A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro-tumor necrosis factor alpha (proTNFalpha) processing has been identified and named TACE (TNFalpha converting enzyme). In experiments with TACE deficient (TACE-/-) fibroblasts we found that 4beta-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleukin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive release of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloproteases might be involved. PMA-induced shedding of IL-6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNFalpha we generated chimeric IL-6R and proTNFalpha proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFalpha and IL-6R, respectively. Interestingly, proTNFalpha chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNFalpha CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFalpha CS is similar to that of proTNFalpha itself. We conclude that the amino-acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNFalpha chimeras containing the IL-6R CS and which were resistant to shedding.