Beverly J. Castner

An Essential Role for Ectodomain Shedding in Mammalian Development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability.

We previously implicated tumor necrosis factor-α converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-α (TGF-α), pro-TGF-α. Here we examined TGF-α processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-α as compared with wild-type cultures and that TGF-α cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-α and TACE cDNAs increased shedding of mature TGF-α with concomitant conversion of cell-associated pro-TGF-α to a processed form. Purified TACE accurately cleaved pro-TGF-α in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-α containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often thanTace +/+ Egfr wa-2 / wa-2 counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.

TACE/ADAM17 Processing of EGFR Ligands Indicates a Role as a Physiological Convertase

Abstract: EGF family growth factors, including transforming growth factor‐alpha (TGFα), amphiregulin (AR), and heparin‐binding EGF (HB‐EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble growth factor. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis factor‐alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFα from its transmembrane precursor, proTGFα. Here, we review our findings that primary keratinocytes from TaceΔZn/ΔZn mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFα compared to their normal counterparts, even though TGFα mRNA and cell‐associated protein levels were similar in the two cell populations. Restoration of TACE activity in TaceΔZn/ΔZn cells increased shedding of TGFα species, including the mature, 6‐kDa protein. Further, exogenous TACE enzyme accurately cleaved the N‐terminal processing site of proTGFα in cell lysates, as well as both physiologic sites of a soluble proTGFα ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB‐EGF proteins from TaceΔZn/ΔZn cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open‐eye defect of Egfrwa‐2/wa‐2 newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

Further evidence for a common mechanism for shedding of cell surface proteins

Pro‐TNFα, Steel factor, type II IL‐1R and IL‐2Rα were expressed in COS‐7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate‐based inhibitor of metalloproteases. COS‐7 cell membranes were found to cleave various synthetic pro‐TNFα peptides with the same specificity as a partially purified TNFα converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS‐7 cell shedding activity.

Stoichiometry of the complex of human interleukin‐4 with its receptor

A large number of cytokines have been shown to possess a four‐helix bundle structure with a unique up‐up‐down‐down connectivity. The receptors for this family of cytokines have been shown to be homologous as well, each possessing two tandem repeats of a fibronectin type III‐like domain. The crystal structure of human growth hormone bound to the soluble portion of its receptor has served as the only experimentally‐determined example of the interaction between the four‐helix bundle cytokines and their receptors: Two identical receptor subunits bind to different epitopes on the same growth hormone ligand. We have conducted a series of experiments to determine if this structural paradigm is true for interleukin‐4 and interleukin‐4 receptor. Native polyacrylamide gel electrophoresis and gel filtration chromatography reveal that interleukin‐4 forms a tight 1:1 complex with the soluble portion of interleukin‐4 receptor and is thus unlike the growth hormone system.