Non-canonical interleukin 23 receptor complex assembly: p40 protein recruits interleukin 12 receptor β1 via site II and induces p19/interleukin 23 receptor interaction via site III

Abstract

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 β1 (IL-12Rβ1). 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-12Rβ1 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-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. 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-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 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.

Keywords: Cytokine; Cytokine Action; Interleukin; Interleukin 12 Receptor; Interleukin 23; Interleukin 23 Receptor; Molecular Modeling; Mutagenesis; p19; p40.

FIGURE 1.

Binding of p19 to IL-12Rβ1 and IL-23R is dependent on p40. A, IL-23_p40 (green, domains D1-D3) and IL-23_p19 (blue) bind to IL-12Rβ1 (orange, domains D1 and D2) and IL-23R (pink, domain D1). B, coimmunoprecipitation of FLAG-tagged p40 or p19 and Fc-tagged sIL-12Rβ1 or sIL-23R using protein A-agarose. One of three independent experiments is shown. L, lysates; +, coimmunoprecipitates; C, controls (without sIL-12Rβ1 or sIL-23R); WB, Western blot. C, Western blotting of secreted p40 and/or p19 from transfected COS-7 cells. D, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with increasing amounts of the indicated cytokine-conditioned supernatants (CS). Data are mean ± S.D. The concentration of p19/p40 and p40-p19-Fp was 200 ng/ml and 685 ng/ml, respectively, as determined by p40-ELISA.

FIGURE 2.

Characterization of site I in p19. A, model of site I of p19 (blue) toward p40 (green). B, Western blotting (WB) of secreted p40 and/or p19_R179E, K66A, I176E, A178W, and R179A from transfected COS-7 cells. C, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ± S.D. D, co-IP of c-myc-tagged p40 and FLAG-tagged p19 or p19_R179E, K66A, I176E, A178W, and R179A. L, lysates; +, coimmunoprecipitates; C, controls (without mAbs).

FIGURE 3.

Targeted site I mutations in p40 prevent the formation of IL-23. A, model of p40 (green) toward site I of p19 (blue). B, Western blotting (WB) of secreted p19, p40_Y265K, and p40_Y318K. One of three independent experiments is shown. C, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ± S.D. D, co-IP of c-myc-tagged p19 and FLAG-tagged p40 or the p40 mutants Y265K and Y318K. L, lysates; +, coimmunoprecipitates; C, controls (without mAbs). One of three independent experiments is shown.

FIGURE 4.

Binding of deletion variants of p40 to p19. A, Western blotting (WB) of secreted p19 and p40, p40_D1, p40_D2, p40_D3, p40_D1D2, and p40_D2D3. One of three independent experiments is shown. B, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ±S.D. C, co-IP of FLAG-tagged p40 variants and c-myc-tagged p19. L, lysates; +, coimmunoprecipitates; C, controls (without mAbs). One of three independent experiments is shown.

FIGURE 5.

Characterization of p19 site III. A, model of the predicted site III of p19 (blue) toward IL-23R (pink). B, Western blotting (WB) of secreted p40 and p19 or p19_W157A, p40, p40-p19-Fp, or p40-p19_W157A-Fp. One of three independent experiments is shown. C, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ± S.D. D, co-IP of FLAG-tagged p40 and p19 or p19_W157A and Fc-tagged sIL-12Rβ1 or sIL-23R. One of three independent experiments is shown. E, co-IP of FLAG-tagged p40-p19_wt and _W157A fusion proteins and Fc-tagged sIL-12Rβ1 or sIL-23R. L, lysates; +, coimmunoprecipitates; C, controls (without sIL-12Rβ1-Fc or sIL-23R-Fc). One of three independent experiments is shown.

FIGURE 6.

Absence of a classical site II in p19. A, model of the predicted site II of p19 (blue) toward IL-12Rβ1 (orange). B, Western blotting (WB) of secreted p40 and p19_L36E, p19_L135E, or p19_R38E. One of three independent experiments is shown. C, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ± S.D. D, co-IP of FLAG-tagged p40 and p19, p19_L36E, p19_L135E, or p19_R38E with Fc-tagged sIL-12Rβ1 or sIL-23R. L, lysates; +, coimmunoprecipitates; C, controls (without sIL-12Rβ1-Fc or sIL-23R-Fc). One of three independent experiments is shown. E, Western blotting of secreted p40-p19-Fp and p40-p19_L36E_R38E_L135E-Fp. F, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells stimulated with the indicated cytokines (10% conditioned cell culture supernatant). One of three independent experiments is shown. Data are mean ± S.D. G, co-IP of FLAG-tagged p40-p19-Fp or p40-p19_L36E_L135E_R38E-Fp with Fc-tagged sIL-12Rβ1 or sIL-23R. One of three independent experiments is shown.

FIGURE 7.

p40 substitutes for the site II interaction of p19 toward IL-12Rβ1. A, co-IP of FLAG-tagged p40 variants and Fc-tagged sIL-12Rβ1. One of three independent experiments is shown. WB, Western blot. B, co-IP of FLAG-tagged p40 and Fc-tagged sIL-12Rβ1 deletion variants. One of three independent experiments is shown. C, model of the predicted site II of p19 (blue) toward IL-12Rβ1 (orange). D, co-IP of FLAG-tagged p40 and Fc-tagged sIL-12Rβ1 point mutation variants. One of three independent experiments is shown. L, p40 lysate; +, coimmunoprecipitates; C, control (without sIL-12Rβ1 variant).

FIGURE 8.

p40_D2D3-p19 specifically interacts with IL-23R but not with IL-12Rβ1 and acts as an agonist independently of IL-12Rβ1. A, co-IP of FLAG-tagged p40_D2D3 and p19 and Fc-tagged sIL-12Rβ1 or sIL-23R. L, lysates; +, coimmunoprecipitates; C, controls (without sIL-12Rβ1-Fc or sIL-23R-Fc); WB, Western blot. One of three independent experiments is shown. B, co-IP of the fusion protein p40_D2D3-p19-Fp with Fc-tagged sIL-12Rβ1 or sIL-23R. One of three independent experiments is shown. C and D, Coomassie staining (C) and Western blotting (D) of purified p40_D2D3-p19Fc. E, co-IP of myc-tagged sIL-23R and recombinant p40_D2D3-p19Fc. L, lysates; +, coimmunoprecipitates; C, controls (without sIL-23R). One of three independent experiments is shown. F–H, cell viability assay of Ba/F3-IL-12Rβ1-IL-23R cells (F and G), Ba/F3-IL-12Rβ1 cells (H), and Ba/F3-IL-23R cells (H) stimulated with the indicated recombinant cytokines. One of three independent experiments is shown. Data are mean ± S.D.

FIGURE 9.

Predicted and experimentally validated model of the IL-23 receptor complex. A, predicted model with canonical sites I, II, and III of p19. B, experimentally validated model with sites I and III in p19 and major interaction of p40 with IL-12Rβ1. The proposed binding site II of p19 did not contribute to IL-12Rβ1 binding. Instead, the interaction of p40_D1D2 with IL-12Rβ1 was sufficient for receptor complex formation. Shown are p40 (green, D1-D3), p19 (blue, site I), IL-12Rβ1 (orange, domains D1 and D2), and IL-23R (pink, D1).