Deciphering site 3 interactions of interleukin 12 and interleukin 23 with their cognate murine and human receptors.

Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common ß subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rß1·IL-12Rß2 receptor complex, and IL-23 uses also IL-12Rß1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rß1 is mediated by p40, and binding to IL-12Rß2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rß2 in a cross-species manner. Although Trp157 is conserved between murine and human IL-23p19 (Trp156 in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rß1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rß2 and IL-23R in a murine/human cross-species manner.

Defining the functional binding sites of interleukin 12 receptor ß1 and interleukin 23 receptor to Janus kinases.

The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor ß1 (IL-12Rß1) as one component of their receptor signaling complexes, with IL-12Rß2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rß1, whereas Jak2 binds to IL-23R and also to IL-12Rß2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rß1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rß1 and Jak2 by IL­23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable.

Human and Murine Interleukin 23 Receptors Are Novel Substrates for A Disintegrin and Metalloproteases ADAM10 and ADAM17.

IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor ß1 (IL-12Rß1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17.

Activated ErbB3 Translocates to the Nucleus via Clathrin-independent Endocytosis, Which Is Associated with Proliferating Cells.

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin ß1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues.

Insights into IL-23 biology: From structure to function.

Interleukin (IL-)23 is a central cytokine controlling TH17 development. Overshooting IL-23 signaling contribute to autoimmune diseases. Moreover, GWAS studies have identified several SNPs within the IL-23 receptor, which are associated with autoimmune diseases. IL-23 is a member of the IL-12-type cytokine family and consists of IL-23p19 and p40. Within the IL-12 family, IL-12 and IL-23 share the p40 cytokine subunit and the IL-12Rß1 as one chain of the receptor complex. For signaling, IL-23 triggers heterodimerization of IL-12Rß1 and the IL-23R. Subsequently, signal transduction pathways including JAK/STAT, MAPK and PI3K are activated. Most studies have investigated the biological relevance of IL-23 in the development of TH17 cells and autoimmunity, whereas less is known about the molecular context of IL-23 biology. Therefore, we focused on IL-23 receptor complex assembly, signal transduction and functional relevance of IL-23R SNPs in the context of IL-23-inhibitory principles.

Non-canonical interleukin 23 receptor complex assembly: p40 protein recruits interleukin 12 receptor ß1 via site II and induces p19/interleukin 23 receptor interaction via site III.

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor ß1 (IL-12Rß1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rß1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rß1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rß1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rß1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rß1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rß1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.

Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rß2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rß2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

Identification of canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the intracellular domain of the interleukin 23 receptor.

Signaling of interleukin 23 (IL-23) via the IL-23 receptor (IL-23R) and the shared IL-12 receptor ß1 (IL-12Rß1) controls innate and adaptive immune responses and is involved in the differentiation and expansion of IL-17-producing CD4(+) T helper (TH17) cells. Activation of signal transducer and activator of transcription 3 (STAT3) appears to be the major signaling pathway of IL-23, and STAT binding sites were predicted in the IL-23R but not in the IL-12Rß1 chain. Using site-directed mutagenesis and deletion variants of the murine and human IL-23R, we showed that the predicted STAT binding sites (pYXXQ; including Tyr-504 and Tyr-626 in murine IL-23R and Tyr-484 and Tyr-611 in human IL-23R) mediated STAT3 activation. Furthermore, we identified two uncommon STAT3 binding/activation sites within the murine IL-23R. First, the murine IL-23R carried the Y(542)PNFQ sequence, which acts as an unusual Src homology 2 (SH2) domain-binding protein activation site of STAT3. Second, we identified a non-canonical, phosphotyrosine-independent STAT3 activation motif within the IL-23R. A third predicted site, Tyr-416 in murine and Tyr-397 in human IL-23R, is involved in the activation of PI3K/Akt and the MAPK pathway leading to STAT3-independent proliferation of Ba/F3 cells upon stimulation with IL-23. In contrast to IL-6-induced short term STAT3 phosphorylation, cellular activation by IL-23 resulted in a slower but long term STAT3 phosphorylation, indicating that the IL-23R might not be a major target of negative feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. In summary, we characterized IL-23-dependent signal transduction with a focus on STAT3 phosphorylation and identified canonical tyrosine-dependent and non-canonical tyrosine-independent STAT3 activation sites in the IL-23R.