Abstract
Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.
Highlights
The TNF-␣1 converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that releases soluble tumor necrosis factor-␣ (TNF) from cells by cleaving within the extracellular domain of membrane-bound pro-TNF [1, 2]
Reconstitution of Other Shedding Events in tace⌬Zn/⌬Zn EC-2 Cells by Transfection with TACE cDNA—We investigated whether the tace⌬Zn/⌬Zn EC-2 cells could be used to examine other TACE-mediated shedding events. tace⌬Zn/⌬Zn monocytes are defective in shedding p75 TNFR [3], and we found that tace⌬Zn/⌬Zn EC-2 cells transfected with cDNA encoding p75 TNFR or p55 TNFR shed these proteins inefficiently (Table III). (As with TNF release, TNFR shedding activity is expressed as the ratio of soluble TNFR in the culture medium to cell-associated TNFR (S:C) after the 2-h incubation.) Co-transfection of tace⌬Zn/⌬Zn EC-2 cells with TACE cDNA increased the S:C ratios of p75 TNFR and p55 TNFR 16- and 6-fold, respectively, suggesting that TACE is involved in shedding both p75 TNFR and p55 TNFR in these cells
In addition to these observations regarding the role of the TACE Cys-rich domains in IL-1R-II shedding, we consistently found that catalytic TACE/dis-epidermal growth factor-like domain (EGF) ADAM-10 B and to a lesser extent catalytic dis-TACE/EGF ADAM-10 B exhibited a decreased sensitivity of TNF shedding to IC-3 (Table IV)
Summary
Fibroblast cell line derived from the tace⌬Zn/⌬Zn mice to study the function of different domains of the TACE protein. We address the potential roles of membrane anchoring, the cytoplasmic domain, and the cysteine-rich sequences in the shedding of three different TACE substrates. We found that expression of TACE with a membrane anchor reconstitutes shedding in these cells, whereas expression of secreted TACE does not. We found that the cytoplasmic domain of TACE is not required for any of the shedding events examined and that the cysteine-rich domain of TACE is required for shedding of one of the substrates while affecting the inhibitor sensitivity of another substrate’s shedding
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