Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. We have now purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. Our results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins.

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.

The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-cleavage). The enzymes responsible for these activities have not been unambiguously identified. By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor ␣ converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated ␣-cleavage of APP. Our data suggest that TACE may be the ␣-secretase responsible for the majority of regulated ␣-cleavage in cultured cells. Furthermore, we show that inhibiting this enzyme affects both APP secretion and A␤ formation in cultured cells.The amyloid protein, A␤, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP) 1 (1-3). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the A␤ domain (␤-cleavage), one within the A␤ domain (␣-cleavage), and one at the carboxyl terminus of the A␤ domain (␥-cleavage). The enzymes responsible for these activities have not been unambiguously identified.In most cells in culture, a fraction (10 -30%) of all APP undergoes ␣-cleavage (4 -7). This results in the secretion of the large extracellular domain of APP into the medium. This secreted APP (APP s ) is a major APP-related species found in cerebrospinal fluid and brain homogenates (8,9) and is thought to interact with components of the extracellular matrix and with receptors on cells. In cultured cells it has been shown that the fraction of APP that is converted to APP s can be increased by activating second messenger cascades including those involving protein kinase C, protein kinase A, mitogen-activated protein kinase, protein phosphatase 1, protein phosphatase 2B (calcineurin), and calcium (4 -6, 10 -14). In most cells in culture, activating protein kinase C causes the majority (80 -95%) of the APP to undergo ␣-cleavage ("regulated" ␣-cleavage). Stimulation of APP s formation and secretion by activating second messenger cascades is not due to the phosphorylation of APP (15, 16) but may be due to protein phosphorylation leading to alterations in the trafficking of APP (17) or in the activity of an ␣-secretase. Importantly, stimulating ␣-cleavage of APP leads to a significant decrease in A␤ formation (18 -20).The potential importance of regulated cleavage of APP is indicated by the ability of acetylcholine, a critical neurotransmitter altered in Alzheimer's disease, working through muscarinic receptors, to stimulate regulated cleavage (4,10,21,22). Activation of other metabotropic receptors also leads to activation of regulated secretion of APP (4,21,23,24). Regulated cleavage of APP appears to occur in vivo under conditions in which protein ...

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)-␣ 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 membraneanchoring 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.

cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin‐1 (IL‐1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL‐1 receptor (type II receptor). The mature type II IL‐1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin‐like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL‐1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL‐1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL‐1 receptor can bind all three forms of IL‐1 (IL‐1 alpha, IL‐1 beta and IL‐1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL‐1 receptor.