AbstractLimited proteolysis of the Interleukin-6 Receptor (IL-6R) leads to the release of the IL-6R ectodomain. Binding of the cytokine IL-6 to the soluble IL-6R (sIL-6R) results in an agonistic IL-6/sIL-6R complex, which activates cells via gp130 irrespective of whether the cells express the IL-6R itself. This signaling pathway has been termed trans-signaling and is thought to mainly account for the pro-inflammatory properties of IL-6. A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 are the major proteases that cleave the IL-6R. We have previously shown that deletion of a ten amino acid long stretch within the stalk region including the cleavage site prevents ADAM17-mediated cleavage, whereas the receptor retained its full biological activity. In the present study, we show that deletion of a triple serine (3S) motif (Ser-359 to Ser-361) adjacent to the cleavage site is sufficient to prevent IL-6R cleavage by ADAM17, but not ADAM10. We find that the impaired shedding is caused by the reduced distance between the cleavage site and the plasma membrane. Positioning of the cleavage site in greater distance towards the plasma membrane abrogates ADAM17-mediated shedding and reveals a novel cleavage site of ADAM10. Our findings underline functional differences in IL-6R proteolysis by ADAM10 and ADAM17.