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


transduction system, (b) a novel pathway of signal transduction that mediates
biologic r...
IFNγ RECEPTOR SIGNALING              565

   This concept was further refined by independent reports in 1987–1988 of the

IFNγ induced the tyrosine phosphorylation of the IFNγ receptor α chain lead-
ing to the ...
Figure 2 Crystal structure of a complex between IFNγ and the soluble IFNγ receptor α chain
extracellular domain. The two s...
IFNγ RECEPTOR SIGNALING              567

action of mononuclear phagocytes. In addition, the cytokine regulates humoral

Table 1 Properties of the IFNγ receptor α and β subunits

IFNγ RECEPTOR SIGNALING              569

to be a result of IFNγ -dependent receptor β chain downregulation and was not

Figure 1 Polypeptide chain structure of the human IFNγ receptor. The IFNγ receptor c...
IFNγ RECEPTOR SIGNALING              571

identified throughout the extracellular domain that contributed to the species

another and remain 27 A apart. This distance is much greate...
IFNγ RECEPTOR SIGNALING              573

play the dominant functional role within this sequence (49). The second is a five...

Figure 3 Structure of STAT proteins and their utilization by cytokine receptors. Th...
IFNγ RECEPTOR SIGNALING                     575

Figure 4 Structure of Janus family kinases and their utilization by cy...

protocol that involved both positive and negative selection and thereby gener-
ated eigh...
IFNγ RECEPTOR SIGNALING              577

These sequences, termed box1 and box2, were originally defined as PXXPXP
and LEVL...

was monitored. Cells expressing the human receptor β chain–Jak2 chimera
were capable of ...
IFNγ RECEPTOR SIGNALING              579

In this study, immunoprecipitates derived from untreated, detergent-solubilized

IFNγ receptor α chain sequence also required the presence of two additional
residues, D4...
IFNγ RECEPTOR SIGNALING              581

STAT recruitment from one cytokine receptor to another, they did not show the

Figure 5 Proposed signaling mechanism of the IFNγ receptor. The details of this model...
IFNγ RECEPTOR SIGNALING              583

The results discussed above can n...

with neutralizing IFNγ -specific monoclonal antibodies (99, 103). IFNγ re-
ceptor α chain...
IFNγ RECEPTOR SIGNALING              585

IFNγ Receptor α Chain Deficiencies in Cancer
Overexpression of a truncated murine...

that occur within developing tumor cells favor the pathologic outgrowth of the
IFNγ RECEPTOR SIGNALING                      587

      mosome region 6q16 to 6q22. J. Immunol.               human IFNγ r...

26. Ealick SE, Cook WJ, Vijay-Kumar S, Car-      37. Bach EA, Szabo SJ, Dighe AS, Ashken...
IFNγ RECEPTOR SIGNALING                    589

      is specifically required for class I major            258:1808–12

70. Shuai K, Ziemiecki A, Wilks AF, Harpur             Quelle FW, Nosaka T, Vignali DAA,...
IFNγ RECEPTOR SIGNALING                      591

90. Greenlund AC, Morales MO, Viviano                   S, Bluethmann H,...
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  1. 1. Annu. Rev. Immunol. 1997. 15:563–91 Copyright c 1997 by Annual Reviews Inc. All rights reserved THE IFNγ RECEPTOR: A Paradigm for Cytokine Receptor Signaling Erika A. Bach, Michel Aguet∗ and Robert D. Schreiber Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110; e-mail:; ∗ Swiss Institute for Experimental Cancer Research (ISREC), Ch. des Boveresses, CH-1066 Epalinges, Lausanne, Switzerland; e-mail 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 recently culminated in the elucidation of a comprehensive molecular understanding of the events that underlie IFNγ -induced cellular responses. The structure and function of the IFNγ receptor have been defined. The mechanism of IFNγ signal trans- duction has been largely elucidated, and the physiologic relevance of this process validated. Most recently, the molecular events that link receptor ligation to sig- nal transduction have been established. Together these insights have produced a model of IFNγ signaling that is nearly complete and that serves as a paradigm for signaling by other members of the cytokine receptor superfamily. INTRODUCTION The elucidation of the molecular understanding of the IFNγ receptor has been an odyssey that has evolved over the past 15 years. Our understanding of this receptor system has been the result of combined discoveries from several lab- oratories working on different portions of the IFNγ signaling pathway. These discoveries have led to the formulation of a cytokine receptor signaling model that is currently one of the most complete. In addition, these studies have provided information that has facilitated the understanding of many other cy- tokine receptor systems. Specifically, the study of the IFNγ receptor has been critical in revealing (a) tyrosine phosphorylation of the cytokine receptor intra- cellular domain as a mechanism that couples the activated receptor to its signal 563 0732-0582/97/0410-0563$08.00
  2. 2. 564 BACH, AGUET & SCHREIBER transduction system, (b) a novel pathway of signal transduction that mediates biologic responses of many different cytokine receptors, and (c) the molecular basis of specificity for the induction of many cytokine-dependent cellular re- sponses. The purpose of this review is to summarize the major advances that have produced the current comprehensive model of IFNγ receptor signaling. HISTORICAL PERSPECTIVES The current understanding of the IFNγ receptor system represents the synthesis of two distinct experimental approaches aimed at analyzing IFNγ -dependent gene induction. One focused on the cell surface with the intention of proceeding into the cell nucleus, while the other focused on the nucleus of IFNγ -treated cells and tracked the molecular trail back to the membrane. The meeting of these two experimental approaches occurred at the inner leaflet of the plasma membrane and resulted in the establishment of a clear molecular model of the IFNγ 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 a variety of different cell types (1). These experiments showed that most primary and cultured cells expressed a moderate level of high affinity binding sites for IFNγ . The interaction of IFNγ with its receptor was not inhibited by other interferon classes, which explained the basis for the biologic specificity of IFNγ . In addition, human and murine IFNγ 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 subunits of the functionally active IFNγ receptor and in determining the structure-function relationships operative within each subunit. A major step forward in defining the subunit composition of IFNγ receptors came 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 random assortment of human chromosomes. All hybrids that contained human chro- mosome 6 bound human IFNγ with high affinity, an observation later explained by the presence of the human IFNγ receptor α 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 to the hypothesis that functionally active human or murine IFNγ receptors consist of 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 is a species-matched subunit that is required for induction of biologic responses.
  3. 3. IFNγ RECEPTOR SIGNALING 565 This concept was further refined by independent reports in 1987–1988 of the purification 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 the murine homologue (9–13). When the ligand-binding chains of the human or murine IFNγ receptor were expressed at high levels in murine or human cells, respectively, they 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 IFNγ -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 (14–16). These observations thus added significant support to the concept that functionally active IFNγ 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 IFNγ receptors were simultaneously identified by the Pestka and Aguet laboratories using complementation cloning approaches (17, 18). The nomenclature for the IFNγ receptor subunits has not been formally established by the investigators in the field. Currently, the ligand-binding com- ponent of the IFNγ receptor is referred to as either the IFNγ receptor α chain, IFNγ R1, or CDw119. The second subunit has been designated the IFNγ re- ceptor β chain, accessory factor-1 (AF-1) or IFNγ R2. For purposes of clarity in this review and to maintain consistency with the nomenclature for other cy- tokine receptors, we shall use only the designations IFNγ receptor α and β chains 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 conducted independently in the laboratories of James Darnell, Ian Kerr, George Stark, and James Ihle that identified a novel signaling pathway activated following treatment of cells with either IFNα or IFNγ (reviewed in 19–21). This work resulted in the identification of two classes of signaling proteins that partici- pated in this pathway. One was a family of latent cytosolic transcription factors that eventually became known as STAT proteins (for signal transducers and activators of transcription). The other was a family of structurally distinct protein tyrosine kinases known as Janus family kinases or JAKs. The unique feature of this signaling pathway, now known as the JAK-STAT pathway, was that receptor ligation resulted in the activation of specific cytosolic STAT pro- teins that dimerized and translocated directly from the membrane to the nucleus and effected transcriptional activation of specific target genes. However, the events linking receptor ligation with signal transduction remained ill-defined. This missing step was filled in 1994 when the Schreiber laboratory showed that
  4. 4. 566 BACH, AGUET & SCHREIBER IFNγ induced the tyrosine phosphorylation of the IFNγ receptor α chain lead- ing to the formation of a docking site on the activated receptor for a particular STAT, namely Stat1 (22). This observation thus bridged the two experimental approaches and brought into focus the past 15 years of IFNγ receptor research. THE LIGAND Interferons were originally described as agents capable of protecting cells from viral infection (1). Based on criteria such as their cellular source, general biologic properties, and gene structure, interferon family members have been segregated into two categories. Type I IFN is induced primarily as a result of viral infection of cells and has been divided into two classes based on the cell of origin. IFNα is a family of 17 related proteins encoded by distinct genes that are synthesized largely by leukocytes. IFNβ is a single protein encoded by a distinct gene that is produced largely by fibroblasts. In contrast, Type II IFN is induced by immune and inflammatory stimuli, is synthesized exclusively by T lymphocytes and natural killer cells, and is commonly known as IFNγ . IFNγ bears no structural resemblance to IFNα or IFNβ at the protein level, and the chromosomal location of the IFNγ gene is distinct from that of the Type I IFN locus. The human IFNγ molecule is a noncovalent homodimer that consists of two identical 17-kDa polypeptide chains (23, 24). During biosynthesis the polypep- tides are variably N-glycosylated, giving rise to a mature form of the molecule that exhibits a predominant molecular mass of 50 kDa (25). The crystal struc- ture of IFNγ confirms its dimeric nature and reveals that the two polypeptides self-associate in an antiparallel fashion, producing a molecule that exhibits a twofold axis of symmetry (26). This observation has led to the suggestion that a single IFNγ homodimer can bind two IFNγ receptor molecules. Experimental support for this prediction has been derived from results demonstrating that full biologic 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 pleiotropic actions have been well studied (1). These include effects that promote both specific and nonspecific mechanisms of host defense against infectious agents and tumors. Like the other members of the interferon family, IFNγ can protect cells from viral infection and can exert profound antiproliferative effects on a variety of normal and neoplastic cells. However, IFNγ is acknowledged to play a more comprehensive role in immunomodulation compared to the Type I interferons. IFNγ is one of the major cytokines responsible for upregulating MHC class I protein expression and for inducing MHC class II proteins on a variety of leukocytes and epithelial cells. IFNγ has also been shown to be the major cytokine responsible for activating or otherwise regulating the
  5. 5. Figure 2 Crystal structure of a complex between IFNγ and the soluble IFNγ receptor α chain extracellular 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 receptor α chain. Upper panel: The view is perpendicular to the twofold axis of symmetry. The putative position of the cell membrane is at the bottom of the page. Lower panel: The complex as viewed parallel to the twofold axis of symmetry. The putative site of interaction of the IFNγ receptor β chain indicated by arrows. [Reprinted with permis- sion from Nature 376: 230-235, copyright 1995, Macmillian Magazines, Ltd (Reference 45).]
  6. 6. IFNγ RECEPTOR SIGNALING 567 action of mononuclear phagocytes. In addition, the cytokine regulates humoral immune responses by effecting IgG heavy chain switching in either a direct or an indirect manner. Finally, IFNγ regulates the production of a variety of other immunomodulatory or proinflammatory cytokines such as IL-12 and TNFα. GENERAL STRUCTURE OF THE IFNγ RECEPTOR IFNγ Receptor Subunit Gene Structure, Regulation, and Life Cycle In the last decade the chromosomal locations of the genes encoding the human and murine IFNγ receptor polypeptides have been identified and their structures characterized. In addition, the expression patterns of these genes have been defined. Recent studies indicate that the expression of the two IFNγ receptor subunits differs significantly. Specifically, the receptor α chain is expressed at moderate levels on the surface of nearly all cells. Receptor α chain gene expression appears to be constitutive, and analysis of the promoter of this gene reveals a structure resembling that found in housekeeping genes. In contrast, the receptor β chain is constitutively expressed at extremely low levels, but expression can be regulated in certain cell types by external stimuli. Regulation of the receptor β chain gene thus becomes a critical factor in determining IFNγ responsiveness in certain cells. The human IFNγ receptor α chain is encoded by a 30-kb gene located on the long arm of chromosome 6 (Table 1) (3). The murine homologue is a 22-kb gene present on chromosome 10 (27). The 5 flanking regions of these genes contain a GC-rich region with no TATA box like that of promoters for noninducible housekeeping genes (G Merlin, Z Dembic, personal communica- tion). This observation suggests that expression of the IFNγ receptor α chain is not regulated by external stimuli, a result that has been largely confirmed experimentally. Both genes consist of 7 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 intracellular domain. Transcription of the human and murine IFNγ receptor α chain genes gives rise to mRNA transcripts of 2.3 kb (1). The receptor α chain polypeptide is synthesized in the endoplasmic reticulum (ER) and is posttranslationally modified as it moves from the ER to the Golgi by the addition of N-linked carbohydrates (28, 29). Although expression of the fully mature protein at the plasma membrane varies widely between tissues (200–25,000 sites/cell), there does not appear to be a direct correlation between the extent of receptor α chain expression and the magnitude of IFNγ -induced responses in cells (1). Following IFNγ receptor ligation, the receptor-ligand
  7. 7. 568 BACH, AGUET & SCHREIBER Table 1 Properties of the IFNγ receptor α and β subunits α chain β chain Property Human Murine Human Murine Primary sequence Signal peptide 17aa 26aa 21aa 18aa Mature form 472aa 451aa 316aa 314aa Homology 52% 58% Chromosomal localization 6 10 21 16 Domain structure Extracellular 228aa 228aa 226aa 224aa Transmembrane 23aa 23aa 24aa 24aa Intracellular 221aa 200aa 66aa 66aa Potential N-linked glycosylation sites 5 5 5 6 Predicted Mr (kDa) 52.5 49.8 34.8 35.6 Mr (kDa) 90 90 61–67 60–65 Intracellular 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 lysosome where it is degraded. In many cells, such as fibroblasts and macrophages, the uncoupled receptor α chain enters a large intracellular pool of α subunits and eventually 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 the cell surface (15, 28, 30–32). The human IFNγ receptor β chain gene has been localized to chromosome 21q22.1 (17, 33). The murine homologue resides on chromosome 16 (Table 1) (4). These syntenic chromosomal regions also contain the genes of several other IFN receptor family members, including the subunits of the IFNα/β receptor (IFNAR1 and IFNAR2) and the orphan IFN receptor family member denoted CRF2–4 (33, 34). Transcriptional activation of the IFNγ receptor β chain gene results in the generation of an mRNA transcript of 1.8 kb in human cells or 2 kb in mouse cells (17, 18). At the present time, structural data is available only for the mouse IFNγ receptor β chain gene. This 17-kb gene appears to consist of 7 exons and contains, within the 5 flanking region, several potential binding sites for a variety of externally regulated activated transcription factors. The latter observation suggested that transcription of the β chain gene may be tightly regulated, a hypothesis that has recently been strengthened experi- mentally. Based on the observation that different CD4+ T helper cell subsets differed 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 cellular expression of IFNγ receptor β chain (36, 37). Unresponsiveness was shown
  8. 8. IFNγ RECEPTOR SIGNALING 569 to be a result of IFNγ -dependent receptor β chain downregulation and was not linked to T cell differentiation (37). In this system, Th1 cells, which produce IFNγ , were found to lack the receptor β subunit and were IFNγ unresponsive. In contrast, Th2 cells, which do not produce IFNγ , expressed the receptor β chain and were IFNγ responsive (37). However, receptor β chain downregula- tion was induced in murine Th2 cells, as well as in human peripheral blood T cells, upon exposure to IFNγ (37, 38). Interestingly, ligand-induced receptor β chain downregulation did not occur in certain fibroblast cell lines. Thus, IFNγ appears to regulate expression of its own receptor β chain on certain cell types and thereby determines the ability of these cells to respond to subsequent exposure to IFNγ . Recently, treatment of T cells with phorbol esters or with CD3 antibodies has been shown to effect induction of receptor β chain mRNA (38). Taken together, these results demonstrate that β chain expression can be regulated either positively or negatively in a stimulus-specific manner. Structure of the IFNγ Receptor Polypeptides The human and murine IFNγ receptor α chains are organized in a similar manner and are symmetrically oriented around a single transmembrane domain (Table 1 and Figure 1). However, despite this organizational similarity the two polypeptides exhibit only 52.5% overall sequence identity. This modest level of identity extends throughout both the extracellular and intracellular domains of the polypeptides. The IFNγ receptor α chain is a member of the class 2 cytokine receptor family, which includes tissue factor, IFNAR1 and IFNAR2, the ligand-binding component of the IL-10 receptor, and CRF2–4 (39). Like all members of the class 2 cytokine receptor family, the intracellular domain of this subunit is devoid of intrinsic kinase or phosphatase activities. Like the IFNγ receptor α chain, the receptor β chain is also a member of the class 2 cytokine receptor family. The human and murine IFNγ receptor β subunits are also structurally similar to one another (Table 1 and Figure 1). Although human and murine receptor β chains exhibit 58% identity overall, this value increases to 73% when their cytoplasmic domains are compared (17, 18). Structure-Function Analyses of the IFNγ Receptor Subunit Extracellular Domains Immunochemical and radioligand binding experiments indicate that the IFNγ receptor α 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 (residues 6–227) was required for expression of ligand-binding activity (40). However, by exchanging corresponding regions between the human and murine IFNγ re- ceptor α chain extracellular domains, several important internal sequences were
  9. 9. 570 BACH, AGUET & SCHREIBER Figure 1 Polypeptide chain structure of the human IFNγ receptor. The IFNγ receptor consists of two species-matched polypeptides. The IFNγ receptor α chain is required for ligand binding and signaling. The IFNγ receptor β chain is required primarily for signaling and plays only a minor role in ligand binding. The intracellular domain of the receptor α chain contains two functionally importance sequences: (1) an LPKS sequence required for α chain association with the tyrosine kinase Jak1, and (2) a YDKPH sequence that, when phosphorylated, forms the docking site for latent Stat1. The intracellular domain of the receptor β chain contains a functionally important box1/box2 sequence required for Jak2 association.
  10. 10. IFNγ RECEPTOR SIGNALING 571 identified throughout the extracellular domain that contributed to the species specificity of the ligand-binding process (41). Moreover, this study also re- vealed the presence of distinct regions within the receptor α chain that played an obligate role in biologic response induction but not in ligand binding. One ex- planation for the latter observation is that the functionally important sequences may contribute to the interaction between the IFNγ receptor α and β subunits. Recent studies in the general field of receptor biology have established the paradigm that a ligand can effect the activation of its cellular receptor by induc- ing association or oligomerization of the appropriate receptor subunits. Among cytokine receptors, this process was first described in studies of the receptor for the monomeric ligand growth hormone (42). In the case of IFNγ , ligand- induced receptor dimerization was anticipated due to the suspected bivalent nature of the ligand. Experimental support for this possibility was provided by studies that analyzed the ligand-binding characteristics of a soluble human IFNγ receptor α subunit (43, 44). By means of ligand-binding assays, sucrose density gradient ultracentrifugation, and HPLC gel filtration chromatography, sECD and ligand were shown to form stable complexes in free solution that consisted of one mole of ligand and two moles of soluble receptor. Formation of the 2:1 (receptor : ligand) complex was also demonstrated on cell surfaces using either chemical cross-linking or immunochemical approaches. Structural confirmation of the nature of the IFNγ : IFNγ receptor complex came in 1995 when the crystal structure of human IFNγ bound to the solu- ble human IFNγ receptor α chain extracellular domain was solved to 2.9 A ˚ (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 bound IFNγ was similar but not identical to that determined for the unbound cytokine. The only major differences occurred within the AB loops and C-termini, which are flexible and have little or no secondary structure in unbound IFNγ , but which appear well ordered in receptor-bound IFNγ . The core structure of the ligated IFNγ receptor α chain extracellular domain indicates that it forms a rod-like molecule which is folded into two domains, denoted D1 (membrane distal) and D2 (membrane proximal). Each domain is folded into two β-strands consisting of β-pleated sheets. The domains are separated by an 11 amino acid linker and are oriented at an angle of 120◦ relative to one another. The membrane proximal 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 to IFNγ . Receptor binding thus orients the symmetrical IFNγ molecule perpen- dicular to the cell membrane and thereby allows for equivalent interactions to occur between the second binding site of IFNγ and a second receptor α chain. In their dimerized form, the IFNγ receptor α chains do not interact with one
  11. 11. 572 BACH, AGUET & SCHREIBER ˚ another and remain 27 A apart. This distance is much greater than would have been predicted by the crystal structure of the complex of growth hormone bound to its receptor (42). This characteristic becomes important because, un- like the ligated growth hormone receptor, the ligated IFNγ receptor α chain must interact with an additional receptor subunit in order to effect initiation of the intracellular signaling process. Given the structural restraints apparent in the core structure of the IFNγ /IFNγ receptor α chain complex, it is possible to envision that symmetrical binding sites for the IFNγ receptor β chain are gener- ated during ligand-induced receptor α chain dimerization. The crystal structure thus supports the concept that ligand induces the assembly of an activated IFNγ receptor complex that consists of two receptor α chains and two β chains. Another study has defined the contribution of the IFNγ receptor β chain to the ligand-binding process (46). Using an experimental system where the two human IFNγ receptor subunits were expressed either individually or together in murine fibroblasts, no direct interaction was detected between human IFNγ and the human IFNγ receptor β subunit. However, when the human β subunit was present at high levels on murine cells that also expressed the human IFNγ receptor α subunit, IFNγ binding was increased fourfold over that observed on cells expressing only the human receptor α chain. Thus, one function of the IFNγ receptor β chain is to stabilize the complex formed between ligand and the receptor α subunit. Structure-Function Analyses of the IFNγ Receptor Subunit Intracellular Domains The human IFNγ receptor α chain was one of the first cytokine receptor subunits subjected to a detailed structure-function analysis of its intracellular domain. Using a combination of deletion and substitution mutagenesis approaches, three distinct sequences within the subunit’s 221 amino acid intracellular domain were identified as being required for specific receptor functions (15, 22, 47–49). The first was a leucine-isoleucine sequence residing at positions 270 and 271 of the mature polypeptide that plays a critical role in directing receptor trafficking through the cell (1, 15). Deletion or alanine substitution of this sequence resulted in a receptor mutant that was deficient in its ability to internalize ligand and that accumulated at the cell surface. However, this receptor mutant was still capable of supporting IFNγ -induced biologic responses, thereby dissociating the ligand trafficking and signaling functions of this subunit. More important, these analyses revealed the presence of two topographically distinct, intracellular domain sequences that were required for induction of IFNγ -dependent cellular responses. The first is a membrane proximal Leu-Pro- Lys-Ser (LPKS) sequence residing at positions 266–269 (15, 22). On the basis of alanine scanning analysis, the proline residue at position 267 was found to
  12. 12. IFNγ RECEPTOR SIGNALING 573 play the dominant functional role within this sequence (49). The second is a five amino acid region that is located at positions 440–444 near the carboxy terminus of the receptor α subunit and that contains the residues Tyr-Asp-Lys-Pro-His (YDKPH) (15, 47). Mutational analysis of these five residues demonstrated that only Y440 , D441 , and H444 were functionally important. Receptor α chains harboring alanine substitutions at any of these three residues failed to induce IFNγ -dependent cellular responses (47). The particular functional importance of Y440 was confirmed by two additional observations. First, receptor α chains that contained a conservative phenylalanine substitution at Y440 were also func- tionally inactive (47). Second, mutation or deletion of any other tyrosine residue within the receptor’s intracellular domain (denoted the 4XYF mutant) did not ablate receptor activity (22). The physiologic importance of these two intra- cellular domain regions was demonstrated in experiments in which receptor α chains containing mutations within the LPKS and/or YDKPH sequences were overexpressed either in cultured cell lines or in specific tissues of transgenic mice. Analysis of these cells and/or tissues revealed that the overexpression of functionally inactive mutant receptor α chains inhibited biologic responses induced by endogenous IFNγ receptors in a dominant negative manner (50–52). Recently, a structure-function analysis of the IFNγ receptor β chain intra- cellular domain has been completed (53). This study, together with another that used a cytoplasmically truncated form of the receptor β chain, showed that only the membrane proximal half of the 66 amino acid intracellular do- main was required for β chain function (53, 54). Moreover, within this portion of the intracellular domain, two closely spaced sequences (263 PPSIP267 and 270 IEEYL274 ) 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 receptor β chain function. However, no single amino acid within this subregion of the receptor β chain intracellular domain could be identified as playing a dominant functional role. Thus, only a restricted amount of 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 Pathway Whereas the aforementioned experiments identified the IFNγ receptor subunits on a molecular basis and characterized the key functional regions within these proteins, they did not define the mechanism(s) of IFNγ receptor signaling. This process was largely elucidated in experiments, conducted concomitantly with the IFNγ receptor characterization studies, that identified two distinct protein classes involved in mediating IFN-dependent cellular responses. The
  13. 13. 574 BACH, AGUET & SCHREIBER Figure 3 Structure of STAT proteins and their utilization by cytokine receptors. There are currently seven members of the signal transducers and activators of transcription (STAT) family that are activated in a specific manner by distinct cytokine receptors. Receptor specificity has been defined using gene targeted mice (76–82). first class, termed STAT proteins, was initially identified in seminal biochemical studies performed in the laboratory of James Darnell (Figure 3). The second consisted of a group of unusual protein tyrosine kinases, termed Janus family kinases (JAKs), the participation of which in IFN signaling was defined in elegant genetic studies conducted in the laboratories of Ian Kerr and George Stark (Figure 4). The combination of these two sets of observations led to the definition of a novel signal transduction pathway, now known as the JAK-STAT pathway, 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 not dependent on new protein synthesis (55). Analysis of the promoter regions of these immediate-early ISGs revealed the presence of two classes of conserved nucleotide sequences that directed the rapid transcriptional activation of IFN- inducible genes (19, 20). The first element, termed the interferon-stimulated response 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-interferon activation site (GAS), was a 9 nucleotide site with a consensus sequence of TTNCNNNAA that effected transcriptional activation of IFNγ -induced genes.
  14. 14. IFNγ RECEPTOR SIGNALING 575 Figure 4 Structure of Janus family kinases and their utilization by cytokine receptors. There are currently four members of the Janus (JAK) family of protein tyrosine kinases. They associate with cytokine receptor intracellular domains and are required to form the STAT docking sites on ligated cytokine receptors and to activate STAT proteins. Receptor utilization has been defined using gene targeted mice (74, 75). Synthetic oligonucleotides that contained ISRE elements were used to iso- late and characterize two novel transcription factors of molecular masses 91 and 113 (p91 and p113) (56, 57). These transcription factors, now known as Stat1α (p91) and Stat2 (p113), were highly unusual because they contained src homology 2 (SH2) domains (19–21, 58). The importance of the SH2 domain was revealed when the mechanism of IFN-induced STAT protein activation was established. In unstimulated cells, STAT proteins were present in the cytosol in a latent monomeric form (59). However, upon addition of IFN to cells, the STATs 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 Stat1α and Stat2 [which then combined with a 48-kDa protein to form the transcrip- tionally active complex known as ISGF3 (57)], IFNγ effected activation only of Stat1α (59). Subsequent studies revealed that Stat1 and Stat2 represent the founding 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 variety of different cytokines and growth factors (Figure 3) (19–21) . At the same time that these experiments were conducted, other labs were pioneering the investigation of IFNα and IFNγ signaling mechanisms predominantly using genetic approaches. These studies used a mutagenesis
  15. 15. 576 BACH, AGUET & SCHREIBER 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-unresponsive complementation group (denoted U1A), a single gene was identified that com- plemented the genetic deficiency (63). Partial sequence analysis of this gene revealed 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 a small family of ubiquitously expressed kinases known as Janus kinases (Fig- ure 4) (64). At the time, this family was thought to contain two additional members known as Jak1 and Jak2 (64, 65). By means of cDNAs encoding the three Janus family members and the panel of IFN-unresponsive cellular mutants, it was shown that IFNα signaling required the concomitant presence of both Tyk2 and Jak1, whereas IFNγ signaling required the dual presence of Jak1 and Jak2 (66, 67). Recently, a fourth family member, Jak3, has been identified and displays a restricted expression pattern largely limited to cells of hematopoietic origin (68, 69). However, Jak3 does not play a role in mediating IFN biologic responses. Additional work from several laboratories has demon- strated that distinct combinations of Janus family members are involved in the signaling pathways of many cytokine and growth factor receptors that possess intracellular domains devoid of endogenous kinase activity (Figure 4) (19–21). The functional connection between the JAK and STAT protein families was made when it became apparent that the JAKs were the enzymes responsible for effecting 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 a regulated signal transduction pathway. The general physiologic relevance of the JAK-STAT pathway has been unequivocally demonstrated by two sets of observations. First, humans lacking Jak3 are unable to respond to lymphocyte growth factors such as IL-7 and IL-2 and display severe defects in their ability to produce T lymphocytes (72, 73). Second, mice with targeted disruptions of specific JAK (74, 75) or STAT (76–82) genes display distinct defects in immune system development and/or function. The Link Between the IFNγ Receptor and the JAK-STAT Pathway THE JAK CONNECTION In 1991, studies on the signal transducing molecule of the IL-6 receptor system, gp130, revealed that two functionally critical se- quences within the membrane proximal intracellular domain of this polypeptide were conserved among the members of the cytokine receptor superfamily.
  16. 16. IFNγ RECEPTOR SIGNALING 577 These sequences, termed box1 and box2, were originally defined as PXXPXP and LEVL, respectively (83, 84). Subsequent studies revealed that box1/box2 sequences were important in mediating the interaction between certain cytokine receptor cytoplasmic domains and Janus kinases (85, 86). This information proved to be useful in defining the interactions of the IFNγ receptor subunits with specific members of the JAK family. Sequence comparisons indicated that the functionally critical, membrane proximal, intracellular domain sequences within the IFNγ receptor α and β subunits 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 or IFNγ . In unstimulated cells, the IFNγ receptor α chain was found to associate with an inactive form of Jak1 (49, 87). This interaction was specific because it did not occur with other Janus family members. Using an alanine scanning mutagenesis approach, Jak1 binding to the IFNγ receptor α chain intracellular domain was found to be dependent on the presence of the intact, function- ally important 266 LPKS269 sequence (49). In IFNγ -treated cells, the receptor α chain–associated Jak1 molecules became activated through tyrosine phosphory- lation (49, 87). These results indicated that whereas inactive Jak1 constitutively associates with the IFNγ receptor α chain, its activation is ligand dependent. Similar studies have been conducted on the IFNγ receptor β subunit. Using coprecipitation/western blot analyses, three groups have demonstrated that the IFNγ receptor β chain cytoplasmic domain associates with Jak2 in a constitutive and specific manner (53, 54, 88). Structure-function analysis of the receptor β chain demonstrated that this association is mediated through a functionally critical, 12 amino acid box1/box2-like sequence 263 PPSIPLQIEEYL274 located 13 amino acids away from the membrane in the β chain intracellular domain (53). Mutation within this region produced a receptor β subunit that was unable to interact with Jak2 and failed to support Stat1 activation or IFNγ -dependent biologic response induction (53). The elucidation of the structure-function relationships of the JAK association sites present on the IFNγ receptor polypeptides led to subsequent experiments that examined the issue of Janus kinase substrate specificity. A key question that needed to be addressed was whether the apparent obligate pairing of Jak1 and 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 chimeric molecules were generated in which the human IFNγ receptor β chain cytoplas- mic domain was replaced by either Jak2 or c-src (53). These chimeras were then expressed in murine cells that also expressed the human IFNγ receptor α sub- unit, and the ability of these murine transfectants to respond to human IFNγ
  17. 17. 578 BACH, AGUET & SCHREIBER was monitored. Cells expressing the human receptor β chain–Jak2 chimera were capable of manifesting a full complement of biologic responses to hu- man IFNγ (53). In contrast, cells expressing the receptor β chain–src chimera were unresponsive to the human ligand. These results showed that the sole obligate function of the intracellular domain of the IFNγ receptor β subunit is to chaperone Jak2 into the ligand-activated receptor complex. The results also suggested that Janus kinases may display a certain level of substrate specificity that obligates their participation in the JAK-STAT pathway and that cannot be replaced by other classes of protein tyrosine kinases. In a second study, a different family of human IFNγ receptor β chain chimeras were generated, in which the β chain cytoplasmic domain was replaced with other cytokine receptor cytoplasmic domains that utilize Janus kinases other than Jak2 (89). When expressed in hamster cells that also expressed the human IFNγ receptor α chain, all chimeric molecules were functionally competent to respond to hu- man IFNγ , although some quantitative differences were noted (89). This result implies that the specificity displayed by the Janus family kinases resides at the level of enzyme-receptor association. This concept has been strengthened by the recent generation and characterization of mice with a targeted disruption of the Jak1 gene (SJ Rodig, M Aguet, RD Schreiber, unpublished observa- tions). Taken together these results suggest that the Janus kinases are critical for 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 that are responsible for mediating the association between the receptor polypep- tides, Jak1 and Jak2, they did not account for how the receptor-associated JAK kinases were activated. One possible mechanism that could account for the ligand-induced activation of IFNγ receptor-associated kinases was that ligand could effect oligomerization of the two IFNγ receptor subunits and thereby permit Jak1 and Jak2 to transactivate one another. However, until recently, data supporting this concept has been difficult to obtain. One study suggested that ligand induces the association of the IFNγ receptor α and β chains (46). Using chemical cross-linking and immunoprecipitation approaches, a complex con- taining two IFNγ receptor α chains and one to two β chains was detected only in cells that had been exposed to IFNγ . In contrast, another study claimed that the IFNγ receptor subunits preassociate with one another in the absence of ligand (88). However, the latter study employed a digitonin lysis step that failed to fully solubilize cellular membranes and generated large pieces of membrane that con- tained a variety of irrelevant integral membrane proteins. The final resolution of this issue came when ligand-induced complex formation was shown to oc- cur under physiologic conditions in the absence of chemical cross-linkers (53).
  18. 18. IFNγ RECEPTOR SIGNALING 579 In this study, immunoprecipitates derived from untreated, detergent-solubilized cells and formed with nonblocking monoclonal antibodies specific for the IFNγ receptor α chain, contained the α subunit and Jak1 but not the β subunit, Jak2, or irrelevant membrane proteins (53). In contrast, α chain immunoprecipitates from IFNγ -treated cells contained both receptor subunits and activated forms of both Jak1 and Jak2. These results unequivocally demonstrated that the re- ceptor α and β subunits are not strongly preassociated with one another on the surface of unstimulated cells but are induced to associate upon exposure to lig- and. Thus, ligand-induced receptor subunit association leads to transactivation of Jak1 and Jak2. THE STAT CONNECTION Whereas the importance of Stat1 activation in medi- ating many IFNγ -dependent responses had been established, the mechanism that coupled ligation of the IFNγ receptor to the JAK-STAT pathway remained ill defined. A clue that led to the resolution of this issue was derived from the structure-function mutagenesis analyses described above that pointed to the critical importance of a single tyrosine residue residing at position 440 in the hu- man IFNγ receptor α chain cytoplasmic domain (47). Mechanistic insights into the importance of Y440 came with the demonstration that ligation of the IFNγ receptor leads to rapid and reversible tyrosine phosphorylation of the receptor α chain intracellular domain (22, 87). Tyrosine phosphorylation was observed in cells expressing the wild-type receptor α chain, the YF440 mutant, or the 4XYF mutant (22). Thus, whereas none of the tyrosine residues were important for ligand-induced JAK activation, one residue (Y440 ) was identified that served both as a substrate site for these enzymes and as a key element in the induction of IFNγ -dependent biologic responses. The breakthrough in this area came from our observation that the YF440 mutant also failed to support IFNγ -dependent Stat1 activation (22). This result suggested a direct link between ligand-induced receptor tyrosine phosphorylation and activation of the JAK-STAT pathway. Proof of this concept was provided by studies showing that small 5–12 amino acid phosphopeptides derived from the IFNγ receptor α chain that contained the minimal sequence 440 Y(PO4 )DKPH444 were capable of directly binding to Stat1 and blocking its subsequent activation by IFNγ in a cell-free assay sys- tem (22, 90). Importantly, these phosphopeptides not only precipitated a latent form of Stat1 from crude cell lysates but also interacted with highly purified, recombinant Stat1 in the absence of additional proteins. This interaction was of moderate affinity (KD = 137 nM) and was specific because (a) Stat1 did not bind either to nonphosphorylated forms of the peptides or to irrelevant phosphopep- tides, and (b) Y440 -containing phosphopeptides did not interact strongly with irrelevant STAT molecules, such as Stat2, that are not activated by IFNγ (22, 90). Additional experiments revealed that Stat1 binding to the phosphorylated
  19. 19. 580 BACH, AGUET & SCHREIBER IFNγ receptor α chain sequence also required the presence of two additional residues, D441 and H444 , the only two other residues in this region that are also required for receptor function (47). No other receptor α chain residues were identified that played an obligate role in formation of the Stat1 docking site on the receptor. On the basis of surface plasmon resonance experiments, Stat1 binding to its phosphorylated receptor docking site was determined to be 110 times stronger than its ability to bind to a Stat1 phosphopeptide contain- ing Y701 (90). These results thus showed that IFNγ -induced Stat1 activation was an ordered, affinity-driven process. Moreover, this study represented the first demonstration that STAT proteins bind in a specific and direct manner to distinct docking sites on ligated, tyrosine-phosphorylated cytokine receptors. Parallel experiments revealed that the SH2 domain of STAT proteins was responsible for directing STAT binding to the receptor. This concept was de- rived from two types of experiments. First, antibodies specific for the Stat1 SH2 domain inhibited binding of Stat1 to the active IFNγ receptor α chain phosphopeptide (90). Second, transfer of the SH2 domain of one STAT protein to 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 directly recruited by the activated IFNα receptor, whereas a Stat2 chimera containing the Stat1 SH2 domain was directly recruited by the activated IFNγ receptor (91). Taken together, these data further refined the general model by showing that STAT proteins were specifically recruited to activated cytokine receptors based on the ability of the SH2 domain of the STAT protein to bind specifically to a ligand-induced receptor docking site. The aforementioned data suggested that similar STAT recruiting regions may be present in the intracellular domains of other cytokine receptors. This possi- bility was first confirmed in studies showing that IL-4 effects the recruitment and activation of another STAT protein, Stat6, via the IL-4 receptor α chain in 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 receptor docking sites were identified and had the sequences 577 GY(PO4 )KAFS582 and 605 GY(PO4 )KPFQ610 . Subsequent studies by several groups defined receptor docking sites for Stats 2, 3, and 5 (93–96). Critical support for the physiologic relevance of this process has come from the demonstration that STAT recruit- ment by activated cytokine receptors can be transferred from one receptor to another by transfer of STAT docking sites between receptors. This process was illustrated in experiments in which Stat 3 recruiting activity was transferred to a truncated erythropoietin receptor, which normally recruits Stat5, by trans- fer of the Stat3 docking site from the IL-6 receptor family signal transducer gp130 (94). Whereas these experiments were the first to show a transfer of
  20. 20. IFNγ RECEPTOR SIGNALING 581 STAT recruitment from one cytokine receptor to another, they did not show the transfer of biologic function. Recently a concomitant transfer of both STAT recruitment and biologic response induction was accomplished using a proto- col whereby STAT docking sites from the IL-2 receptor β chain and the IL-4 receptor α chain were interchanged (97). Thus, the sum total of these exper- iments, which grew out of the IFNγ receptor signaling studies, has led to the development of a molecular model that explains, in part, the molecular basis of signaling specificity of the members of the cytokine receptor superfamily. PHYSIOLOGIC RELEVANCE OF IFNγ -DEPENDENT STAT1 ACTIVATION Although Stat1 was originally identified as a latent cytosolic transcription fac- tor involved in IFNα and IFNγ signaling, it has subsequently been shown, by in vitro gel shift analyses, to be activated by a wide variety of other cytokines and growth factors, including IL-6, IL-10, EGF, PDGF, and growth hormone (19–21). These observations have raised questions about the specificity of the JAK-STAT signaling pathway, since many of these cytokines induce biologic responses in cells that are opposite to those induced by the interferons. How- ever, the specificity issue has largely been resolved by the recent generation of mice with a targeted disruption of the Stat1 gene (76, 77). Stat1 knockout mice were obtained in the expected Mendelian proportions and were able to reproduce. Thus, like the IFNs, Stat1 is required neither for fertility nor em- bryonic development. However, Stat1 gene-targeted mice displayed a global deficiency in their ability to respond to either IFNγ or IFNα. Specifically, cells derived from these animals were unable to initiate transcription of a variety of IFN-inducible genes, such as interferon regulatory factor-1 (IRF-1), guanylate binding protein-1 (GBP-1), MHC class II transactivator (CIITA), and the com- plement protein C3, or to synthesize IFN-induced proteins. Moreover, Stat1 knockout mice were exquisitely sensitive to infection by a variety of micro- bial pathogens (such as Listeria monocytogenes) and viruses (such as vesicular stomatitis virus). Thus, the overall phenotype of the Stat1 knockout mice was one that encompassed the combined phenotypes of mice that are unresponsive to 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 is required for the induction of most, if not all, IFN-dependent biologic responses and demonstrate that, in a physiologic setting, Stat1 plays a dedicated role in signaling only for interferon-mediated biologic effects. Thus, the specificity shown by IFNγ in effecting biologic responses in cells is dependent on two temporally and spatially distinct processes: the specific recruitment of Stat1 to the activated IFNγ receptor at the plasma membrane and the specific induction in the nucleus of IFNγ -activated gene transcription by Stat1 homodimers.
  21. 21. 582 BACH, AGUET & SCHREIBER Figure 5 Proposed signaling mechanism of the IFNγ receptor. The details of this model are described in the text.
  22. 22. IFNγ RECEPTOR SIGNALING 583 THE MODEL OF IFNγ RECEPTOR SIGNAL TRANSDUCTION The results discussed above can now be put together to form one of the most complete models of cytokine receptor signaling to date (Figure 5) (53). In un- stimulated cells, the IFNγ receptor α and β subunits are not preassociated with each other but rather associate through their intracellular domains with inactive forms of specific Janus family kinases. Jak1 and Jak2 constitutively associate with the receptor α and β chains, respectively. Addition of IFNγ , a homod- imeric ligand, to the cells induces the rapid dimerization of receptor α chains, thereby forming a site that is recognized, in a species-specific manner, by the extracellular domain of the receptor β subunit. The ligand-induced assembly of the complete receptor complex containing two α and two β subunits brings into close juxtaposition the intracellular 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 Y440 residue on the receptor α subunit, thereby forming a paired set of Stat1 dock- ing sites on the ligated receptor. Two Stat1 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 Stat1 Y701 residue. Tyrosine-phosphorylated Stat1 molecules dissociate from their recep- tor tether and form homodimeric complexes. The activated Stat1 complex is then 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 translocates to the nucleus and, after binding to a specific sequence in the promoter region of immediate-early IFNγ -inducible genes, effects gene transcription. Thus, IFNγ signaling is an ordered, affinity-driven process that derives its specificity from (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 Stat1 homodimer to specifically activate IFNγ -induced gene transcription. PHYSIOLOGICAL CONSEQUENCES OF IFNγ RECEPTOR DISREGULATION IFNγ Receptor α Chain Deficiencies in Microbial Infection The physiologic consequences of inactivating mutations within the genes en- coding the IFNγ receptor subunits have become evident through the analysis of experimental murine models and naturally occurring human genetic defects. Mice carrying a disruption in the murine IFNγ receptor α chain gene displayed a phenotype consistent with previous in vitro and in vivo studies of mice treated
  23. 23. 584 BACH, AGUET & SCHREIBER with neutralizing IFNγ -specific monoclonal antibodies (99, 103). IFNγ re- ceptor α chain−/− (IFNγ R−/− ) mice exhibited no overt defects in embryonic development and showed normal development of lymphoid compartments and the immune system. These animals displayed a greatly impaired ability to resist infection by a variety of microbial pathogens including Listeria monocytogenes, Leishmania major, and several mycobacteria species, including M. bovis and M. avium, despite the fact that the mice developed normal helper and cytotoxic T cell responses to these pathogens (99, 104). This result demonstrates that the IFNγ receptor plays a critical role in the expression of innate host resistance to microbial infection. In contrast, IFNγ R−/− mice were able to mount a curative response to many viruses, indicating that this receptor system is not the major mediator of antiviral effects in vivo (100). On the basis of these results, the prediction was made that mutations in the human IFNγ receptor α chain gene might result in individuals with recurrent microbial but not viral infections. Recently two groups have simultaneously identified such mutations in children who manifest a severe susceptibility to weakly pathogenic mycobacterial species (105, 106). 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 (105). Genetic analysis of these children revealed an inactivating mutation in the IFNγ receptor α chain gene. In the other study, a Tunisian child was identified with disseminated M. bovis infection following Bacillus Calmette-Gu´ rin (BCG) vaccination (106). e BCG, an attenuated M. bovis strain, is used in many countries as a live vaccine against human tuberculosis and leprosy. In most children, BCG vaccination is innocuous. However, in an extremely small percentage of the population, BCG vaccination results in a disseminated infection that is often fatal. A French na- tional study found that approximately half of the individuals with disseminated BCG 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 receptor α chain gene (106). Subsequently, another unrelated child was identified with a similar but not identical mutation (107). Genetic analysis 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 these patients 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 curative responses to several different viruses (105–107). It will be important in the future to determine why IFNγ receptor defects lead exclusively to enhanced susceptibility to mycobacterial infection.
  24. 24. IFNγ RECEPTOR SIGNALING 585 IFNγ Receptor α Chain Deficiencies in Cancer Overexpression of a truncated murine IFNγ receptor α chain in certain trans- plantable murine tumors rendered the tumors unresponsive to IFNγ in a dom- inant negative manner. These IFNγ unresponsive tumors resisted rejection when transplanted into naive and immune syngeneic hosts (51). These studies clearly identified an important role for IFNγ in promoting tumor immunogenic- ity. These observations also raised the question of whether IFNγ plays a critical role in promoting host surveillance that is capable of controlling the growth of primary tumors. To address this issue, IFNγ receptor α chain knockout mice or wild-type 129/Sv syngeneic control mice were treated with three doses of the chemical carcinogen 3-Methylcolanthrene and were monitored over a 130-day period for tumor development. At every dose tested, IFNγ insensitive animals developed 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). The tumors originating from IFNγ R−/− mice, when explanted and reintroduced into naive control and IFNγ R−/− mice, grew progressively in both types of host. Moreover, when IFNγ signaling was restored in one tumor by expression of the IFNγ receptor α chain, this tumor was now rejected by naive 129/Sv hosts. These data indicate that tumors that develop from IFNγ unresponsive tissues may be able to circumvent detection and rejection by the host immune system. Thus, IFNγ plays a key role in a system of tumor surveillance in immunocompetent hosts. These results predict that some human tumors may develop spontaneous mutations in the IFNγ signaling system that render them IFNγ insensitive. This process would thereby contribute to successful tumor establishment in a naive but immunocompetent host. This possibility has in fact been real- ized in an examination of isolated human lung carcinomas that were tested for defects in IFNγ and IFNα signaling capabilities (DH Kaplan, E Stock- ert, LJ Old, RD Schreiber, unpublished observations). Several tumors of one histologic classification (25% of adenocarcinomas) displayed insensitivity to IFNγ , whereas only one tumor was insensitive to both IFNγ and IFNα. No tumors were found that displayed a selective IFNα insensitivity. Analysis of the different tumors showed that some lacked expression of the IFNγ receptor α chain, some expressed an abnormally active Jak2 enzyme, and the tumor with combined insensitivity to both IFNγ and IFNα did not express Jak1. Re- constitution experiments showed that IFNγ responsiveness was restored in the appropriate tumor cells following replacement of the missing or abnormal re- ceptor α 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
  25. 25. 586 BACH, AGUET & SCHREIBER that occur within developing tumor cells favor the pathologic outgrowth of the neoplastic cells. CONCLUDING REMARKS In this review we have focused on the recent events that have led to the synthesis of a comprehensive model of the IFNγ signaling pathway. Although this work has been ongoing since the early 1980s, the most rapid advances have occurred during the last five years. During this time, most if not all of the components of the IFNγ receptor and IFNγ signal transduction system were identified and the critical molecular interactions defined that established the temporal and topographical relationships that make this an effective and specific signaling pathway. This work has revealed the central role played by a single transcription factor known as Stat1, which now is recognized as the direct link between the IFNγ receptor and IFNγ -inducible genes. Undoubtedly, future work will further refine the IFNγ signaling model. However, even now, this model serves as the general paradigm for signaling through other members of the cytokine receptor superfamily. ACKNOWLEDGMENTS The authors are particularly grateful to Dr. TL Nagabhushan for providing the photographs of the IFNγ -IFNγ receptor crystal structure and, together, with Schering-Plough Research Institute, for supplying the financial support for re- producing this figure. We are also grateful to Drs. Gilles Merlin, Zlatko Dembic and Jean-Laurent Casanova and to Dan Kaplan for sharing their unpublished data. The authors also wish to thank Dan Kaplan and Keith Pinckard and Drs. Paul Allen, Antonio Celada, and Tony Kossiakoff for critical comments. Work in the Schreiber laboratory has been supported by grants from NIH and Genentech, Inc. Visit the Annual Reviews home page at Literature Cited 1. Farrar MA, Schreiber RD. 1993. The feron gamma. Proc. Natl. Acad. Sci. USA molecular cell biology of interferon-γ 84:4151–55 and its receptor. Annu. Rev. Immunol. 3. Pfizenmaier K, Wiegmann K, Scheurich 11:571–611 P, Kr¨ nke M, Merlin G, Aguet M, o 2. Jung V, Rashidbaigi A, Jones C, Tis- Knowles BB, Ucer U. 1988. High affin- chfield JA, Shows TB, Pestka S. 1987. ity human IFN-gamma-binding capacity Human chromosomes 6 and 21 are re- is encoded by a single receptor gene lo- quired for sensitivity to human inter- cated in proximity to c-ras on human chro-
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