Figure 4
Juxtamembrane elongation of the IL-6R stalk differentially influences proteolysis by ADAM10 and ADAM17.
(a) Schematic representation of the IL-6R. The Ig-like domain (‘D1’) is followed by two fibronectin-type-III domains (‘D2’ and ‘D3’), a flexible stalk region, the transmembrane region and the intracellular region. The juxtamembrane amino acid sequences of the wildtype IL-6R and the prolonged variants IL-6R_1xGS and IL-6R_2xGS are shown below. The ADAM17 cleavage site is indicated in bold. (b) HEK293 cells were transfected with expression plasmids encoding wildtype IL-6R, IL-6R_1xGS and IL-6R_2xGS. 48 h later, cell surface expression of the IL-6R variants was determined via flow cytometry: negative control (light gray), wildtype IL-6R (black), IL-6R_1xGS (dark gray), IL-6R_2xGS (gray). (c) HEK293 cells were transfected with expression plasmids encoding either wildtype human IL-6R or IL-6R_1xGS. 48 h later, cells were treated for 2 h with PMA (100 nM) or DMSO as negative control. Generation of the soluble IL-6R was determined via ELISA or sIL-6R was precipitated from the cell supernatant and visualized via Western blotting. Cells were lysed and IL-6R expression determined via Western blotting. β -actin served as loading control. (d) The experiment was performed as described under panel (c), but cells were treated for 1 h with Ionomycin (1 μ M) or DMSO as negative control. (e,f) The experiments were performed as described under panels (c,d), but cells were transfected with either wildtype human IL-6R or IL-6R_2xGS. ELISA data are the mean (± S.D.) of three independent experiments, Western blots and flow cytometry data show one representative experiment out of three performed.
