The length of the interleukin-11 receptor stalk determines its capacity for classic signaling

Abstract

Interleukin (IL)-11 is a multifunctional cytokine that was traditionally recognized for its hematopoietic and anti-inflammatory functions, but has recently been shown also to be involved in tumorigenesis. IL-11 signaling is initiated by binding of the cytokine to the IL-11 receptor (IL-11R), which is not directly involved in signaling but required for IL-11 binding to the signal-transducing receptor glycoprotein (gp) 130. In classic signaling, IL-11 binds to the membrane-bound IL-11R to initiate signal transduction. Additionally, IL-11 signaling can be initiated via soluble IL-11R, known as trans-signaling, and this pathway only requires the three extracellular domains of the IL-11R, but not stalk, transmembrane, or intracellular region. Here, we analyzed the role of the IL-11R stalk region, a 55 amino acid stretch connecting the extracellular domains with the transmembrane helix, in classic IL-11 signaling with the help of cytokine-dependent cell lines. We showed that the stalk region is crucial for IL-11 signaling via the membrane-bound IL-11R. Using different deletion variants, we found that a minimal length of 23 amino acid residues is required for efficient signal transduction. We further found that classic IL-11 signaling depended solely on the length, but not the sequence, of the IL-11R stalk region, suggesting that the stalk functions as a spacer in the signaling complex. We previously described the IL-11R stalk region as determinant of proteolysis and regulator of IL-11 trans-signaling. The results presented here reveal an additional function in classic IL-11 signaling, highlighting the importance of the IL-11R stalk in IL-11 signaling.

Keywords: STAT3; cytokine; gp130; interleukin; interleukin-11; interleukin-11 receptor; proliferation; signal transduction; stalk region.

Figure 1.

IL-11R stalk is essential for classic signaling. A, cell surface expression of IL-11R variants on Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔE323_L372, and Ba/F3-gp130 cells was assessed by flow cytometry. B, equal amounts of Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔE323_L372, and Ba/F3-gp130 cells were stimulated with increasing amounts (0–100 ng/ml) of either IL-11 or Hy–IL-6 for 48 h. Concentration-dependent proliferation was determined as described in “Experimental Procedures.” Error bars represent S.D. of triplicates from one representative experiment. C, STAT3 phosphorylation of Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔE323_L372, and Ba/F3-gp130 cells in response to 10 ng/ml IL-11 or 10 ng/ml Hy–IL-6 stimulation for 15 min was determined by Western blotting. Total STAT3 was determined to ensure equal protein loading.

Figure 2.

Deletions in the IL-11R stalk do not compromise cell surface expression. A, schematic overview of IL-11R stalk deletions. Amino acid residues of the stalk region (spanning Gly-318 to Leu-372) are highlighted in gray. Missing stretches in the scheme indicate deletions. B, cell surface expression of the above-depicted IL-11R deletion variants stably transduced into Ba/F3-gp130 cells.

Figure 3.

Classic IL-11 signaling depends on the stalk length. A–G, equal amounts of the different Ba/F3-gp130 cell lines were stimulated with increasing amounts (0–100 ng/ml) of either IL-11 or Hy–IL-6 for 48 h. Concentration-dependent proliferation was determined as described in “Experimental Procedures.” The analyzed cell line is given above the respective diagram. Error bars represent S.D. of triplicates from one representative experiment. H, STAT3 phosphorylation of Ba/F3–gp130–IL-11RΔP343_L372 and Ba/F3–gp130–IL-11RΔQ333_L372 cells in response to 10 ng/ml IL-11 or 10 ng/ml Hy–IL-6 stimulation for 15 min was determined via Western blotting. Total STAT3 was detected to ensure equal protein loading. The ratio of pSTAT3/STAT3 was determined from three independent experiments.

Figure 4.

A stalk region of 23 amino acid residues is required for classic signaling. A, schematic overview of the four additional IL-11R stalk deletion variants. Amino acid residues of the stalk are highlighted in gray. Missing stretches in the scheme indicate deletions. B, cell surface expression of the above depicted IL-11R deletion variants stably transduced into Ba/F3-gp130 cells. C–F, equal amounts of the different Ba/F3-gp130 cell lines were stimulated with increasing amounts (0–100 ng/ml) of either IL-11 or Hy–IL-6 for 48 h. Concentration-dependent proliferation was determined as described in “Experimental Procedures.” The analyzed cell line is given above the respective diagram. Error bars represent S.D. of triplicates from one representative experiment.

Figure 5.

IL-11-induced STAT3 phosphorylation depends on IL-11R stalk length. A, Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔD341_L372, Ba/F3–gp130–IL-11RΔQ339_L372, Ba/F3–gp130–IL-11RΔE337_L372, and Ba/F3–gp130–IL-11RΔE335_L372 cells were either stimulated with 10 ng/ml IL-11 for 15 min or left untreated. STAT3 phosphorylation was determined by Western blotting. Total STAT3 and IL-11R were visualized within the cell lysate. β-actin was determined to ensure equal protein loading. The ratio of pSTAT3/STAT3 was determined from three independent experiments. B, Ba/F3–gp130–IL-11RΔD341_L372 cells were stimulated with either 10 ng/ml Hy–IL-6, 10 ng/ml IL-11 for the indicated time points, or left untreated. The relative amounts of SOCS3 mRNA were quantified by quantitative real-time PCR. Values were normalized to GAPDH (n = 3 independent experiments, three technical replicates per experiment, mean ± S.D.). C, the experiment was performed with Ba/F3–gp130–IL-11RΔE335_L372 cells as described in B.

Figure 6.

Influence of cytokine concentration and duration of stimulation on IL-11–induced STAT3 phosphorylation. A, Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔD341_L372, Ba/F3–gp130–IL-11RΔQ339_L372, Ba/F3–gp130–IL-11RΔE337_L372, and Ba/F3–gp130–IL-11RΔE335_L372 cells were stimulated with either 10 ng/ml Hy–IL-6, 10 ng/ml IL-11, 100 ng/ml IL-11, or left untreated for 15 min. STAT3 phosphorylation was determined by Western blotting, and total STAT3 was visualized to ensure equal protein loading. The ratio of pSTAT3/STAT3 was determined from three independent experiments. B, Ba/F3–gp130–IL-11R, Ba/F3–gp130–IL-11RΔD341_L372, Ba/F3–gp130–IL-11RΔQ339_L372, Ba/F3–gp130–IL-11RΔE337_L372, and Ba/F3–gp130-IL–11RΔE335_L372 cells were stimulated with 10 ng/ml IL-11 for different time periods (0–60 min). STAT3 phosphorylation was determined by Western blotting, and total STAT3 was visualized to ensure equal protein loading. The ratio of pSTAT3/STAT3 was determined from three independent experiments.

Figure 7.

Classic IL-11 signaling is independent of the stalk sequence. A, stalk sequences of IL-11R, IL-11RΔD341_L372, and IL-11R_random. B, cell surface expression of IL-11R_random was assessed by flow cytometry. C, equal amounts of Ba/F3–gp130–IL-11R_random cells were stimulated with increasing amounts (0–100 ng/ml) of either IL-11 or Hy–IL-6 for 48 h. Concentration-dependent proliferation was determined as described in “Experimental Procedures.” D, STAT3 phosphorylation of Ba/F3–gp130–IL-11R_random in response to 10 ng/ml IL-11 or 10 ng/ml Hy–IL-6 stimulation for 15 min was determined by Western blotting. Total STAT3 was determined to ensure equal protein loading. The ratio of pSTAT3/STAT3 was determined from three independent experiments. E, Ba/F3-gp130, Ba/F3–gp130–IL-11R and Ba/F3–gp130–IL-11R_random cells were stimulated with 10 ng/ml IL-11 for the indicated time points. The relative amounts of SOCS3 mRNA were quantified as described in the legend to Fig. 5B. Values were normalized to GAPDH (n = 3 independent experiments, three technical replicates per experiment, mean ± S.D.). F, sequence alignment of the amino acid residues of the IL-11R stalk sequences from human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), hamster (Mesocricetus auratus), chicken (Gallus gallus), clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio). The predicted stalk region of the human IL-11R has been underlined.