Shedding of interleukin-6 receptor and tumor necrosis factor alpha - Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins

A functionally and structurally diverse group of transmembrane proteins inc luding transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteol ytic activity responsible for pro-tumor necrosis factor a (proTNF alpha) pr ocessing has been identified and named TACE (TNF alpha converting enzyme). In experiments with TACE deficient (TACB(-/-)) fibroblasts we found that 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleu kin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive r elease of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloprot eases might be involved. PMA-induced shedding of IL-6R in TACE deficient mo use fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNF alpha we gen erated chimeric IL-6R and proTNF alpha proteins wherein the endogenous clea vage sites (CS) had been replaced by the corresponding region of proTNF alp ha and IL-6R, respectively. Interestingly, proTNF alpha chimeric proteins s howed only minimal shedding. In contrast, IL-6R chimeras containing the pro TNF alpha CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the pro TNF alpha CS is similar to that of proTNF alpha itself. We conclude that th e amino-acid sequence at the proteolytic CS contributes to the cleavage cha racteristics of a protein. However, this information alone is not sufficien t to transfer cleavability as seen with proTNF alpha chimeras containing th e IL-6R CS and which were resistant to shedding.