FIGURE 4.

The binding site of CANDIS in the IL-6R. A, schematic of IL-6R deletion variant IL-6RΔGlu³¹⁷-Gln³³² (Δ16). TM, transmembrane. B, analysis of the pulldown experiments of wild-type IL-6R and IL-6RΔGlu³¹⁷-Gln³³² (Δ16) by either CANDIS or control peptides, the CANDIS-homologous region of ADAM10 (pA10), and a randomized sequence of CANDIS (cp). C, eligibility of IL-6RΔGlu³¹⁷-Gln³³² (Δ16) as an ADAM17 substrate. HEK293 cells were transfected with the deletion variant or wild-type IL-6R. Shedding was stimulated by addition of PMA. Control cell samples were either treated with dimethyl sulfoxide (DMSO) or with PMA and the metalloprotease inhibitor marimastat (Mari). The shed IL-6R was detected by ELISA. Shedding activity is displayed in relation to the PMA-stimulated wild-type IL-6R sample as described under “Experimental Procedures.” Student's t test was performed with data of three independent experiments of which at least triplicates were performed. p < 0.005 indicates statistical significance. D, impact on CANDIS in IL-1RII shedding. Wild-type ADAM17 or ADAM17p10, comprising an exchange of CANDIS with the respective sequence of ADAM10 (p10) were cotransfected with AP-tagged IL-1RII into ADAM17^ex/ex MEFs. One day after transfection, cells were treated with PMA, and ADAM17 shedding activity was detected and calculated as described under “Experimental Procedures.” Student's t test was used to prove significance and was calculated of four independent experiments performed with triplicates. p < 0.005 was calculated and considered to be statistically significant. S, stalk region; ICD, intracellular domain.