Fig 2: GIL-11 induce JAK/STAT signaling and cellular proliferation via the non-natural gp130:IL-11R:LIFR cytokine receptor complex.a Proliferation of Ba/F3-gp130, Ba/F3-gp130-IL-11R, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-gp130:IL-11R:OSMR, Ba/F3-gp130:IL-6R:LIFR, Ba/F3-gp130:LIFR:IL-11R cells without cytokine (-), with 50 ng/ml HIL-11, 50 ng/ml IL-11, 50 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 500 ng/ml GIL-11. Data represent ±SEM of three independent experiment out of three with three technical replicates each. Two-way ANOVA, including Dunnet’s correction was used for statistics. b STAT3 activation in Ba/F3, Ba/F3-gp130, Ba/F3-gp130_IL-11R, Ba/F3-gp130:LIFR, Ba/F3-gp130:OSMR, Ba/F3-gp130:IL-11R:OSMR, Ba/F3-gp130:IL-6R:LIFR, Ba/F3-gp130:LIFR:IL-11R cells without cytokine (-) and after stimulation with 50 ng/ml HIL-11, 50 ng/ml IL-11, 10 ng/ml LIF, 10 ng/ml OSM, 500 ng/ml GIL-11 for 15 min. Equal amounts of proteins (50 µg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three.

Fig 3: GIL-6 induces JAK/STAT signaling and cellular proliferation via non-natural cytokine receptor complexes.A, proliferation of Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−), with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7. Data represents mean±S.D. of three independent experiments. For statistics the treated groups were compared with the untreated group by two-way ANOVA including Dunnet’s test for correction in multiple comparison. B, STAT3 activation in Ba/F3, Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−) and after stimulation with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7 for 20 min. C, STAT1, STAT3, STAT5, ERK, and Akt activation in Ba/F3-IL-6R:gp130:LIFR cells with the same conditions as for the STAT3 activation. D, STAT3 activation in heart, liver, and spleen after injection of 20 μg GIL-6 or GIO-6. Mice were sacrificed 30 min after intraperitoneal cytokine injection. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. E, schematic illustration of IC7. Site 3 of IL-6 (red) comprising site 3-1, 3-2, and 3-3 was exchanged by site III of CNTF (grey) resulting in IC7. Consequently, IC7 forms tetrameric IC7:IL-6R:gp130:LIFR or IC7:IL-6R:gp130:OSMR complexes.

Fig 4: Design of the cytokimera GIL-6 and GIO-6.A, schematic illustration of LIF within the trimeric LIF:gp130:LIFR complex, IL-6 in the hexameric 2xIL-6:2xIL-6R:2xgp130 complex, and OSM in the trimeric OSM:gp130:OSMR complex. IL-6 (red) was used as a scaffold for the generation of the cytokimera. Site 3 of IL-6 comprising of site 3-1, 3-2, and 3-3 was exchanged by site 3 of either LIF (green) leading to GIL-6 or by site 3 of OSM (blue) resulting in GIO-6. GIL-6 initially binds to D2/D3 of IL-6R via site 1. Subsequently, GIL-6 binds to D2/D3 of gp130 via site 2 of the IL-6 portion before GIL-6 binds to D3/D4 of the LIFR via site 3 of the LIF portion. GIL-6 forms a tetrameric GIL-6:IL-6R:gp130:LIFR complex. GIO-6 binds either to D2/D3 of the OSMR or to D3/D4 of the LIFR via site 3 of OSM, resulting in the tetrameric GIO-6:IL-6R:gp130:OSMR or GIO-6:IL-6R:gp130:LIFR complexes. B, superposed structure in ribbon and surface view of IL-6 (red) (1ALU) including the exchanged regions by LIF (green) or OSM (blue) for GIL-6 left and GIO-6 right, respectively. GIL-6 modelled in complex of the tetrameric GIL-6:IL-6R:gp130:LIFR complex (PDB 1ALU; 2Q7N; 1P9M) in surface view.

Fig 5: CNTF recruits gp130 prior to OSMR.A, Western blotting of co-immunoprecipitation by using Protein A beads to precipitate recombinant Hyper-CNTF-Fc (2 μg), soluble OSMR (1 μg) in the presence or absence of biotinylated gp130 (1 μg). B, binding scheme of Hyper-CNTF. First Hyper-CNTF binds via site 2 to D2/D3 of gp130. The complex of Hyper-CNTF and gp130 binds hypothetically via site 3 to D2/D3 of OSMR. Hyper-CNTF is not able to bind the OSMR before recruiting gp130.