Article Figures & Data
Figures
- FIG 1
Formation of IL-23 receptor complex and IL-23R stalk conservation. (A) The IL-23 receptor complex is comprised of IL-12Rβ1 with five extracellular domains (D1 to D5), IL-23R containing three extracellular regions (D1 to D3), the unstructured stalk region, and the heterodimeric cytokine IL-23 (p19/p40). Upon IL-23 binding, receptor-associated Janus kinases (Tyk2 and Jak2) are activated and induce four signaling pathways (Jak/STAT, PI3K, MAPK, and NF-κB (14). CBD, cytokine binding domain. (B) Aligned amino acid sequences of IL-23R. The first 15 amino acids of the transmembrane domains (TM) of mouse and human IL-23R are underlined and in boldface. The sequence motif WQPWS, which is similar to the cytokine receptor signature WSXWS in D3 of all IL-23Rs, is in boldface. The 20-amino-acid duplication within mIL-23R is shown in italics with the included WQPWS motif in boldface. Identical amino acids are marked with asterisks. Dashes are inserted if the amino acids in one sequence do not have corresponding mates in the other sequences. CAVPO, Cavia porcellus; CANLF, Canis lupus familiaris; FELCA, Felis catus; PANTR, Pan troglodytes.
- FIG 2
The amino acid composition of the IL-23R stalk region is dispensable for biological activity. (A) Schematic overview of stalk regions of murine and human IL-23R and human IL-6R. Identical amino acids within the IL-23R stalks are marked with asterisks. Amino acids of the respective stalk regions are presented in boldface. The 20-amino-acid duplication within mIL-23R is shown in italics. The hIL-6R stalk amino acids, which were transferred into hIL-23R, are underlined. WSXWS motifs are in boldface. A dash is inserted if the amino acid in one sequence does not have a corresponding mate in the other sequence. (B) Histograms of receptor surface expression of Ba/F3-gp130 cell lines. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and the solid lines indicate the respective Ba/F3 cell lines. (C) Analysis of STAT3 activation. Ba/F3 cells were washed, starved, and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3 and STAT3. The Western blot data show the results of one representative experiment out of three. (D) Cellular proliferation of Ba/F3 cells with cDNAs coding for hIL-23R or hIL-23R/hIL-6R-stalk and hIL-12Rβ1. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. Parental Ba/F3-gp130 cells were used as controls. HIL-6–dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent standard deviations (SD) for technical replicates.
- FIG 3
The length of the IL-23R stalk region is critical for biological activity. (A) Schematic overview of the human IL-23R stalk region. Amino acids belonging to the stalk regions are presented in boldface. The WSXWS motif is in boldface. Dashes are inserted if the amino acids in one sequence do not have corresponding mates in the other sequences. (B) Expression of generated human IL-23Rs. COS-7 cells were transiently transfected with cDNAs. The plasmid pEGFP served as a transfection control. Cellular lysates were prepared and analyzed by Western blotting. (C and D) Histograms of hIL-23R (C) and hIL-12Rβ1 (D) surface expression of Ba/F3 cell lines. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and the solid lines indicate the respective Ba/F3 cell lines. (E) Analysis of STAT3 and Erk1/2 activation. Ba/F3 cells were washed, starved, and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, phospho-Erk1/2, STAT3, and Erk1/2. Expression of receptor chains was verified in the lysates by Western blotting using antibodies specific for human IL-23R and IL-12Rβ1. The Western blot data show the results of one representative experiment out of three. (F) Proliferation of Ba/F3 cells expressing stalk deletion variants. The cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. HIL-6–dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (G) Suppression of signal transduction by Jak inhibition. The indicated Ba/F3 cell lines were washed, starved, and pretreated for 60 min with P6 (1 μM) before exposure to HIL-23. Cellular lysates were analyzed as described for panel E.
- FIG 4
Deletion of the murine IL-23R stalk impairs signal transduction. (A) Schematic overview of the murine IL-23R stalk region. Amino acids belonging to the stalk regions are presented in boldface. The WSXWS motif is in boldface. The 20-amino-acid duplication within mIL-23R is shown in italics, and the WQPWS duplication is underlined. Dashes are inserted if the amino acids in one sequence do not have corresponding mates in the other sequences. (B) Expression of generated murine IL-23Rs. COS-7 cells were transiently transfected with cDNAs. The plasmid pEGFP served as a transfection control. Cellular lysates were prepared and analyzed by Western blotting. (C and D) Histograms of mIL-23R (C) and mIL-12Rβ1 (D) surface expression of Ba/F3 cell lines expressing mIL-12Rβ1 and mIL-23R or variants thereof. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and the solid lines indicate the respective Ba/F3 cell lines. (E) Analysis of STAT3 and Erk1/2 activation. Ba/F3 cells were washed, starved, and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, phospho-Erk1/2, STAT3, and Erk1/2. Expression of receptor chains was verified in the lysates by Western blotting using antibodies specific for murine IL-23R and IL-12Rβ1. The Western blot data show the results of one representative experiment out of three. (F) Cellular proliferation of Ba/F3 cells with cDNAs coding for mIL-23R variants and mIL-12Rβ1. Equal numbers of cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. HIL-6-dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (G) Biologically inactive IL-23R still binds IL-23. COS-7 cells were transiently transfected with cDNAs coding for mIL-23R, mIL-23RΔ333–372, or HIL-23Fc. Cellular lysates were prepared, and lysates containing the receptor were mixed with HIL-23Fc lysate or without it as a control. The cytokine-Fc fusion protein was precipitated with protein A-Sepharose. Coimmunoprecipitation of receptors was analyzed by Western blotting. L, total cell lysate; IP, coprecipitates.
- FIG 5
Ligand-independent, autonomous mIL-23R activation is achieved by stalk deletion. (A) Schematic overview of the murine IL-23R stalk region. Amino acids belonging to the stalk regions are presented in boldface. The WSXWS motif is in boldface. The 20-amino-acid duplication within mIL-23R is shown in italics, and the WQPWS duplication is underlined. Dashes are inserted if the amino acids in one sequence do not have corresponding mates in the other sequences. (B and C) Histograms of mIL-23R (B) and mIL-12Rβ1 (C) surface expression of Ba/F3-mIL-12Rβ1-mIL-23RΔ317–336 cells. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (D) Analysis of STAT3 and Erk1/2 activation. Ba/F3 cells were washed, starved, and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, phospho-Erk1/2, STAT3, and Erk1/2. Expression of receptor chains was verified in the lysates by Western blotting using antibodies specific for murine IL-23R and IL-12Rβ1. The Western blot data show the results of one representative experiment out of three. (E) Cellular proliferation of Ba/F3-mIL-12Rβ1-mIL-23RΔ317–336 cells. Equal numbers of cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. HIL-6-dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (F and G) Histograms of mIL-23R (F) and mIL-12Rβ1 (G) surface expression of Ba/F3-mIL-12Rβ1-mIL-23RΔ317–372 cells. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control). (H) Analysis of STAT3 and Erk1/2 activation as described for panel D. The Western blot data show the results of one representative experiment out of three. (I) Cellular proliferation of Ba/F3-mIL-12Rβ1-mIL-23RΔ317–372. The assay was performed as described for panel E. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (J) Autonomous IL-23R still binds IL-23. COS-7 cells were transiently transfected with cDNAs coding for mIL-23RΔ317–372 or HIL-23Fc. Cellular lysates were prepared, and lysates containing the receptor were mixed with HIL-23Fc lysate or without it as a control. The cytokine-Fc fusion protein was precipitated with protein A-Sepharose, and coimmunoprecipitation of receptors was analyzed by Western blotting. L, total cell lysate; IP, coprecipitates.
- FIG 6
IL-12Rβ1 is not necessary for ligand-independent, autonomous IL-23R activation. (A) Histograms of hIL-23R surface expression of stably transduced Ba/F3 cell lines. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and the solid lines indicate the respective Ba/F3 cell lines, as indicated. (B) Analysis of STAT3 activation. Ba/F3 cells expressing hIL-23R or variants were washed, starved, and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3 and STAT3. The Western blot data show the results of one representative experiment out of three. (C) Cellular proliferation of Ba/F3-hIL-23R cells. Equal numbers of cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. HIL-6-dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (D) Histograms of mIL-23R surface expression of stably transduced Ba/F3 cell lines. The gray-shaded areas indicate Ba/F3-gp130 cells (negative control), and the solid lines indicate the respective Ba/F3 cell lines, as indicated. (E) Analysis of STAT3 activation as described for panel B. The Western blot data show the results of one representative experiment out of three. (F) Cellular proliferation of Ba/F3-mIL-23R cells. The assay was performed as described for panel C. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (G) Analysis of Erk1/2 activation as described for panel B. The Western blot data show one representative experiment out of two.
- FIG 7
Ligand-independent, autonomous IL-23R activation occurs in transiently transfected human fibrosarcoma cells. (A) Analysis of STAT3 activation mediated by murine IL-23 receptors. U4C cells were transiently transfected with cDNAs for mIL-23R, mIL-12Rβ1, or mIL-23RΔ317–372 or cotransfected. At 30 h after transfection, the cells were washed and starved overnight in serum-free medium. The cells were stimulated with HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, IL-23R, and IL-12Rβ1. The Western blot data show the results of one representative experiment out of three. Nonspecific bands are indicated by asterisks. (B) Analysis of STAT3 activation mediated by human IL-23 receptors. The assay was performed as described for panel A. The Western blot data show the results of one representative experiment out of two. Nonspecific bands are indicated by the asterisk.
- FIG 8
Ligand-independent, autonomous IL-23R activation is mediated by homodimerization of an IL-23R stalk deletion variant. (A) mIL-23R forms homodimers. CHO-K1 cells were transiently transfected with cDNAs coding for FLAG- and GFP-tagged mIL-23R or mIL-23RΔ317–372. Cellular lysates were prepared, receptors were precipitated with GFP-binding proteins, and coprecipitation was analyzed by Western blotting. Signals for the receptors are indicated by arrows to allow discrimination from nonspecific signals. The results of one representative experiment out of two are shown. L, total cell lysate; IP, coprecipitates. (B) mIL-23R interacts with mIL-23RΔ317–372. The assay was performed as described for panel A. Signals for the receptors are indicated by arrows to allow discrimination from nonspecific signals. The Western blot data show the results of one representative experiment out of two.
- FIG 9
Autonomous IL-23R is independent of gp130. (A) Histogram of mIL-23R surface expression of a stably transduced Ba/F3 cell line. The gray-shaded area indicates Ba/F3 cells (negative control), and the solid line represents the respective Ba/F3-mIL-23RΔ317–372 cell line. (B) Analysis of STAT3 and Erk1/2 activation. Ba/F3 cells were washed, starved, and stimulated with IL-3, HIL-6, and HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, phospho-Erk1/2, STAT3, and Erk1/2. Expression of the mIL-23R chain was verified in the lysates by Western blotting using antibodies specific for murine IL-23R. The Western blot data show the results of one representative experiment out of two. Nonspecific bands are indicated by asterisks. (C) Proliferation of Ba/F3-mIL-23R cells. Equal numbers of cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. The results of one representative experiment out of four are shown. The error bars represent SD for technical replicates.
- FIG 10
Synthetic IL-15–IL-23R fusion receptors are constitutively active. (A) Histogram of synthetic receptor surface expression of Ba/F3-IL-15ER-IL-23RTIR cells. The gray-shaded area indicates Ba/F3-gp130 cells (negative control). (B) Analysis of STAT3 activation. Ba/F3 cells expressing synthetic IL-15ER–IL-23RTIR receptor chimeras were starved and stimulated with 10 ng/ml HIL-23 for 30 min. Cellular lysates were prepared, and equal amounts of total protein (50 μg/lane) were loaded on SDS gels, followed by immunoblotting using specific antibodies for phospho-STAT3, STAT3, phospho-Erk1/2, and Erk1/2. The Western blot data show the results of one representative experiment out of three. (C) Cellular proliferation of Ba/F3-IL-15ER-IL-23RTIR receptor cells. Equal numbers of cells were cultured for 3 days in the presence of 10 ng/ml HIL-6 or 10 ng/ml HIL-23 or without a cytokine. HIL-6-dependent proliferation was set to 100%. The results of one representative experiment out of three are shown. The error bars represent SD for technical replicates. (D) Analysis of STAT3 activation in transiently transfected U4C cells as described for panel B. The Western blot data show the results of one representative experiment out of three. Nonspecific bands are indicated by an asterisk, and synthetic IL-15ER–IL-23RTIR receptor is indicated by the arrow.
