IL-12 Receptor

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IL-12 Receptor Clemens Esche, Michael R. Shurin and Michael T. Lotze* Biological Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA * corresponding author tel: 412-624-9375, fax: 412-624-1172, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14006.

SUMMARY The functional high-affinity IL-12 receptor is composed of at least two -type receptor subunits, each independently exhibiting a low affinity for IL-12. Both subunits are members of the cytokine receptor superfamily. IL-12 p40 interacts primarily with IL12R 1, while IL-12 p35 interacts primarily with the signal-transducing 2 subunit. IL-12 signaling involves activation of the receptor-associated tyrosine kinases JAK2 and TYK2 and downstream tyrosine phosphorylation of the transcription factors STAT3 and STAT4 (the central signal transducer of IL-12). The IL-12R is expressed primarily on activated T and NK cells. Expression of the 2 subunit determines TH1 development since the 2 component is selectively downregulated on TH2 cells. Inhibiting IL-12 responsiveness represents an experimental therapy for TH1-driven inflammatory and autoimmune diseases.

BACKGROUND

Discovery A cDNA encoding a type I transmembrane protein representing a low-affinity component of the functional IL-12 receptor was identified on PHAactivated human PBMCs (Chua et al., 1994). Receptors with this type of cytoplasmic region have been classified as -type cytokine receptors (Stahl and Yancopoulos, 1993). Consequently, the new protein was termed IL-12R (Chua et al., 1994). The corresponding murine cDNA was isolated using crosshybridization (Chua et al., 1995). More recently,

an additional -type IL-12R protein was identified and designated IL-12R 2 (Presky et al., 1996), while the previously reported subunit was reclassified as IL12R 1 (Presky et al., 1996). A recent report suggests the existence of a third chain (Kawashima et al., 1998).

Alternative names IL-12R 1 was originally termed IL-12R (Presky et al., 1996).

Structure The functional high-affinity IL-12R is a heterodimer consisting of a 1 and a 2 chain (Gately et al., 1998).

Main activities and pathophysiological roles The IL-12R is differentially expressed on T cell subsets. NaõÈ ve T cells do not express the IL-12R, while antigen stimulation induces expression of both IL12R subunits. Cells developing along the TH1 pathway continue to express both receptor components, whereas TH2 cells selectively lose the signal-transducing component IL-12R 2 during differentiation. Consequently, expression levels of the 2 subunit represent a therapeutic target for the redirection of ongoing T cell responses. See also Main activities and pathophysiological roles in the IL-12 chapter.

1504 Clemens Esche, Michael R. Shurin and Michael T. Lotze

GENE

Sequence

Accession numbers

See Figure 1. Also see Chua et al. (1994) for human 1, Chua et al. (1995) for murine 1, and Presky et al. (1996) for 2.

GenBank: Human cDNA: U03187 ( 1), U64198 ( 2) Murine cDNA: U23922 ( 1), U64199.1 ( 2)

Description of protein

Chromosome location and linkages Human IL-12R 1 localizes to a region in chromosome 19 at band p13.1, while human IL-12R 2 localizes to chromosome 1 at band p31.2 (Yamamoto et al., 1997).

The 1 subunit is a 662 amino acid type I transmembrane protein with an extracellular domain of 516 amino acids and a cytoplasmic domain of 91 amino acids lacking tyrosine residues (Chua et al., 1994). In contrast, the 2 subunit, which consists of 862 amino acids, has three tyrosine residues in the cytoplasmic domain (Presky et al., 1996).

Relevant homologies and species differences

PROTEIN

Accession numbers GenBank: Human IL-12R: AAA21340.1 ( 1), AAB36675.1 ( 2) Murine IL-12R: AAA87457.1 ( 1), AAB36676.1 ( 2) SwissProt: Human protein: P42701 ( 1)

Both IL-12R subunits are homologous to gp130 (a receptor subunit for IL-6), G-CSF, IL-11, oncostatin M, CNTF and LIF receptors (Chua et al., 1994; Presky et al., 1996; Trinchieri, 1998). Human and murine IL-12R 1 demonstrate a 54% amino acid homology (Chua et al., 1995). Human and murine

Figure 1 Sequencesfor IL-12R 1 and IL-12R 2: Human IL-12Rβ1 1 MEPLVTWVVP 61 ECSWQYEGPT 121 NQTEKSPEVT 181 WKLGDCGPQD 241 VRFSVEQLGQ 301 LHLGKMPYLS 361 RAQSMTYCIE 421 SAHPEKLTLW 481 YVVRCRDEDS 541 VQVSDWLIFF 601 WINPVDFQEE 661 KM Human IL-12Rβ2 1 MAHTFRGCSL 61 HYSRRNKLIL 121 GVAPEQPQNL 181 LDFGINLTPE 241 RCTLYWRDEG 301 YKGSWSDWSE 361 QVTLQELTGG 421 LLAPRQVSAN 481 VSALISENIK 541 WNSIPVQEQM 601 LTAAGESSHG 661 WCSREIPDPA 721 PPCSNWPQRE 781 NPACPWTVLP 841 TFSCGDKLTL

LLFLFLLSRQ AGVSHFLRCC LQLYNSVKYE DDTESCLCPL DGRRRLTLKE GAAYNVAVIS WQPVGQDGGL STVLSTYHFG KQVSEHPVQP ASLGSFLSIL ASLQEALVVE

GAACRTSECC LSSGRCCYFA PPLGDIKVSK EMNVAQEFQL QPTQLELPEG SNQFGPGLNQ ATCSLTAPQD GNASAAGTPH TETQVTLSGL LVGVLGYLGL MSWDKGERTE

FQDPPYPDAD AGSATRLQFS LAGQLRMEWE RRRQLGSQGS CQGLAPGTEV TWHIPADTHT PDPAGMATYS HVSVKNHSLD RAGVAYTVQV NRAARHLCPP PLEKTELPEG

SGSASGPRDL DQAGVSVLYT TPDNQVGAEV SWSKWSSPVC TYRLQLHMLS EPVALNISVG WSRESGAMGQ SVSVDWAPSL RADTAWLRGV LPTPCASSAI APELALDTEL

RCYRISSDRY VTLWVESWAR QFRHRTPSSP VPPENPPQPQ CPCKAKATRT TNGTTMYWPA EKCYYITIFA LSTCPGVLKE WSQPQRFSIE EFPGGKETWQ SLEDGDRCKA

AFMFIITWLL YKFDRRINFH SCIQKGEQGT SPESNFTAKV LVLLNRLRYR SLRAQTPEEE KAMTQNITGH SEGMDNILVT SYICYEIRVY GCLLHYRIYW NEREFCLQGK NSTCAKKYPI KGIQGHQASE AGDLPTHDGY DQLKMRCDSL

IKAKIDACKR HGHSLNSQVT VACTWERGRD TAVNSLGSSS PSNSRLWNMV PTGMLDVWYM TSWTTVIPRT WQPPRKDPSA ALSGDQGGCS KERDSNSQPQ ANWMAFVAPS AEEKTQLPLD KDMMHSASSP LPSNIDDLPS ML

GDVTVKPSHV GLPLGTTLFV THLYTEYTLQ SLPSTFTFLD NVTKAKGRHD KRHIDYSRQQ GNWAVAVSAA VQEYVVEWRE SILGNSKHKA LCEIPYRVSQ ICIAIIMVGI RLLIDWPTPE PPPRALQAES HEAPLADSLE

ILLGSTVNIT CKLACINSDE LSGPKNLTWQ IVRPLPPWDI LLDLKPFTEY ISLFWKNLSV NSKGSSLPTR LHPGGDTQVP PLSGPHINAI NSHPINSLQP FSTHYFQQKV DPEPLVISEV RQLVDLYKVL ELEPQHISLS

CSLKPRQGCF IQICGAEIFV KQCKDIYCDY RIKFQKASVS EFQISSKLHL SEARGKILHY INIMNLCEAG LNWLRSRPYN TEEKGSILIS RVTYVLWMTA FVLLAALRPQ LHQVTPVFRH ESRGSDPKPE VFPSSSLHPL

IL-12 Receptor 1505 IL-12R 2 proteins demonstrate a 68% amino acid sequence identity (Presky et al., 1996). Although similar with respect to their molecular properties, significant differences exist between mice and humans. Ba/F3 cells expressing murine IL-12R 1 exhibited two binding affinities for 125I-mouse IL-12: 50 pM and 470 pM (Chua et al., 1995), while Ba/F3 cells transfected with human IL-12R 1 bound 125I-human IL12 only with very low affinity (> 50 nM) (Presky et al., 1996). These differences should be considered when evaluating potential therapeutic IL-12 antagonists directed against IL-12R in murine models of human disease. Furthermore, expression of IL-12R 2 mRNA is regulated differentially in mice and humans. In mice, IFN treatment of early developing TH2 cells maintains IL-12R 2 expression (Szabo et al., 1997). In humans, IFN induces expression of IL-12R 2 during in vitro T cell differentiation (Rogge et al., 1997).

Affinity for ligand(s) Analysis of steady-state binding data of IL-12 by Scatchard analysis identified a single binding site on PHA-activated human lymphoblasts with an equilibrium dissociation constant of 100±600 pmol/L and 1000±9000 sites per cell (Chizzonite et al., 1992). More recently, three classes of IL-12-binding sites were identified on PHA-activated human T lymphoblasts: high-affinity (Kd ˆ 5±20 pM) (100±1000 sites/ cell), intermediate-affinity (Kd ˆ 50±200 pM) (200± 1000 sites/cell), and low-affinity (Kd ˆ 2±6 nM) (1000± 5000 sites/cell) (Chua et al., 1994; Stern et al., 1997). Both IL-12R 1 and IL-12R 2 bind IL-12 with only low affinity when transfected into COS-7 cells. However, coexpression of both human subunits results in high- (Kd ˆ 55 pM) and low-affinity IL-12binding sites (Kd ˆ 8 nM) and a receptor complex capable of signaling (Presky et al., 1996). Dissociation constants on concanavalin (Con A)-stimulated murine splenocytes were determined to be 40 pM (100±500 sites/cell), 200 pM (600±800 sites/cell) and 7 nM (1000±2000 sites/cell) (Stern et al., 1997).

Cell types and tissues expressing the receptor Presence was detected mostly on activated T cells and NK cells (Desai et al., 1992). Dendritic cells express a single class of high-affinity IL-12R (Grohmann et al., 1998). In addition, IL-12R 1 was detected on human B cell lines and was upregulated on activated human

peripheral blood or tonsillar B lymphoblasts that, however, failed to bind measurable amounts of IL-12 (Benjamin et al., 1996; Wu et al., 1996). TH1 cells express both the 1 and 2 subunits, whereas TH2 cells express only the 1 subunit due to selective downregulation of 2 (Rogge et al., 1997; Szabo et al., 1997). Consequently, only TH1 cells are capable of signaling in response to IL-12. Murine B10.D2 T cells express IL-12R 2 at sufficient levels to allow functional responses to IL-12, whereas Balb/c T cells express low levels of 2 and demonstrate limited functional IL-12-induced STAT4 phosphorylation (Guler et al., 1997).

Regulation of receptor expression IL-4 rapidly extinguishes IL-12R 2 chains in vitro (Szabo et al., 1997; Wu et al., 1997a) and in vivo (Himmelrich et al., 1998), while IFN maintains the ability to signal through IL-12R 2 both in vitro (Szabo et al., 1997) and in vivo (Himmelrich et al., 1998). Thus, IFN and IL-4 promote the development of TH1 and TH2 responses by differential regulation of IL-12 receptor expression (Gollob et al., 1997). TGF and IL-10 prevent TH1 responses by nearly completely suppressing expression of highaffinity IL-12-binding sites, whereas expression of IL12R 1 is only partially inhibited (Wu et al., 1997a). Cholera toxin inhibits the expression of both 1 and 2 chains (Braun et al., 1999). T cells from patients with Sezary syndrome express little or no message for the IL-12R 2 subunit and exhibit highly reduced levels of STAT4 (Showe et al., 1999). Prostaglandin E2 (PGE2) and dexamethasone inhibit IL-12R 1 expression and mRNA for IL-12R 2 (Wu et al., 1998). In contrast, IL-2, IL-7, IL-15, phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies induce IL-12R 1 expression (Wu et al., 1997a). The natural killer T (NKT) cell ligand -galactosylceramide (-GalCer) induces mRNA for both IL-12R 1 and IL-12R 2 (Kitamura et al., 1999). Upregulation of the receptor correlates with the ability of the cells to proliferate in response to IL-12 (Chizzonite et al., 1992; Desai et al., 1992).

SIGNAL TRANSDUCTION

Associated or intrinsic kinases IL-12 receptor stimulation activates the receptorassociated tyrosine kinases JAK2 and TYK2 (Bacon et al., 1995a). In addition, an isoform of MAP kinase

1506 Clemens Esche, Michael R. Shurin and Michael T. Lotze is phosphorylated (Pignata et al., 1994). IL-12 also induces tyrosine phosphorylation of the src family lck tyrosine kinase (Pignata et al., 1995).

Cytoplasmic signaling cascades IL-12 binding to its receptor on T and NK cells induces simultaneous activation of two members of the Janus kinase (JAK) family of protein tyrosine kinases, JAK2 and TYK2 (Bacon et al., 1995a). TYK2 interacts with the 1 subunit of the IL-12 receptor and JAK2 interacts with the 2 subunit (Zou et al., 1997). Both JAK2 and TYK2 also transduce signals via other cytokine receptors including type 1 (Velazquez et al., 1992) and type 2 IFN (Watling et al., 1993), IL3 (Silvennoinen et al., 1993), IL-6 (Stahl et al., 1994), and GM-CSF (Quelle et al., 1994). NOS2-derived nitric oxide is a prerequisite for IL-12-induced activation of TYK2 in NK cells (expressing NOS2) but not in T cells (lacking NOS2) (Diefenbach et al., 1999). JAK kinases phosphorylate the IL-12 receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for transcription factors termed signal transducers and activators of transcription (STAT). IL-12 binding to its receptor results in activation of three members of the STAT family. STAT1 can dimerize with either STAT3 or STAT4 and STAT4 can dimerize with STAT3 (Jacobson et al., 1995; Yu et al., 1996). Only STAT4, but neither STAT1 nor STAT3 is activated directly through the IL-12 receptor (Naeger et al., 1999). Only IL-12 and IFN induce tyrosine phosphorylation and DNA binding of STAT4 (Bacon et al., 1995b; Cho et al., 1996). This signaling pathway is therefore relatively specific to IL-12. Deletion of the STAT4 gene in knockout mice results in defective responses specific to IL-12 (Thierfelder et al., 1996; Kaplan et al., 1996). Tyrosine phosphorylation of STAT proteins induces their dimerization and subsequent translocation to the nucleus where they bind to related DNA sequences and regulate transcription (Darnell et al., 1994). Signaling induced by IL-12 varies with the particular cell evaluated.

DOWNSTREAM GENE ACTIVATION

Transcription factors activated STAT1, STAT3, STAT4 (Trinchieri, 1998), NFB and c-Jun (Zhang et al., 1999) are all activated by IL-12R.

Genes induced IL-12 induces upregulation of IFN (with costimulus of IL-18), the IL-18R (Xu et al., 1998), the IL-12R 2, IRF-1 (Coccia et al., 1999), ERM (Ouyang et al., 1999) and other, yet to be identified genes.

Promoter regions involved Early reports suggested that IL-12 alone does not activate IFN promoter activity (Jacobson et al., 1995). However, c-Jun represents at least one transcription factor induced by IL-12 that binds to the IFN promoter and can stimulate transcription (Zhang et al., 1999). In addition, IL-12 strongly induces IFN promoter activity in the presence of costimulatory signals provided by soluble antibodies to CD3 and CD28 (Barbulescu et al., 1998). Both STAT4 and AP-1 are required for IFN promoter activation (Barbulescu et al., 1998). Activated STAT4 binds to the GAS element in the IRF-1 promoter (Coccia et al., 1999). The precise mechanism by which STAT proteins mediate transcriptional activation remain to be elucidated (Hoey and Grusby, 1999).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Antibodies that activate the receptor have not been identified.

Phenotypes of receptor knockouts and receptor overexpression mice IL-12R 1-deficient (IL-12R 1ÿ/ÿ) mice are grossly indistinguishable from their control littermates (Wu et al., 1997b). However, splenocytes from IL-12Rÿ/ÿ are deficient in all IL-12-induced biologic activities including proliferation, IFN secretion, TH1 development and enhancement of NK lytic activity (Wu et al., 1997b). In addition, IL-12R 1ÿ/ÿ mice exhibit a severe defect in their ability to generate IFN producing TH1 cells, whereas generation of TH2 cells is moderately enhanced (Wu et al., 1997b).

IL-12 Receptor 1507

Human abnormalities Recessive mutations in the gene encoding the IL12R 1 subunit have been identified in seven unrelated individuals with idiopathic mycobacterial and Salmonella infections (Altare et al., 1998; de Jong et al., 1998). Their cells were deficient in IL-12R signaling and IFN production and their remaining T cell responses were independent of endogenous IL-12. Thus, the lack of IL-12R 1 expression results in a human immunodeficiency and demonstrates the essential role of IL-12 in resistance to infections due to intracellular bacteria.

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors Monoclonal antibodies against IL-12R 1 inhibit IL-12-induced proliferation of activated T cells, IL12-induced secretion of IFN by resting PBMCs, and IL-12-mediated lymphokine-activated killer cell activation (Wu et al., 1996). Thus, the IL-12R 1 chain appears to be an essential component of the functional IL-12R on both T and NK cells.

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IL-12 Receptor 1509

ACKNOWLEDGEMENTS

LICENSED PRODUCTS IL-12R has not been licensed. R&D Systems (www.rndsystems.com), (www.sigma-aldrich.com). Antibodies: Flow cytometry: PharMingen (www.pharmingen.com)

Sigma

This work has been supported in part by the 1999 Advanced Polymer Systems Research Fellowship of the Dermatology Foundation to CE, NIH Grant CA 80126 to MRS, and NIH Grants CA 68067 and CA 73743 to MTL. We apologize to all colleagues whose contributions have not been directly referenced in this chapter.