 is itself an immediate-early gene that gives rise to a protein that functions by binding directly to DNA sequences in the promoters of IFN-induced genes. In contrast, CIITA is an intermediate gene induced by a collaboration between STAT1, IRF-1, and a ubiquitous transcription factor (USF-1), and functions, by binding to other transcription factors that make contact with DNA sequences within the X, X2, and Y boxes of the promoters in MHC class II genes.<br />BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY<br />Phenotypes of receptor knockouts and receptor overexpression mice<br />The physiologic consequences of global in vivo inacti-vation of the IFNy signaling pathway in mice were originally uncovered using neutralizing monoclonal antibodies specific for IFNy (Buchmeier and Schreiber, 3985). However, the physiologic role of IFNy has been more fully elucidated using mice with engineered disruptions in either the IFNy structural gene (Dalton et a!., 1993), the genes encoding the two IFNy receptor subunits (IFNGR1 and IFNGR2) (Huang et ai, 1993; Kamijo et ai, 1993; Lu et ai, 1998). or the genes encoding the three signaling proteins required by IFNy for biologic response induction, i.e. STAT1 (Durbin ct ai, 1996; Meraz et ai., 1996), JAK1 (Rodig et ai, 3998), and JAK2 (Neubauer et ai, 1998; Paraganas et ai, 1998). As a group, these mice display a greatly impaired ability to resist infection by a variety of microbial pathogens, including Listeria monocytogenes, L. major, and several mycobacterial species, including Mycobacterium bovis and M. avium, Moreover, mice that cannot respond to IFNy display enhanced tumor development when challenged with chemical carcinogens (Dighe et ai, 1994; Kaplan el ai, 1998). Importantly, mice lacking either IFNGR1 or IFNGR2 are able to mount a curative response to many viruses, while mice lacking the IFNa/,/9 receptor or STAT1, and<br />1832 Viiay Shankaran and Robert D. Schreiber<br /> <br />cells of mice deficient in JAK1 are not. These results thus demonstrate that under physiologic conditions the majority of the antiviral effects of the iFN system are largely mediated via type I interferons (Muller el al. 1994). Mice have also been generated which overcxpress a truncated dominant-negative mutant form of IFNGRI in cither the T cell or macrophage compartments and thereby represent mice that display a tissue-targeted IFNy inscnsitivity (Dighe el al., 1995). These types of mice will be useful in defining ihe tissue-specific actions of IFNy in protective host responses against pathogens and neoplastic cells.<br />Human abnormalities<br />Two research groups initially identified children from three unrelated families with inactivating mutations of 1FNGR1 who manifest a severe susceptibility to weakly pathogenic mycobaclerial species (Jouanguy ei al., 1996; Newport et a!., 1996). Genetic analysis of these patients' families revealed that susceptibility to atypical mycobaclerial infection was inherited in an autosomal recessive manner. Sequence analysis of the patients' IFNGR1 aileies identified missense mutations in genetic regions coding for the extracellular domain of the IFNGR1 polypeptide, leading to the production of truncated receptor proteins that were not retained at the cell surface. The clinical syndromes of these patients were similar. In one study, a group of related Maltese children were identified that showed extreme susceptibility to infection with M. fortuitum, M. avium, and M. chelonei (Newport et al., 1996). In another study, a Tunisian child was identified with disseminated M. bovis infection following bacillus Calmette-Guerin (BCG) vaccination (Jouanguy et a/., 1996). A third study identified a child of distinct ancestry who had a similar irnmuno-compromised phenotype (Pierre-Audigier el al., 1997). Biopsies from these patients revealed the presence of multibacillary, poorly defined granulomas, which contained scattered rnacrophages, but lacked epithe-lioid and giant cells and surrounding lymphocytes. Importantly, these patients showed enhanced susceptibility to mycobacteria and occasionally to salmonella but not to other typical bacteria or other common microbial pathogens or fungi. Moreover, in all three kindred, the patients were able to mount antibody and/or curative responses to several different viruses. Subsequently, a number of patients with other IFNy receptor mutations have been identified. A child with severe, disseminated infections due to M. fortuitum and M. avium was identified who lacked mutations in •<br />the IFNGRI gene (Dorman and Holland, 1998). This child had a homozygous mutation in the IFNGR2 gene. The mutation resulted in a premature stop codon in the extracellular domain-encoding region, and led to the production of IFNGR2 proteins that were not expressed at the cell surface. The clinical and histopathological phenotype of this patient closely resembled that of patients lacking expression of the IFNGR1 chain.<br />Other patients have been identified who develop less severe mycobacteria! disease than the children described above. Upon analysis of their IFNy receptor subunil genes, some of the patients were found to display distinct mutations in the IFNGR1 gene, leading to reduced but not ablated receptor function. Two patients were homozygous for a point mutation in the extracellular domain-encoding region of the IFNGRS subunit gene that produced an isoleucine-to-threonine amino acid substitution. The mutant receptors were found to require 100- to 1000-fold higher concentrations of IFNy than normal receptors in order to activate STAT1 (Jouanguy et al,, 1997). These patients responded io high-dose IFNy therapy.<br />Another set of 19 patients from 12 unrelated families were found to inherit partial IFNy insensi-tivity in an autosomal dominant manner (Jouanguy et al., 1999). All of these patients were found to be heterozygous for a wild-type IFNGRI allele and an IFNGRI allele with a frameshift mutation that produced an IFNGRI protein that Sacked most of the intracellular domain, including the JAK1- and STATi-binding sites. Interestingly, in this group of patients, there were 12 independent mutation events at a single site, defining a small deletion hotspot in the IFNGRI gene. The truncated receptor chain accumulated on cell surfaces and was shown to act in a dominant negative manner to inhibit IFN-y responses in cells. The definition of the molecular basis for this defect was facilitated by the observation that these patients phenotypicaliy resembled IFNy-insensitive cells and transgenic mice which overexpressed a genetically engineered truncated IFNGRI subunit (Dighe et al., 1993, 1994). In all of these patients, defects in IFNy responsiveness were partial, and cells from the patients retained some degree of sensitivity to IFNy. This phenotype correlated with the milder infections in these patients, and their positive responses to exogenous IFNy therapy.<br />References<br />Ague:. M., and Merlin, G. (1987). Purification of human gamma interieron receptors by sequential affinity chromatography on<br />

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