THE IFN³ RECEPTOR: A Paradigm for Cytokine Receptor Signaling

P1: RPK/vks/plb/rsk P2: MBL/PLB QC: RPK

February 1, 1997 18:20 Annual Reviews BACHCHPT.DUN AR26-22

Annu. Rev. Immunol. 1997. 15:563–91Copyright c© 1997 by Annual Reviews Inc. All rights reserved

THE IFNγ RECEPTOR: A Paradigmfor Cytokine Receptor Signaling

Erika A. Bach, Michel Aguet∗ and Robert D. SchreiberCenter for Immunology and Department of Pathology, Washington University Schoolof Medicine, St. Louis, Missouri 63110; e-mail: [email protected];∗Swiss Institute for Experimental Cancer Research (ISREC), Ch. des Boveresses,CH-1066 Epalinges, Lausanne, Switzerland; e-mail [email protected]

KEY WORDS: signal transduction, JAK-STAT pathway, transcription factors, tyrosine kinases,Stat1

ABSTRACT

During the last several years, the mechanism of IFNγ -dependent signal trans-duction has been the focus of intense investigation. This research has recentlyculminated in the elucidation of a comprehensive molecular understanding of theevents that underlie IFNγ -induced cellular responses. The structure and functionof the IFNγ receptor have been defined. The mechanism of IFNγ signal trans-duction has been largely elucidated, and the physiologic relevance of this processvalidated. Most recently, the molecular events that link receptor ligation to sig-nal transduction have been established. Together these insights have produced amodel of IFNγ signaling that is nearly complete and that serves as a paradigmfor signaling by other members of the cytokine receptor superfamily.

INTRODUCTION

The elucidation of the molecular understanding of the IFNγ receptor has beenan odyssey that has evolved over the past 15 years. Our understanding of thisreceptor system has been the result of combined discoveries from several lab-oratories working on different portions of the IFNγ signaling pathway. Thesediscoveries have led to the formulation of a cytokine receptor signaling modelthat is currently one of the most complete. In addition, these studies haveprovided information that has facilitated the understanding of many other cy-tokine receptor systems. Specifically, the study of the IFNγ receptor has beencritical in revealing (a) tyrosine phosphorylation of the cytokine receptor intra-cellular domain as a mechanism that couples the activated receptor to its signal

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564 BACH, AGUET & SCHREIBER

transduction system, (b) a novel pathway of signal transduction that mediatesbiologic responses of many different cytokine receptors, and (c) the molecularbasis of specificity for the induction of many cytokine-dependent cellular re-sponses. The purpose of this review is to summarize the major advances thathave produced the current comprehensive model of IFNγ receptor signaling.

HISTORICAL PERSPECTIVES

The current understanding of the IFNγ receptor system represents the synthesisof two distinct experimental approaches aimed at analyzing IFNγ -dependentgene induction. One focused on the cell surface with the intention of proceedinginto the cell nucleus, while the other focused on the nucleus of IFNγ -treatedcells and tracked the molecular trail back to the membrane. The meeting ofthese two experimental approaches occurred at the inner leaflet of the plasmamembrane and resulted in the establishment of a clear molecular model of theIFNγ signaling system.

The IFNγ receptor was initially characterized in the early 1980s in radio-ligand binding studies conducted in several laboratories, including our own, on avariety of different cell types (1). These experiments showed that most primaryand cultured cells expressed a moderate level of high affinity binding sitesfor IFNγ . The interaction of IFNγ with its receptor was not inhibited by otherinterferon classes, which explained the basis for the biologic specificity of IFNγ .In addition, human and murine IFNγ bound to their respective receptors in astrictly species-specific manner and thereby induced biologic responses only inspecies-matched cells. The latter observation proved to be critical in definingthe subunits of the functionally active IFNγ receptor and in determining thestructure-function relationships operative within each subunit.

A major step forward in defining the subunit composition of IFNγ receptorscame from key genetic experiments conducted by Pestka and associates in 1987(2). These studies employed a family of stable murine:human somatic cell hy-brids that contained the full complement of murine chromosomes and a randomassortment of human chromosomes. All hybrids that contained human chro-mosome 6 bound human IFNγ with high affinity, an observation later explainedby the presence of the human IFNγ receptorα chain gene on this chromosome(3). However, biologic responsiveness to human IFNγ was found only in hy-brids that contained both human chromosomes 6 and 21. These observations,together with similar studies using hamster:murine somatic cell hybrids, led tothe hypothesis that functionally active human or murine IFNγ receptors consistof two (or more) species-matched subunits (2, 4). The first is the receptor sub-unit responsible for binding ligand in a species-specific manner. The second isa species-matched subunit that is required for induction of biologic responses.



IFNγ RECEPTOR SIGNALING 565

This concept was further refined by independent reports in 1987–1988 of thepurification of the ligand-binding component of the human IFNγ receptor (5–7)and the subsequent cloning of its gene initially by Aguet and colleagues (8).This event was followed one year later by the isolation of the gene encoding themurine homologue (9–13). When the ligand-binding chains of the human ormurine IFNγ receptor were expressed at high levels in murine or human cells,respectively, they bound human or murine ligand in a manner that was identicalto endogenous receptors expressed on homologous cells. However, treatmentof the transfected cells with heterologous ligand failed to effect induction ofcellular responses. In contrast, when the human IFNγ -binding protein wasexpressed in murine cells that also contained human chromosome 21, thesecells not only bound the human ligand but also responded to it (14–16). Theseobservations thus added significant support to the concept that functionallyactive IFNγ receptors require a second, species-specific subunit. Definitiveproof of this concept came in 1994 when the second subunit of both the humanand murine IFNγ receptors were simultaneously identified by the Pestka andAguet laboratories using complementation cloning approaches (17, 18).

The nomenclature for the IFNγ receptor subunits has not been formallyestablished by the investigators in the field. Currently, the ligand-binding com-ponent of the IFNγ receptor is referred to as either the IFNγ receptorα chain,IFNγR1, or CDw119. The second subunit has been designated the IFNγ re-ceptorβ chain, accessory factor-1 (AF-1) or IFNγR2. For purposes of clarityin this review and to maintain consistency with the nomenclature for other cy-tokine receptors, we shall use only the designations IFNγ receptorα andβchains to refer to the two receptor subunits.

At the same time that the IFNγ receptor was being identified on a molecu-lar level, seminal biochemical and genetic experiments were being conductedindependently in the laboratories of James Darnell, Ian Kerr, George Stark,and James Ihle that identified a novel signaling pathway activated followingtreatment of cells with either IFNα or IFNγ (reviewed in 19–21). This workresulted in the identification of two classes of signaling proteins that partici-pated in this pathway. One was a family of latent cytosolic transcription factorsthat eventually became known as STAT proteins (for signal transducers andactivators of transcription). The other was a family of structurally distinctprotein tyrosine kinases known as Janus family kinases or JAKs. The uniquefeature of this signaling pathway, now known as the JAK-STAT pathway, wasthat receptor ligation resulted in the activation of specific cytosolic STAT pro-teins that dimerized and translocated directly from the membrane to the nucleusand effected transcriptional activation of specific target genes. However, theevents linking receptor ligation with signal transduction remained ill-defined.This missing step was filled in 1994 when the Schreiber laboratory showed that




IFNγ induced the tyrosine phosphorylation of the IFNγ receptorα chain lead-ing to the formation of a docking site on the activated receptor for a particularSTAT, namely Stat1 (22). This observation thus bridged the two experimentalapproaches and brought into focus the past 15 years of IFNγ receptor research.

THE LIGAND

Interferons were originally described as agents capable of protecting cells fromviral infection (1). Based on criteria such as their cellular source, generalbiologic properties, and gene structure, interferon family members have beensegregated into two categories. Type I IFN is induced primarily as a result ofviral infection of cells and has been divided into two classes based on the cellof origin. IFNα is a family of 17 related proteins encoded by distinct genes thatare synthesized largely by leukocytes. IFNβ is a single protein encoded by adistinct gene that is produced largely by fibroblasts. In contrast, Type II IFN isinduced by immune and inflammatory stimuli, is synthesized exclusively by Tlymphocytes and natural killer cells, and is commonly known as IFNγ . IFNγbears no structural resemblance to IFNα or IFNβ at the protein level, and thechromosomal location of the IFNγ gene is distinct from that of the Type I IFNlocus.

The human IFNγ molecule is a noncovalent homodimer that consists of twoidentical 17-kDa polypeptide chains (23, 24). During biosynthesis the polypep-tides are variably N-glycosylated, giving rise to a mature form of the moleculethat exhibits a predominant molecular mass of 50 kDa (25). The crystal struc-ture of IFNγ confirms its dimeric nature and reveals that the two polypeptidesself-associate in an antiparallel fashion, producing a molecule that exhibits atwofold axis of symmetry (26). This observation has led to the suggestion that asingle IFNγ homodimer can bind two IFNγ receptor molecules. Experimentalsupport for this prediction has been derived from results demonstrating that fullbiologic activity is only manifest by the homodimeric form of the protein (1).

IFNγ induces varied effects on a wide range of target cells, and its pleiotropicactions have been well studied (1). These include effects that promote bothspecific and nonspecific mechanisms of host defense against infectious agentsand tumors. Like the other members of the interferon family, IFNγ can protectcells from viral infection and can exert profound antiproliferative effects ona variety of normal and neoplastic cells. However, IFNγ is acknowledged toplay a more comprehensive role in immunomodulation compared to the TypeI interferons. IFNγ is one of the major cytokines responsible for upregulatingMHC class I protein expression and for inducing MHC class II proteins ona variety of leukocytes and epithelial cells. IFNγ has also been shown tobe the major cytokine responsible for activating or otherwise regulating the

December 21, 1997 14:14 Annual Reviews temp AR26-22

Figure 2 Crystal structure of a complex between IFNγ and the soluble IFNγ receptorα chainextracellular domain. The two single chains that comprise the biologically active IFNγ

homodimer are shown in blue and magenta. Each IFNγ monomer contacts one soluble receptor,shown in yellow and green, and thus one IFNγ homodimer dimerizes the IFNγ receptorα chain.

Upper panel: The view is perpendicular to the twofold axis of symmetry. The putative positionof the cell membrane is at the bottom of the page.

Lower panel: The complex as viewed parallel to the twofold axis of symmetry. The putativesite of interaction of the IFNγ receptorβ chain indicated by arrows. [Reprinted with permis-sion from Nature 376: 230-235, copyright 1995, Macmillian Magazines, Ltd (Reference 45).]




action of mononuclear phagocytes. In addition, the cytokine regulates humoralimmune responses by effecting IgG heavy chain switching in either a direct oran indirect manner. Finally, IFNγ regulates the production of a variety of otherimmunomodulatory or proinflammatory cytokines such as IL-12 and TNFα.

GENERAL STRUCTURE OF THE IFNγ RECEPTOR

IFNγ Receptor Subunit Gene Structure, Regulation,and Life CycleIn the last decade the chromosomal locations of the genes encoding the humanand murine IFNγ receptor polypeptides have been identified and their structurescharacterized. In addition, the expression patterns of these genes have beendefined. Recent studies indicate that the expression of the two IFNγ receptorsubunits differs significantly. Specifically, the receptorα chain is expressedat moderate levels on the surface of nearly all cells. Receptorα chain geneexpression appears to be constitutive, and analysis of the promoter of this genereveals a structure resembling that found in housekeeping genes. In contrast,the receptorβ chain is constitutively expressed at extremely low levels, butexpression can be regulated in certain cell types by external stimuli. Regulationof the receptorβ chain gene thus becomes a critical factor in determining IFNγ

responsiveness in certain cells.The human IFNγ receptorα chain is encoded by a 30-kb gene located on

the long arm of chromosome 6 (Table 1) (3). The murine homologue is a22-kb gene present on chromosome 10 (27). The 5′ flanking regions of thesegenes contain a GC-rich region with no TATA box like that of promoters fornoninducible housekeeping genes (G Merlin, Z Dembic, personal communica-tion). This observation suggests that expression of the IFNγ receptorα chainis not regulated by external stimuli, a result that has been largely confirmedexperimentally. Both genes consist of 7 exons. Exons 1–5 encode the receptorextracellular domain; exon 6 encodes a small portion of the membrane proximalregion of the extracellular domain and the transmembrane domain; and exon 7encodes the entire intracellular domain. Transcription of the human and murineIFNγ receptorα chain genes gives rise to mRNA transcripts of 2.3 kb (1). Thereceptorα chain polypeptide is synthesized in the endoplasmic reticulum (ER)and is posttranslationally modified as it moves from the ER to the Golgi bythe addition of N-linked carbohydrates (28, 29). Although expression of thefully mature protein at the plasma membrane varies widely between tissues(200–25,000 sites/cell), there does not appear to be a direct correlation betweenthe extent of receptorα chain expression and the magnitude of IFNγ -inducedresponses in cells (1). Following IFNγ receptor ligation, the receptor-ligand




Table 1 Properties of the IFNγ receptorα andβ subunits

α chain β chainProperty Human Murine Human Murine

Primary sequenceSignal peptide 17aa 26aa 21aa 18aaMature form 472aa 451aa 316aa 314aaHomology 52% 58%

Chromosomal localization 6 10 21 16Domain structure

Extracellular 228aa 228aa 226aa 224aaTransmembrane 23aa 23aa 24aa 24aaIntracellular 221aa 200aa 66aa 66aa

Potential N-linked glycosylation sites 5 5 5 6Predicted Mr (kDa) 52.5 49.8 34.8 35.6Mr (kDa) 90 90 61–67 60–65Intracellular conserved tyrosines 5 3

complex is internalized and enters an acidified compartment. Within this com-partment, the complex dissociates and free IFNγ is trafficked to the lysosomewhere it is degraded. In many cells, such as fibroblasts and macrophages, theuncoupled receptorα chain enters a large intracellular pool ofα subunits andeventually recycles back to the cell surface. In most cells, the size of the intra-cellular pool is approximately 2–4 times that of the receptors expressed at thecell surface (15, 28, 30–32).

The human IFNγ receptorβ chain gene has been localized to chromosome21q22.1 (17, 33). The murine homologue resides on chromosome 16 (Table 1)(4). These syntenic chromosomal regions also contain the genes of several otherIFN receptor family members, including the subunits of the IFNα/β receptor(IFNAR1 and IFNAR2) and the orphan IFN receptor family member denotedCRF2–4 (33, 34). Transcriptional activation of the IFNγ receptorβ chain generesults in the generation of an mRNA transcript of 1.8 kb in human cells or 2 kbin mouse cells (17, 18). At the present time, structural data is available onlyfor the mouse IFNγ receptorβ chain gene. This 17-kb gene appears to consistof 7 exons and contains, within the 5′ flanking region, several potential bindingsites for a variety of externally regulated activated transcription factors.

The latter observation suggested that transcription of theβ chain gene maybe tightly regulated, a hypothesis that has recently been strengthened experi-mentally. Based on the observation that different CD4+ T helper cell subsetsdiffered in their ability to respond to IFNγ (35), two independent groups demon-strated in 1995 that the IFNγ unresponsive state was due to a lack of cellularexpression of IFNγ receptorβ chain (36, 37). Unresponsiveness was shown




to be a result of IFNγ -dependent receptorβ chain downregulation and was notlinked to T cell differentiation (37). In this system, Th1 cells, which produceIFNγ , were found to lack the receptorβ subunit and were IFNγ unresponsive.In contrast, Th2 cells, which do not produce IFNγ , expressed the receptorβchain and were IFNγ responsive (37). However, receptorβ chain downregula-tion was induced in murine Th2 cells, as well as in human peripheral blood Tcells, upon exposure to IFNγ (37, 38). Interestingly, ligand-induced receptorβ chain downregulation did not occur in certain fibroblast cell lines. Thus,IFNγ appears to regulate expression of its own receptorβ chain on certain celltypes and thereby determines the ability of these cells to respond to subsequentexposure to IFNγ . Recently, treatment of T cells with phorbol esters or withCD3 antibodies has been shown to effect induction of receptorβ chain mRNA(38). Taken together, these results demonstrate thatβ chain expression can beregulated either positively or negatively in a stimulus-specific manner.

Structure of the IFNγ Receptor PolypeptidesThe human and murine IFNγ receptorα chains are organized in a similarmanner and are symmetrically oriented around a single transmembrane domain(Table 1 and Figure 1). However, despite this organizational similarity the twopolypeptides exhibit only 52.5% overall sequence identity. This modest levelof identity extends throughout both the extracellular and intracellular domainsof the polypeptides. The IFNγ receptorα chain is a member of the class 2cytokine receptor family, which includes tissue factor, IFNAR1 and IFNAR2,the ligand-binding component of the IL-10 receptor, and CRF2–4 (39). Likeall members of the class 2 cytokine receptor family, the intracellular domainof this subunit is devoid of intrinsic kinase or phosphatase activities. Like theIFNγ receptorα chain, the receptorβ chain is also a member of the class 2cytokine receptor family. The human and murine IFNγ receptorβ subunitsare also structurally similar to one another (Table 1 and Figure 1). Althoughhuman and murine receptorβ chains exhibit 58% identity overall, this valueincreases to 73% when their cytoplasmic domains are compared (17, 18).

Structure-Function Analyses of the IFNγ ReceptorSubunit Extracellular DomainsImmunochemical and radioligand binding experiments indicate that the IFNγ

receptorα chain binds ligand with a single high affinity (Ka) of 109–1010 M−1

(1). Deletion mutagenesis analysis of the receptor soluble extracellular do-main (sECD) showed that the majority of the extracellular domain (residues6–227) was required for expression of ligand-binding activity (40). However,by exchanging corresponding regions between the human and murine IFNγ re-ceptorα chain extracellular domains, several important internal sequences were




Figure 1 Polypeptide chain structure of the human IFNγ receptor. The IFNγ receptor consists oftwo species-matched polypeptides. The IFNγ receptorα chain is required for ligand binding andsignaling. The IFNγ receptorβ chain is required primarily for signaling and plays only a minorrole in ligand binding. The intracellular domain of the receptorα chain contains two functionallyimportance sequences: (1) an LPKS sequence required forα chain association with the tyrosinekinase Jak1, and (2) a YDKPH sequence that, when phosphorylated, forms the docking site forlatent Stat1. The intracellular domain of the receptorβ chain contains a functionally importantbox1/box2 sequence required for Jak2 association.




identified throughout the extracellular domain that contributed to the speciesspecificity of the ligand-binding process (41). Moreover, this study also re-vealed the presence of distinct regions within the receptorα chain that playedan obligate role in biologic response induction but not in ligand binding. One ex-planation for the latter observation is that the functionally important sequencesmay contribute to the interaction between the IFNγ receptorα andβ subunits.

Recent studies in the general field of receptor biology have established theparadigm that a ligand can effect the activation of its cellular receptor by induc-ing association or oligomerization of the appropriate receptor subunits. Amongcytokine receptors, this process was first described in studies of the receptorfor the monomeric ligand growth hormone (42). In the case of IFNγ , ligand-induced receptor dimerization was anticipated due to the suspected bivalentnature of the ligand. Experimental support for this possibility was providedby studies that analyzed the ligand-binding characteristics of a soluble humanIFNγ receptorα subunit (43, 44). By means of ligand-binding assays, sucrosedensity gradient ultracentrifugation, and HPLC gel filtration chromatography,sECD and ligand were shown to form stable complexes in free solution thatconsisted of one mole of ligand and two moles of soluble receptor. Formationof the 2:1 (receptor : ligand) complex was also demonstrated on cell surfacesusing either chemical cross-linking or immunochemical approaches.

Structural confirmation of the nature of the IFNγ : IFNγ receptor complexcame in 1995 when the crystal structure of human IFNγ bound to the solu-ble human IFNγ receptorα chain extracellular domain was solved to 2.9A(Figure 2). This study confirmed the 2:1 stoichiometry of the receptor : IFNγ

complex and represented the first solved crystal structure of a ligand-occupied,class 2 cytokine receptor (45). Within this complex, the core structure of boundIFNγ was similar but not identical to that determined for the unbound cytokine.The only major differences occurred within the AB loops and C-termini, whichare flexible and have little or no secondary structure in unbound IFNγ , but whichappear well ordered in receptor-bound IFNγ . The core structure of the ligatedIFNγ receptorα chain extracellular domain indicates that it forms a rod-likemolecule which is folded into two domains, denoted D1 (membrane distal) andD2 (membrane proximal). Each domain is folded into twoβ-strands consistingof β-pleated sheets. The domains are separated by an 11 amino acid linkerand are oriented at an angle of 120◦ relative to one another. The membraneproximal D2 domain is positioned at a 60◦ angle relative to the cell membrane,thereby causing the D1 domain to assume an angle that is complementary toIFNγ . Receptor binding thus orients the symmetrical IFNγ molecule perpen-dicular to the cell membrane and thereby allows for equivalent interactions tooccur between the second binding site of IFNγ and a second receptorα chain.In their dimerized form, the IFNγ receptorα chains do not interact with one




another and remain 27A apart. This distance is much greater than wouldhave been predicted by the crystal structure of the complex of growth hormonebound to its receptor (42). This characteristic becomes important because, un-like the ligated growth hormone receptor, the ligated IFNγ receptorα chainmust interact with an additional receptor subunit in order to effect initiation ofthe intracellular signaling process. Given the structural restraints apparent inthe core structure of the IFNγ /IFNγ receptorα chain complex, it is possible toenvision that symmetrical binding sites for the IFNγ receptorβ chain are gener-ated during ligand-induced receptorα chain dimerization. The crystal structurethus supports the concept that ligand induces the assembly of an activated IFNγ

receptor complex that consists of two receptorα chains and twoβ chains.Another study has defined the contribution of the IFNγ receptorβ chain to

the ligand-binding process (46). Using an experimental system where the twohuman IFNγ receptor subunits were expressed either individually or togetherin murine fibroblasts, no direct interaction was detected between human IFNγ

and the human IFNγ receptorβ subunit. However, when the humanβ subunitwas present at high levels on murine cells that also expressed the human IFNγ

receptorα subunit, IFNγ binding was increased fourfold over that observed oncells expressing only the human receptorα chain. Thus, one function of theIFNγ receptorβ chain is to stabilize the complex formed between ligand andthe receptorα subunit.

Structure-Function Analyses of the IFNγ ReceptorSubunit Intracellular DomainsThe human IFNγ receptorα chain was one of the first cytokine receptor subunitssubjected to a detailed structure-function analysis of its intracellular domain.Using a combination of deletion and substitution mutagenesis approaches, threedistinct sequences within the subunit’s 221 amino acid intracellular domain wereidentified as being required for specific receptor functions (15, 22, 47–49). Thefirst was a leucine-isoleucine sequence residing at positions 270 and 271 ofthe mature polypeptide that plays a critical role in directing receptor traffickingthrough the cell (1, 15). Deletion or alanine substitution of this sequenceresulted in a receptor mutant that was deficient in its ability to internalize ligandand that accumulated at the cell surface. However, this receptor mutant was stillcapable of supporting IFNγ -induced biologic responses, thereby dissociatingthe ligand trafficking and signaling functions of this subunit.

More important, these analyses revealed the presence of two topographicallydistinct, intracellular domain sequences that were required for induction ofIFNγ -dependent cellular responses. The first is a membrane proximal Leu-Pro-Lys-Ser (LPKS) sequence residing at positions 266–269 (15, 22). On the basisof alanine scanning analysis, the proline residue at position 267 was found to




play the dominant functional role within this sequence (49). The second is a fiveamino acid region that is located at positions 440–444 near the carboxy terminusof the receptorα subunit and that contains the residues Tyr-Asp-Lys-Pro-His(YDKPH) (15, 47). Mutational analysis of these five residues demonstratedthat only Y440, D441, and H444 were functionally important. Receptorα chainsharboring alanine substitutions at any of these three residues failed to induceIFNγ -dependent cellular responses (47). The particular functional importanceof Y440 was confirmed by two additional observations. First, receptorα chainsthat contained a conservative phenylalanine substitution at Y440 were also func-tionally inactive (47). Second, mutation or deletion of any other tyrosine residuewithin the receptor’s intracellular domain (denoted the 4XYF mutant) did notablate receptor activity (22). The physiologic importance of these two intra-cellular domain regions was demonstrated in experiments in which receptorα

chains containing mutations within the LPKS and/or YDKPH sequences wereoverexpressed either in cultured cell lines or in specific tissues of transgenicmice. Analysis of these cells and/or tissues revealed that the overexpressionof functionally inactive mutant receptorα chains inhibited biologic responsesinduced by endogenous IFNγ receptors in a dominant negative manner (50–52).

Recently, a structure-function analysis of the IFNγ receptorβ chain intra-cellular domain has been completed (53). This study, together with anotherthat used a cytoplasmically truncated form of the receptorβ chain, showedthat only the membrane proximal half of the 66 amino acid intracellular do-main was required forβ chain function (53, 54). Moreover, within this portionof the intracellular domain, two closely spaced sequences (263PPSIP267 and270IEEYL274) were identified that played an obligate role in response induction(53). Substitution of either of these five amino acid sequence blocks with ala-nine residues abrogated receptorβ chain function. However, no single aminoacid within this subregion of the receptorβ chain intracellular domain could beidentified as playing a dominant functional role. Thus, only a restricted amountof intracellular domain sequence within each of the two subunits of the IFNγ

receptor is required for induction of IFNγ -specific cellular responses.

SIGNALING

The JAK-STAT PathwayWhereas the aforementioned experiments identified the IFNγ receptor subunitson a molecular basis and characterized the key functional regions within theseproteins, they did not define the mechanism(s) of IFNγ receptor signaling.This process was largely elucidated in experiments, conducted concomitantlywith the IFNγ receptor characterization studies, that identified two distinctprotein classes involved in mediating IFN-dependent cellular responses. The




Figure 3 Structure of STAT proteins and their utilization by cytokine receptors. There are currentlyseven members of the signal transducers and activators of transcription (STAT) family that areactivated in a specific manner by distinct cytokine receptors. Receptor specificity has been definedusing gene targeted mice (76–82).

first class, termed STAT proteins, was initially identified in seminal biochemicalstudies performed in the laboratory of James Darnell (Figure 3). The secondconsisted of a group of unusual protein tyrosine kinases, termed Janus familykinases (JAKs), the participation of which in IFN signaling was defined inelegant genetic studies conducted in the laboratories of Ian Kerr and GeorgeStark (Figure 4). The combination of these two sets of observations led to thedefinition of a novel signal transduction pathway, now known as the JAK-STATpathway, that is responsible for mediating the activation of many, if not all,IFN-inducible genes (19–21).

This research was made possible by the identification of a family of genes(termed interferon-stimulated genes or ISGs) that were induced rapidly (i.e.within 15 to 30 min) in IFN-treated cells and the transcription of which was notdependent on new protein synthesis (55). Analysis of the promoter regions ofthese immediate-early ISGs revealed the presence of two classes of conservednucleotide sequences that directed the rapid transcriptional activation of IFN-inducible genes (19, 20). The first element, termed the interferon-stimulatedresponse element (ISRE), was a 12–15 nucleotide site with a consensus se-quence of AGTTTCNNTTTCNC/T that was responsible for driving expres-sion of IFNα/β inducible genes. The second, termed the gamma-interferonactivation site (GAS), was a 9 nucleotide site with a consensus sequence ofTTNCNNNAA that effected transcriptional activation of IFNγ -induced genes.




Figure 4 Structure of Janus family kinases and their utilization by cytokine receptors. There arecurrently four members of the Janus (JAK) family of protein tyrosine kinases. They associate withcytokine receptor intracellular domains and are required to form the STAT docking sites on ligatedcytokine receptors and to activate STAT proteins. Receptor utilization has been defined using genetargeted mice (74, 75).

Synthetic oligonucleotides that contained ISRE elements were used to iso-late and characterize two novel transcription factors of molecular masses 91and 113 (p91 and p113) (56, 57). These transcription factors, now known asStat1α (p91) and Stat2 (p113), were highly unusual because they contained srchomology 2 (SH2) domains (19–21, 58). The importance of the SH2 domainwas revealed when the mechanism of IFN-induced STAT protein activation wasestablished. In unstimulated cells, STAT proteins were present in the cytosolin a latent monomeric form (59). However, upon addition of IFN to cells, theSTATs were activated by tyrosine phosphorylation, formed homo- or hetero-dimers, translocated to the nucleus, and bound to the promoter regions of ISGs,effecting gene induction (59–62). Whereas IFNα was found to activate Stat1αand Stat2 [which then combined with a 48-kDa protein to form the transcrip-tionally active complex known as ISGF3 (57)], IFNγ effected activation onlyof Stat1α (59). Subsequent studies revealed that Stat1 and Stat2 represent thefounding members of a larger protein family that currently consists of seven dis-tinct gene products. These family members, Stat1, Stat2, Stat3, Stat4, Stat5a,Stat5b, and Stat6, play key roles in mediating the biologic effects of a varietyof different cytokines and growth factors (Figure 3) (19–21) .

At the same time that these experiments were conducted, other labswere pioneering the investigation of IFNα and IFNγ signaling mechanismspredominantly using genetic approaches. These studies used a mutagenesis




protocol that involved both positive and negative selection and thereby gener-ated eight cellular complementation groups that displayed distinctive combina-tions of IFNα and/or IFNγ signaling defects (19). For one IFNα-unresponsivecomplementation group (denoted U1A), a single gene was identified that com-plemented the genetic deficiency (63). Partial sequence analysis of this generevealed that it encoded a previously identified protein tyrosine kinase of un-known function known as Tyk2. Tyk2 was a structurally unusual protein ty-rosine kinase because it contained two kinase-like domains. It belongs to asmall family of ubiquitously expressed kinases known as Janus kinases (Fig-ure 4) (64). At the time, this family was thought to contain two additionalmembers known as Jak1 and Jak2 (64, 65). By means of cDNAs encodingthe three Janus family members and the panel of IFN-unresponsive cellularmutants, it was shown that IFNα signaling required the concomitant presenceof both Tyk2 and Jak1, whereas IFNγ signaling required the dual presenceof Jak1 and Jak2 (66, 67). Recently, a fourth family member, Jak3, has beenidentified and displays a restricted expression pattern largely limited to cells ofhematopoietic origin (68, 69). However, Jak3 does not play a role in mediatingIFN biologic responses. Additional work from several laboratories has demon-strated that distinct combinations of Janus family members are involved in thesignaling pathways of many cytokine and growth factor receptors that possessintracellular domains devoid of endogenous kinase activity (Figure 4) (19–21).

The functional connection between the JAK and STAT protein families wasmade when it became apparent that the JAKs were the enzymes responsible foreffecting cytokine-dependent STAT phosphorylation and activation (70, 71).This point was demonstrated by showing that cells deficient in either Jak1,Jak2, or Tyk2 were unable to activate Stat1 following treatment with IFN (66,67). These results demonstrated that these two classes of proteins formed aregulated signal transduction pathway. The general physiologic relevance ofthe JAK-STAT pathway has been unequivocally demonstrated by two sets ofobservations. First, humans lacking Jak3 are unable to respond to lymphocytegrowth factors such as IL-7 and IL-2 and display severe defects in their abilityto produce T lymphocytes (72, 73). Second, mice with targeted disruptions ofspecific JAK (74, 75) or STAT (76–82) genes display distinct defects in immunesystem development and/or function.

The Link Between the IFNγ Receptorand the JAK-STAT PathwayTHE JAK CONNECTION In 1991, studies on the signal transducing moleculeof the IL-6 receptor system, gp130, revealed that two functionally critical se-quences within the membrane proximal intracellular domain of this polypeptidewere conserved among the members of the cytokine receptor superfamily.




These sequences, termed box1 and box2, were originally defined as PXXPXPand LEVL, respectively (83, 84). Subsequent studies revealed that box1/box2sequences were important in mediating the interaction between certain cytokinereceptor cytoplasmic domains and Janus kinases (85, 86). This informationproved to be useful in defining the interactions of the IFNγ receptor subunitswith specific members of the JAK family.

Sequence comparisons indicated that the functionally critical, membraneproximal, intracellular domain sequences within the IFNγ receptorα andβsubunits were similar to box1/box2 motifs. To examine whether these se-quences functioned in a similar manner within the IFNγ receptor polypeptides,coprecipitation studies were performed on cells treated with either buffer orIFNγ . In unstimulated cells, the IFNγ receptorα chain was found to associatewith an inactive form of Jak1 (49, 87). This interaction was specific becauseit did not occur with other Janus family members. Using an alanine scanningmutagenesis approach, Jak1 binding to the IFNγ receptorα chain intracellulardomain was found to be dependent on the presence of the intact, function-ally important266LPKS269 sequence (49). In IFNγ -treated cells, the receptorαchain–associated Jak1 molecules became activated through tyrosine phosphory-lation (49, 87). These results indicated that whereas inactive Jak1 constitutivelyassociates with the IFNγ receptorα chain, its activation is ligand dependent.

Similar studies have been conducted on the IFNγ receptorβ subunit. Usingcoprecipitation/western blot analyses, three groups have demonstrated that theIFNγ receptorβ chain cytoplasmic domain associates with Jak2 in a constitutiveand specific manner (53, 54, 88). Structure-function analysis of the receptorβ chain demonstrated that this association is mediated through a functionallycritical, 12 amino acid box1/box2-like sequence263PPSIPLQIEEYL274 located13 amino acids away from the membrane in theβ chain intracellular domain(53). Mutation within this region produced a receptorβ subunit that was unableto interact with Jak2 and failed to support Stat1 activation or IFNγ -dependentbiologic response induction (53).

The elucidation of the structure-function relationships of the JAK associationsites present on the IFNγ receptor polypeptides led to subsequent experimentsthat examined the issue of Janus kinase substrate specificity. A key questionthat needed to be addressed was whether the apparent obligate pairing of Jak1and Jak2 for IFNγ signaling reflected the specificity of the kinase-receptor sub-unit interaction or the enzymatic substrate specificity of the kinases themselves.Two approaches have been utilized to address this issue. In one study chimericmolecules were generated in which the human IFNγ receptorβ chain cytoplas-mic domain was replaced by either Jak2 or c-src (53). These chimeras were thenexpressed in murine cells that also expressed the human IFNγ receptorα sub-unit, and the ability of these murine transfectants to respond to human IFNγ




was monitored. Cells expressing the human receptorβ chain–Jak2 chimerawere capable of manifesting a full complement of biologic responses to hu-man IFNγ (53). In contrast, cells expressing the receptorβ chain–src chimerawere unresponsive to the human ligand. These results showed that the soleobligate function of the intracellular domain of the IFNγ receptorβ subunit isto chaperone Jak2 into the ligand-activated receptor complex. The results alsosuggested that Janus kinases may display a certain level of substrate specificitythat obligates their participation in the JAK-STAT pathway and that cannotbe replaced by other classes of protein tyrosine kinases. In a second study,a different family of human IFNγ receptorβ chain chimeras were generated,in which theβ chain cytoplasmic domain was replaced with other cytokinereceptor cytoplasmic domains that utilize Janus kinases other than Jak2 (89).When expressed in hamster cells that also expressed the human IFNγ receptorα chain, all chimeric molecules were functionally competent to respond to hu-man IFNγ , although some quantitative differences were noted (89). This resultimplies that the specificity displayed by the Janus family kinases resides at thelevel of enzyme-receptor association. This concept has been strengthened bythe recent generation and characterization of mice with a targeted disruptionof the Jak1 gene (SJ Rodig, M Aguet, RD Schreiber, unpublished observa-tions). Taken together these results suggest that the Janus kinases are criticalfor JAK-STAT pathway activation but are not a source of pathway specificity.

LIGAND-INDUCED ASSEMBLY AND ACTIVATION OF THE IFNγ RECEPTOR Where-as these studies defined the critical sites on the IFNγ receptor subunits thatare responsible for mediating the association between the receptor polypep-tides, Jak1 and Jak2, they did not account for how the receptor-associated JAKkinases were activated. One possible mechanism that could account for theligand-induced activation of IFNγ receptor-associated kinases was that ligandcould effect oligomerization of the two IFNγ receptor subunits and therebypermit Jak1 and Jak2 to transactivate one another. However, until recently, datasupporting this concept has been difficult to obtain. One study suggested thatligand induces the association of the IFNγ receptorα andβ chains (46). Usingchemical cross-linking and immunoprecipitation approaches, a complex con-taining two IFNγ receptorα chains and one to twoβ chains was detected only incells that had been exposed to IFNγ . In contrast, another study claimed that theIFNγ receptor subunits preassociate with one another in the absence of ligand(88). However, the latter study employed a digitonin lysis step that failed to fullysolubilize cellular membranes and generated large pieces of membrane that con-tained a variety of irrelevant integral membrane proteins. The final resolutionof this issue came when ligand-induced complex formation was shown to oc-cur under physiologic conditions in the absence of chemical cross-linkers (53).




In this study, immunoprecipitates derived from untreated, detergent-solubilizedcells and formed with nonblocking monoclonal antibodies specific for the IFNγ

receptorα chain, contained theα subunit and Jak1 but not theβ subunit, Jak2,or irrelevant membrane proteins (53). In contrast,α chain immunoprecipitatesfrom IFNγ -treated cells contained both receptor subunits and activated formsof both Jak1 and Jak2. These results unequivocally demonstrated that the re-ceptorα andβ subunits are not strongly preassociated with one another on thesurface of unstimulated cells but are induced to associate upon exposure to lig-and. Thus, ligand-induced receptor subunit association leads to transactivationof Jak1 and Jak2.

THE STAT CONNECTION Whereas the importance of Stat1 activation in medi-ating many IFNγ -dependent responses had been established, the mechanismthat coupled ligation of the IFNγ receptor to the JAK-STAT pathway remainedill defined. A clue that led to the resolution of this issue was derived fromthe structure-function mutagenesis analyses described above that pointed to thecritical importance of a single tyrosine residue residing at position 440 in the hu-man IFNγ receptorα chain cytoplasmic domain (47). Mechanistic insights intothe importance of Y440 came with the demonstration that ligation of the IFNγreceptor leads to rapid and reversible tyrosine phosphorylation of the receptorα

chain intracellular domain (22, 87). Tyrosine phosphorylation was observed incells expressing the wild-type receptorα chain, the YF440 mutant, or the 4XYFmutant (22). Thus, whereas none of the tyrosine residues were important forligand-induced JAK activation, one residue (Y440) was identified that servedboth as a substrate site for these enzymes and as a key element in the induction ofIFNγ -dependent biologic responses. The breakthrough in this area came fromour observation that the YF440 mutant also failed to support IFNγ -dependentStat1 activation (22). This result suggested a direct link between ligand-inducedreceptor tyrosine phosphorylation and activation of the JAK-STAT pathway.

Proof of this concept was provided by studies showing that small 5–12 aminoacid phosphopeptides derived from the IFNγ receptorα chain that containedthe minimal sequence440Y(PO4)DKPH444 were capable of directly binding toStat1 and blocking its subsequent activation by IFNγ in a cell-free assay sys-tem (22, 90). Importantly, these phosphopeptides not only precipitated a latentform of Stat1 from crude cell lysates but also interacted with highly purified,recombinant Stat1 in the absence of additional proteins. This interaction was ofmoderate affinity (KD = 137 nM) and was specific because (a) Stat1 did not bindeither to nonphosphorylated forms of the peptides or to irrelevant phosphopep-tides, and (b) Y440-containing phosphopeptides did not interact strongly withirrelevant STAT molecules, such as Stat2, that are not activated by IFNγ (22,90). Additional experiments revealed that Stat1 binding to the phosphorylated




IFNγ receptorα chain sequence also required the presence of two additionalresidues, D441 and H444, the only two other residues in this region that arealso required for receptor function (47). No other receptorα chain residueswere identified that played an obligate role in formation of the Stat1 dockingsite on the receptor. On the basis of surface plasmon resonance experiments,Stat1 binding to its phosphorylated receptor docking site was determined to be110 times stronger than its ability to bind to a Stat1 phosphopeptide contain-ing Y701 (90). These results thus showed that IFNγ -induced Stat1 activationwas an ordered, affinity-driven process. Moreover, this study represented thefirst demonstration that STAT proteins bind in a specific and direct manner todistinct docking sites on ligated, tyrosine-phosphorylated cytokine receptors.

Parallel experiments revealed that the SH2 domain of STAT proteins wasresponsible for directing STAT binding to the receptor. This concept was de-rived from two types of experiments. First, antibodies specific for the Stat1SH2 domain inhibited binding of Stat1 to the active IFNγ receptorα chainphosphopeptide (90). Second, transfer of the SH2 domain of one STAT proteinto another transferred its ability to be recruited from one receptor to another(91). In the latter experiments STAT recruitment by the IFNγ and IFNα recep-tors could be exchanged by interchanging the SH2 domains of Stat1 and Stat2.Specifically, a Stat1 chimera containing the Stat2 SH2 domain could be directlyrecruited by the activated IFNα receptor, whereas a Stat2 chimera containingthe Stat1 SH2 domain was directly recruited by the activated IFNγ receptor(91). Taken together, these data further refined the general model by showingthat STAT proteins were specifically recruited to activated cytokine receptorsbased on the ability of the SH2 domain of the STAT protein to bind specificallyto a ligand-induced receptor docking site.

The aforementioned data suggested that similar STAT recruiting regions maybe present in the intracellular domains of other cytokine receptors. This possi-bility was first confirmed in studies showing that IL-4 effects the recruitmentand activation of another STAT protein, Stat6, via the IL-4 receptorα chainin a manner analogous to that shown for Stat1 recruitment by the IFNγ re-ceptor (92). However, in the case of Stat6, two phosphorylated IL-4 receptordocking sites were identified and had the sequences577GY(PO4)KAFS582 and605GY(PO4)KPFQ610. Subsequent studies by several groups defined receptordocking sites for Stats 2, 3, and 5 (93–96). Critical support for the physiologicrelevance of this process has come from the demonstration that STAT recruit-ment by activated cytokine receptors can be transferred from one receptor toanother by transfer of STAT docking sites between receptors. This process wasillustrated in experiments in which Stat 3 recruiting activity was transferredto a truncated erythropoietin receptor, which normally recruits Stat5, by trans-fer of the Stat3 docking site from the IL-6 receptor family signal transducergp130 (94). Whereas these experiments were the first to show a transfer of




STAT recruitment from one cytokine receptor to another, they did not show thetransfer of biologic function. Recently a concomitant transfer of both STATrecruitment and biologic response induction was accomplished using a proto-col whereby STAT docking sites from the IL-2 receptorβ chain and the IL-4receptorα chain were interchanged (97). Thus, the sum total of these exper-iments, which grew out of the IFNγ receptor signaling studies, has led to thedevelopment of a molecular model that explains, in part, the molecular basis ofsignaling specificity of the members of the cytokine receptor superfamily.

PHYSIOLOGIC RELEVANCE OF IFNγ -DEPENDENTSTAT1 ACTIVATION

Although Stat1 was originally identified as a latent cytosolic transcription fac-tor involved in IFNα and IFNγ signaling, it has subsequently been shown, byin vitro gel shift analyses, to be activated by a wide variety of other cytokinesand growth factors, including IL-6, IL-10, EGF, PDGF, and growth hormone(19–21). These observations have raised questions about the specificity of theJAK-STAT signaling pathway, since many of these cytokines induce biologicresponses in cells that are opposite to those induced by the interferons. How-ever, the specificity issue has largely been resolved by the recent generationof mice with a targeted disruption of the Stat1 gene (76, 77). Stat1 knockoutmice were obtained in the expected Mendelian proportions and were able toreproduce. Thus, like the IFNs, Stat1 is required neither for fertility nor em-bryonic development. However, Stat1 gene-targeted mice displayed a globaldeficiency in their ability to respond to either IFNγ or IFNα. Specifically, cellsderived from these animals were unable to initiate transcription of a variety ofIFN-inducible genes, such as interferon regulatory factor-1 (IRF-1), guanylatebinding protein-1 (GBP-1), MHC class II transactivator (CIITA), and the com-plement protein C3, or to synthesize IFN-induced proteins. Moreover, Stat1knockout mice were exquisitely sensitive to infection by a variety of micro-bial pathogens (such asListeria monocytogenes) and viruses (such as vesicularstomatitis virus). Thus, the overall phenotype of the Stat1 knockout mice wasone that encompassed the combined phenotypes of mice that are unresponsiveto IFNγ and to IFNα (98–100). In contrast, Stat1 knockout mice displayed nor-mal responses to IL-6, IL-10, EGF, and GH. These studies reveal that Stat1 isrequired for the induction of most, if not all, IFN-dependent biologic responsesand demonstrate that, in a physiologic setting, Stat1 plays a dedicated role insignaling only for interferon-mediated biologic effects. Thus, the specificityshown by IFNγ in effecting biologic responses in cells is dependent on twotemporally and spatially distinct processes: the specific recruitment of Stat1 tothe activated IFNγ receptor at the plasma membrane and the specific inductionin the nucleus of IFNγ -activated gene transcription by Stat1 homodimers.




Figure 5 Proposed signaling mechanism of the IFNγ receptor. The details of this model aredescribed in the text.




THE MODEL OF IFNγ RECEPTOR SIGNALTRANSDUCTION

The results discussed above can now be put together to form one of the mostcomplete models of cytokine receptor signaling to date (Figure 5) (53). In un-stimulated cells, the IFNγ receptorα andβ subunits are not preassociated witheach other but rather associate through their intracellular domains with inactiveforms of specific Janus family kinases. Jak1 and Jak2 constitutively associatewith the receptorα andβ chains, respectively. Addition of IFNγ , a homod-imeric ligand, to the cells induces the rapid dimerization of receptorα chains,thereby forming a site that is recognized, in a species-specific manner, by theextracellular domain of the receptorβ subunit. The ligand-induced assemblyof the complete receptor complex containing twoα and twoβ subunits bringsinto close juxtaposition the intracellular domains of these proteins togetherwith the inactive JAK enzymes that they carry. In this complex, Jak1 and Jak2transactivate one another and then phosphorylate the functionally critical Y440

residue on the receptorα subunit, thereby forming a paired set of Stat1 dock-ing sites on the ligated receptor. Two Stat1 molecules then associate with thepaired docking sites, are brought into close proximity with receptor-associatedactivated JAK enzymes, and are activated by phosphorylation of the Stat1 Y701

residue. Tyrosine-phosphorylated Stat1 molecules dissociate from their recep-tor tether and form homodimeric complexes. The activated Stat1 complex isthen phosphorylated on a specific C-terminal serine residue (S723) (101). Re-cent reports suggest that the serine phosphorylation is mediated by an as-yet-undefined MAP-kinase-like enzyme (101, 102). Activated Stat1 translocatesto the nucleus and, after binding to a specific sequence in the promoter regionof immediate-early IFNγ -inducible genes, effects gene transcription. Thus,IFNγ signaling is an ordered, affinity-driven process that derives its specificityfrom (a) the specific binding of a particular STAT protein to a defined, ligand-induced docking site on the activated receptor and (b) the ability of the Stat1homodimer to specifically activate IFNγ -induced gene transcription.

PHYSIOLOGICAL CONSEQUENCES OF IFNγRECEPTOR DISREGULATION

IFNγ Receptorα Chain Deficiencies in Microbial InfectionThe physiologic consequences of inactivating mutations within the genes en-coding the IFNγ receptor subunits have become evident through the analysisof experimental murine models and naturally occurring human genetic defects.Mice carrying a disruption in the murine IFNγ receptorα chain gene displayed aphenotype consistent with previous in vitro and in vivo studies of mice treated




with neutralizing IFNγ -specific monoclonal antibodies (99, 103). IFNγ re-ceptorα chain−/− (IFNγR−/−) mice exhibited no overt defects in embryonicdevelopment and showed normal development of lymphoid compartments andthe immune system. These animals displayed a greatly impaired ability to resistinfection by a variety of microbial pathogens includingListeria monocytogenes,Leishmania major, and several mycobacteria species, includingM. bovisandM. avium,despite the fact that the mice developed normal helper and cytotoxicT cell responses to these pathogens (99, 104). This result demonstrates that theIFNγ receptor plays a critical role in the expression of innate host resistance tomicrobial infection. In contrast, IFNγR−/− mice were able to mount a curativeresponse to many viruses, indicating that this receptor system is not the majormediator of antiviral effects in vivo (100).

On the basis of these results, the prediction was made that mutations in thehuman IFNγ receptorα chain gene might result in individuals with recurrentmicrobial but not viral infections. Recently two groups have simultaneouslyidentified such mutations in children who manifest a severe susceptibility toweakly pathogenic mycobacterial species (105, 106). In one study, a group ofrelated Maltese children were identified that showed extreme susceptibility toinfection withM. fortuitum, M. aviumandM. chelonei(105). Genetic analysisof these children revealed an inactivating mutation in the IFNγ receptorα chaingene. In the other study, a Tunisian child was identified with disseminatedM.bovisinfection following Bacillus Calmette-Gu´erin (BCG) vaccination (106).BCG, an attenuatedM. bovisstrain, is used in many countries as a live vaccineagainst human tuberculosis and leprosy. In most children, BCG vaccination isinnocuous. However, in an extremely small percentage of the population, BCGvaccination results in a disseminated infection that is often fatal. A French na-tional study found that approximately half of the individuals with disseminatedBCG infection have some form of classical immunodeficiency (106). However,the other half of the population was regarded as idiopathic because no immun-odeficiency could be identified as the causal agent underlying their condition.Analysis of an idiopathic patient revealed the presence of an inactivating muta-tion in the IFNγ receptorα chain gene (106). Subsequently, another unrelatedchild was identified with a similar but not identical mutation (107). Geneticanalysis of the families of these patients has revealed that the mutations are in-herited in an autosomal recessive manner (105–107). It is noteworthy that thesepatients only showed enhanced susceptibility to mycobacterium, but not to typ-ical bacteria or other more common microbial pathogens or fungi. Moreover,in all three kindred, the patients were able to mount antibody and/or curativeresponses to several different viruses (105–107). It will be important in thefuture to determine why IFNγ receptor defects lead exclusively to enhancedsusceptibility to mycobacterial infection.




IFNγ Receptorα Chain Deficiencies in CancerOverexpression of a truncated murine IFNγ receptorα chain in certain trans-plantable murine tumors rendered the tumors unresponsive to IFNγ in a dom-inant negative manner. These IFNγ unresponsive tumors resisted rejectionwhen transplanted into naive and immune syngeneic hosts (51). These studiesclearly identified an important role for IFNγ in promoting tumor immunogenic-ity. These observations also raised the question of whether IFNγ plays a criticalrole in promoting host surveillance that is capable of controlling the growth ofprimary tumors. To address this issue, IFNγ receptorα chain knockout mice orwild-type 129/Sv syngeneic control mice were treated with three doses of thechemical carcinogen 3-Methylcolanthrene and were monitored over a 130-dayperiod for tumor development. At every dose tested, IFNγ insensitive animalsdeveloped tumors more frequently than did wild-type controls, and at the low-est carcinogen doses, only the knockout mice developed tumors (DH Kaplan,AS Dighe, E Richards, LJ Old, RD Schreiber, unpublished observations). Thetumors originating from IFNγR−/− mice, when explanted and reintroducedinto naive control and IFNγR−/− mice, grew progressively in both types ofhost. Moreover, when IFNγ signaling was restored in one tumor by expressionof the IFNγ receptorα chain, this tumor was now rejected by naive 129/Svhosts. These data indicate that tumors that develop from IFNγ unresponsivetissues may be able to circumvent detection and rejection by the host immunesystem. Thus, IFNγ plays a key role in a system of tumor surveillance inimmunocompetent hosts.

These results predict that some human tumors may develop spontaneousmutations in the IFNγ signaling system that render them IFNγ insensitive.This process would thereby contribute to successful tumor establishment ina naive but immunocompetent host. This possibility has in fact been real-ized in an examination of isolated human lung carcinomas that were testedfor defects in IFNγ and IFNα signaling capabilities (DH Kaplan, E Stock-ert, LJ Old, RD Schreiber, unpublished observations). Several tumors of onehistologic classification (25% of adenocarcinomas) displayed insensitivity toIFNγ , whereas only one tumor was insensitive to both IFNγ and IFNα. Notumors were found that displayed a selective IFNα insensitivity. Analysis ofthe different tumors showed that some lacked expression of the IFNγ receptorα chain, some expressed an abnormally active Jak2 enzyme, and the tumorwith combined insensitivity to both IFNγ and IFNα did not express Jak1. Re-constitution experiments showed that IFNγ responsiveness was restored in theappropriate tumor cells following replacement of the missing or abnormal re-ceptorα chain, Jak2, or Jak1. Thus, in contrast to the case of the human IFNγ

receptor mutations that resulted in decreased host effector function against cer-tain mycobacterial infections, genetic defects within the IFNγ receptor system




that occur within developing tumor cells favor the pathologic outgrowth of theneoplastic cells.

CONCLUDING REMARKS

In this review we have focused on the recent events that have led to the synthesisof a comprehensive model of the IFNγ signaling pathway. Although this workhas been ongoing since the early 1980s, the most rapid advances have occurredduring the last five years. During this time, most if not all of the componentsof the IFNγ receptor and IFNγ signal transduction system were identified andthe critical molecular interactions defined that established the temporal andtopographical relationships that make this an effective and specific signalingpathway. This work has revealed the central role played by a single transcriptionfactor known as Stat1, which now is recognized as the direct link betweenthe IFNγ receptor and IFNγ -inducible genes. Undoubtedly, future work willfurther refine the IFNγ signaling model. However, even now, this model servesas the general paradigm for signaling through other members of the cytokinereceptor superfamily.

ACKNOWLEDGMENTS

The authors are particularly grateful to Dr. TL Nagabhushan for providing thephotographs of the IFNγ -IFNγ receptor crystal structure and, together, withSchering-Plough Research Institute, for supplying the financial support for re-producing this figure. We are also grateful to Drs. Gilles Merlin, Zlatko Dembicand Jean-Laurent Casanova and to Dan Kaplan for sharing their unpublisheddata. The authors also wish to thank Dan Kaplan and Keith Pinckard andDrs. Paul Allen, Antonio Celada, and Tony Kossiakoff for critical comments.Work in the Schreiber laboratory has been supported by grants from NIH andGenentech, Inc.

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THE IFN³ RECEPTOR: A Paradigm for Cytokine Receptor Signaling

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