Shedding of interleukin-6 receptor and tumor necrosis factor α

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 α (proTNFα) processing has been identified and named TACE (TNFα converting enzyme). In experiments with TACE deficient (TACE-/-) fibroblasts we found that 4β-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of IL-6R and proTNFα we generated chimeric IL-6R and proTNFα proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNFα and IL-6R, respectively. Interestingly, proTNFα chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNFα CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNFα CS is similar to that of proTNFα 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 proTNFα chimeras containing the IL-6R CS and which were resistant to shedding.