Carl P. Blobel

ADAMs: key components in EGFR signalling and development

ADAM (a disintegrin and metalloprotease) proteins are membrane-anchored metalloproteases that process and shed the ectodomains of membrane-anchored growth factors, cytokines and receptors. ADAMs also have essential roles in fertilization, angiogenesis, neurogenesis, heart development and cancer. Research on ADAMs and their role in protein ectodomain shedding is emerging as a fertile ground for gathering new insights into the functional regulation of membrane proteins.

Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor α, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17− /− knockout mice corroborated the essential role of adam17− /− in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

Evidence for a role of a tumor necrosis factor-α (TNF-α)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a member of the TNF family, is a dendritic cell survival factor and is essential for osteoclastogenesis and osteoclast activation. In this report we demonstrate (i) that TRANCE, like TNF-α, is made as a membrane-anchored precursor, which is released from the plasma membrane by a metalloprotease; (ii) that soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-α convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8 fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed from cells into the supernatant. We propose that the TRANCE ectodomain is released from cells by TACE or a related metalloprotease-disintegrin, and that this release is an important component of the function of TRANCE in bone and immune homeostasis.

Adam meets Eph : An ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.

Metalloprotease-Disintegrins: Links to Cell Adhesion and Cleavage of TNFα and Notch

Why are such functionally distinct protein modules as a metalloprotease domain and a disintegrin domain combined in MDC proteins? With respect to the metalloprotease functions, one possibility is that the disintegrin domain might be used to target the metalloprotease to another cell in trans via an integrin (see Figure 1Figure 1). Alternatively, the disintegrin domain, or other protein domains such as the EGF repeat or cysteine-rich region, might be used to increase the efficiency of the protease by binding the substrate directly or indirectly in cis or in trans. It is also possible that removal of the metalloprotease domain may be used to regulate the function of the disintegrin domain, as suggested by two independent studies. In sperm fertilin β, removal of the noncatalytic metalloprotease-domain during sperm maturation correlates with the acquisition of fertilization competence and exposes an epitope that is recognized by a function-blocking monoclonal antibody. For meltrin α, which plays a role in muscle fusion, overexpression of a truncated form of the protein lacking the metalloprotease domain leads to an increase in muscle fusion, whereas overexpression of the full-length protein leads to a decrease in the observed fusion (Yagami-Hiromasa et al. 1995xYagami-Hiromasa, T., Sato, T., Kurisaki, T., Kamijo, K., Nabeshima, Y., and Fujisawa-Sehara, A. Nature. 1995; 377: 652–656Crossref | PubMedSee all ReferencesYagami-Hiromasa et al. 1995). Since only a small percentage of the detectable meltrin α lacks the metalloprotease domain in C2C12 mouse myoblasts, one interesting possibility is that only this small pool of processed protein may promote muscle cell binding and fusion. MDC proteins have been proposed to mediate cell–cell fusion directly (seeWolfsberg and White 1996xWolfsberg, T.G. and White, J.M. Dev. Biol. 1996; 180: 389–401Crossref | PubMed | Scopus (205)See all ReferencesWolfsberg and White 1996, for details), although it should be noted that the studies that point toward a role of fertilin and meltrin α in membrane fusion can also be explained by simply invoking a critical binding step via the disintegrin domain that is a prerequisite for fusion to occur.The concepts of targeting the metalloprotease domain via the disintegrin domain and modulation of the disintegrin domain function by removal of the metalloprotease domain illustrate two of several conceivable means of MDC protein regulation that are not necessarily mutually exclusive. Different MDC proteins may employ these protein modules in different ways, and any particular protein might also have distinct functions depending on the stage of development, the tissue, or even the subcellular localization that it is expressed in. The Drosophila metalloprotease-disintegrin KUZ, which is currently the only MDC protein for which a mutant phenotype has been reported, is required in the early embryo for neural inhibition, and is later involved in eye development, neural-promoting and -inhibiting processes (Rooke et al. 1996xRooke, J., Pan, D., Xu, T., and Rubin, G.M. Science. 1996; 273: 1227–1230Crossref | PubMedSee all ReferencesRooke et al. 1996), and axon extension (Fambrough et al. 1996xFambrough, D., Pan, D., Rubin, G.M., and Goodman, C.S. Proc. Natl. Acad. Sci. USA. 1996; 93: 13233–13238Crossref | PubMed | Scopus (141)See all ReferencesFambrough et al. 1996). A fascinating indication for a potentially distinct mechanism of the neural promoting and inhibiting activity mediated by KUZ is described by Rooke et al. 1996xRooke, J., Pan, D., Xu, T., and Rubin, G.M. Science. 1996; 273: 1227–1230Crossref | PubMedSee all ReferencesRooke et al. 1996. In the cuticle of adult mosaic Drosophila, clusters of sensory bristles appear at the boundary of kuz− and wildtype cells instead of the single sensory bristle that normally develops, whereas no sensory bristles are found in the interior of a mutant cell patch. Apparently a non-cell-autonomous neural-promoting signal can be supplied to the mutant cells by adjacent wildtype cells, and not by kuz− cells. Yet once kuz− cells have adopted a neural fate, they are unable to laterally inhibit the neural fate of other mutant cells, resulting in the formation of additional bristles only at the boundary of the mutant cell patch.Pan and Rubin 1997xPan, D. and Rubin, G.M. Cell. 1997; 90: 271–280Abstract | Full Text | Full Text PDF | PubMed | Scopus (299)See all ReferencesPan and Rubin 1997 have now provided strong genetic and biochemical evidence that the lateral inhibition mediated by KUZ involves a specific cleavage event in the extracellular domain of the transmembrane receptor Notch. Cleavage of Notch receptors in the extracellular domain appears to be an evolutionarily conserved feature, and the subcellular location of human Notch2 processing has been narrowed down to the trans-Golgi network (Blaumueller et al. 1997xBlaumueller, C.M., Qi, H., Zagouras, P., and Artavanis-Tsakonas, S. Cell. 1997; 90: 281–291Abstract | Full Text | Full Text PDF | PubMed | Scopus (416)See all ReferencesBlaumueller et al. 1997). Processing of the full-length ∼300 kDa human Notch2 yields a 110 kDa fragment containing the transmembrane domain and cytoplasmic tail, and a disulfide-linked 180 kDa fragment that most likely corresponds to the extracellular domain. Cell surface labeling experiments indicate that only cleaved but not full-length Notch2 emerges on the cell surface. The cleaved 110 kDa membrane-anchored fragment of human Notch2 resembles the ∼100 kDa processed form of Drosophila Notch. Since the ∼100 kDa form of Notch is not detectable in kuz− embryosPan and Rubin 1997xPan, D. and Rubin, G.M. Cell. 1997; 90: 271–280Abstract | Full Text | Full Text PDF | PubMed | Scopus (299)See all ReferencesPan and Rubin 1997 propose that KUZ mediates the extracellular cleavage of Notch and that this cleavage is necessary for Notch to mediate lateral inhibition. Taken together, the two papers support a model where KUZ or its mammalian homolog MDC/ADAM10 is responsible for maturation and functional activation of Notch receptors in the secretory pathway (4xBlaumueller, C.M., Qi, H., Zagouras, P., and Artavanis-Tsakonas, S. Cell. 1997; 90: 281–291Abstract | Full Text | Full Text PDF | PubMed | Scopus (416)See all References, 11xPan, D. and Rubin, G.M. Cell. 1997; 90: 271–280Abstract | Full Text | Full Text PDF | PubMed | Scopus (299)See all References). It should be noted that the extracellular domain cleavage of Notch discussed here is distinct from a putative cytoplasmic cleavage that might allow the cytoplasmic domain to enter the nucleus (Blaumueller et al. 1997xBlaumueller, C.M., Qi, H., Zagouras, P., and Artavanis-Tsakonas, S. Cell. 1997; 90: 281–291Abstract | Full Text | Full Text PDF | PubMed | Scopus (416)See all ReferencesBlaumueller et al. 1997).If the processing of Notch receptors is evolutionarily conserved, then one might expect the Notch processing protease to be functionally conserved as well. IndeedPan and Rubin 1997xPan, D. and Rubin, G.M. Cell. 1997; 90: 271–280Abstract | Full Text | Full Text PDF | PubMed | Scopus (299)See all ReferencesPan and Rubin 1997 show that expression of a mouse dominant negative KUZ lacking the metalloprotease domain (KUZDN) in both Drosophila and Xenopus laevis results in an increased number of neurogenic cells, presumably due to a lack of lateral inhibition. Furthermore, expression of a Drosophila KUZDN in Drosophila neurons mimics the defect in axon extension reported by Fambrough et al. 1996xFambrough, D., Pan, D., Rubin, G.M., and Goodman, C.S. Proc. Natl. Acad. Sci. USA. 1996; 93: 13233–13238Crossref | PubMed | Scopus (141)See all ReferencesFambrough et al. 1996, confirming the idea that the metalloprotease domain of KUZ, as opposed to other domains, is responsible for axon extension. The substrates of KUZ during axonal extension have not been identified but could be different from Notch, such as matrix proteins or cytokines.In conclusion, it is clear that metalloprotease-disintegrins are involved in a remarkably diverse set of tasks, ranging from a role in fertilization and muscle fusion, TNFα release from the plasma membrane, intracellular cleavage and activation of Notch, and other essential functions in Drosophila development. The nature of these diverse tasks further suggests that MDC proteins may function at different subcellular locations, such as on the cell surface (fertilin, TACE?), or intracellularly in the secretory pathway (KUZ?). It seems likely that the proteins discussed here, and the more than 20 family members of presently unknown function, which have been found in organisms ranging from C. elegans to mammals (references can be found inWolfsberg and White 1996xWolfsberg, T.G. and White, J.M. Dev. Biol. 1996; 180: 389–401Crossref | PubMed | Scopus (205)See all ReferencesWolfsberg and White 1996) will unveil further exciting secrets. Due to an increasing interest in MDC proteins, a better understanding should soon begin to emerge about the mechanism of MDC protein function, of the specific functions of different family members in development and disease, and of the interactions with other proteins that govern MDC protein activity.

Metalloprotease-Disintegrin MDC9: Intracellular Maturation and Catalytic Activity

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-α. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-α convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-α-convertase.

In search of partners: linking extracellular proteases to substrates

Proteases function as molecular switches in signalling circuits at the cell surface and in the extracellular milieu. In light of the many proteases that are encoded by the genome, and the even larger number of bioactive substrates, it is crucial to identify which proteases cleave a particular substrate and which substrates individual proteases cleave. Elucidating the substrate degradomes of proteases will help us to understand the function of proteases in development and disease and to validate proteases as drug targets.

Remarkable roles of proteolysis on and beyond the cell surface

Proteolysis on the cell surface and in the extracellular matrix is essential for normal cellular functions during development and in the adult, but it may also have undesirable consequences, such as promoting cancer, arthritis, and Alzheimers disease. Recent advances highlight the roles of zinc-dependent metalloproteinases (metzincins) in proper skeletal development, in activating EGF-receptor ligands, in Notch-dependent signaling, and in initiating and promoting tumorigenesis.

The Disintegrin/Metalloproteinase ADAM10 Is Essential for the Establishment of the Brain Cortex

The metalloproteinase and major amyloid precursor protein (APP) α-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10 −/− mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that α-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP α-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

Cutting Edge: TNF-α-Converting Enzyme (TACE/ADAM17) Inactivation in Mouse Myeloid Cells Prevents Lethality from Endotoxin Shock

TNF-α, a potent proinflammatory cytokine, is synthesized as a membrane-anchored precursor and proteolytically released from cells. Soluble TNF is the primary mediator of pathologies such as rheumatoid arthritis, Crohn’s disease, and endotoxin shock. The TNF-α converting enzyme (TACE), a disintegrin and metalloprotease 17 (ADAM17), has emerged as the best candidate TNF sheddase, but other proteinases can also release TNF. Because TACE-deficient mice die shortly after birth, we generated conditional TACE-deficient mice to address whether TACE is the relevant sheddase for TNF in adult mice. In this study, we report that TACE inactivation in myeloid cells or temporal inactivation at 6 wk offers strong protection from endotoxin shock lethality in mice by preventing increased TNF serum levels. These findings corroborate that TACE is the major endotoxin-stimulated TNF sheddase in mouse myeloid cells in vivo, thereby further validating TACE as a principal target for the treatment of TNF-dependent pathologies.