ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors

A disintegrin and metalloprotease (ADAM) is a membrane-anchored metalloprotease implicated in the ectodomain shedding of cell surface proteins, including the ligands for epidermal growth factor (EGF) receptors (EGFR)/ErbB. It has been well documented that the transactivation of the EGFR plays critical roles for many cellular functions, such as proliferation and migration mediated through multiple G protein-coupled receptors (GPCRs). Recent accumulating evidence has suggested that ADAMs are the key metalloproteases activated by several GPCR agonists to produce a mature EGFR ligand leading to the EGFR transactivation. In this review, we describe the current knowledge on ADAMs implicated in mediating EGFR transactivation. The major focus of the review will be on the possible upstream mechanisms of ADAM activation by GPCRs as well as downstream signal transduction and the pathophysiological significances of ADAM-dependent EGFR transactivation. ectodomain shedding; angiotensin II     

ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk

All ligands of the epidermal growth factor receptor (EGFR) which has important roles in development and disease, are shed from the plasma membrane by metalloproteases. The ectodomain shedding of EGFR ligands has emerged as a critical component in the functional activation of EGFR in the interreceptor cross-talk. Identification of the sheddases for EGFR ligands using mouse embryonic cells lacking candidate sheddases (a disintegrin and metalloprotease; ADAM) has revealed that ADAM10, -12 and -17 are the sheddases of the EGFR ligands in response to various shedding stimulants such as GPCR agonists, growth factors, cytokines, osmotic stress, wounding and phorbol ester. Among the EGFR ligands, heparin-binding EGF-like growth factor (HB-EGF) is a representative ligand to understand the pathophysiological roles of the ectodomain shedding in wound healing, cardiac diseases, etc. Here we focus on the ectodomain shedding of HB-EGF by ADAMs, which is not only a key event of receptor cross-talk but also a novel intercellular signaling by the carboxy-terminal fragment (CTF signal).     

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 alpha, 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.     

Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin

A disintegrin and metalloproteinase (ADAM) is a family of enzymes involved in ectodomain shedding of various membrane proteins. However, the molecular mechanism underlying substrate recognition by ADAMs remains unknown. In this study, we successfully captured and analyzed cell surface transient assemblies between the transmembrane amphiregulin precursor (proAREG) and ADAM17 during an early shedding phase, which enabled the identification of cell surface annexins as components of their shedding complex. Annexin family members annexin A2 (ANXA2), A8, and A9 interacted with proAREG and ADAM17 on the cell surface. Shedding of proAREG was increased when ANXA2 was knocked down but decreased with ANXA8 and A9 knockdown, because of enhanced and impaired association with ADAM17, respectively. Knockdown of ANXA2 and A8 in primary keratinocytes altered wound-induced cell migration and ultraviolet B-induced phosphorylation of epidermal growth factor receptor (EGFR), suggesting that annexins play an essential role in the ADAM-mediated ectodomain shedding of EGFR ligands. On the basis of these data, we propose that annexins on the cell surface function as "shedding platform" proteins to determine the substrate selectivity of ADAM17, with possible therapeutic potential in ADAM-related diseases.     

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.     

Regulated cell surface pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.     

Shedding light on sheddases: role in growth and development.

The extracellular domains of several integral membrane proteins are released from the cell surface by a group of enzymes known as "sheddases" through a process called "ectodomain shedding". Because many transmembrane growth and differentiation factors, including members of the epidermal growth factor (EGF) family that play a crucial role in development, require ectodomain shedding for proper action in vivo, proteolysis is now viewed as a regulatory mechanism in the developing embryos. Two recent reports by Zhao et al. provide evidence for the role of cell surface proteolysis by an ADAM (a disintegrin and metalloprotease) in the development of murine lung. Inhibition of tumor necrosis factor-alpha converting enzyme (TACE, ADAM17) by the hydroxamic acid-based metalloprotease inhibitor (TAPI), or a targeted mutation in Zn(2+)-binding domain of TACE, disrupts two essential epithelial functions in lung development: branching morphogenesis and cytodifferentiation. Evidence for the role of ADAMs as sheddases in development and growth factor signaling is discussed.     

PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.     

Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.     

Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are made as transmembrane precursors. Proteolytic processing by ADAMs (a disintegrin and metalloprotease) regulates the bioavailability of several EGFR-ligands, yet little is known about the enzyme responsible for processing the recently identified EGFR ligand, epigen. Here we show that ectodomain shedding of epigen requires ADAM17, which can be stimulated by phorbol esters, phosphatase inhibitors and calcium influx. These results suggest that ADAM17 might be a good target to block the release of bioactive epigen, a highly mitogenic ligand of the EGFR which has been implicated in wound healing and cancer.     

Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion is still not well understood even though it is thought to be related to the protein kinase C/mitogen-activated protein kinase/AP-1 pathway. Recently, TPA was also found to induce epidermal growth factor receptor (EGFR) activity. Here, we investigated whether the EGFR is a necessary component for TPA-induced signal transduction associated with tumor promotion. We demonstrated that potent inhibitors of the EGFR, PD153035 and AG1478, blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs), AP-1 activity, and cell transformation. Egfr gene deficiency blocked TPA-induced ERK activity and AP-1 binding activity. The blocking of the ectodomain of the EGFR by a monoclonal antibody depressed TPA-induced ERK activity and AP-1 DNA binding activity. The use of a neutralizing antibody for heparin-binding EGF, one of the ligands of EGFR, blocked TPA-induced phosphorylation of ERKs. BB-94, a potent inhibitor of matrix metalloproteinases, which are activators of ectodomain shedding of EGFR ligands, also blocked TPA-induced ERK activity, AP-1 DNA binding, and cell transformation but had no effect on EGF-induced signal transduction. Anti-EGFR, anti-heparin-binding EGF, and BB-94 each blocked TPA-induced EGFR phosphorylation, but only anti-EGFR could block EGF-induced EGFR phosphorylation. Based on these results, we conclude that the EGFR is required for mediating TPA-induced signal transduction. EGFR transactivation induced by TPA is a mechanism by which the EGFR mediates TPA-induced tumor promotion-related signal transduction.     

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

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha 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 than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

The epidermal growth factor receptor ligands at a glance

The epidermal growth factor receptor (EGFR) regulates key processes of cell biology, including proliferation, survival, and differentiation during development, tissue homeostasis, and tumorigenesis. Canonical EGFR activation involves the binding of seven peptide growth factors. These ligands are synthesized as transmembrane proteins comprising an N‐terminal extension, the EGF module, a short juxtamembrane stalk, a hydrophobic transmembrane domain, and a carboxy‐terminal fragment. The central structural and functional feature is the EGF module, a sequence containing six cysteines in a conserved spacement which is responsible for binding to the EGFR. While the membrane‐anchored peptide can be biologically active by juxtacrine signaling, in most cases the EGF module is proteolytically cleaved (a process termed ectodomain shedding) to release the soluble growth factor, which may act in an endocrine, paracrine, or autocrine fashion. This review summarizes the structural and functional properties of these fascinating molecules and presents selected examples to illustrate their roles in development, physiology, and pathology. J. Cell. Physiol. 218: 460–466, 2009. © 2008 Wiley‐Liss, Inc.     

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.     

The “A Disintegrin And Metalloprotease” (ADAM) family of sheddases: Physiological and cellular functions

There is an exciting increase of evidence that members of the disintegrin and metalloprotease (ADAM) family critically regulate cell adhesion, migration, development and signalling. ADAMs are involved in “ectodomain shedding” of various cell surface proteins such as growth factors, receptors and their ligands, cytokines, and cell adhesion molecules. The regulation of these proteases is complex and still poorly understood. Studies in ADAM knockout mice revealed their partially redundant roles in angiogenesis, neurogenesis, tissue development and cancer. ADAMs usually trigger the first step in regulated intramembrane proteolysis leading to activation of intracellular signalling pathways and the release of functional soluble ectodomains.     

Cellular roles of ADAM12 in health and disease

ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.     

Cytoplasmic domain phosphorylation of heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a transmembrane precursor protein that is anchored to the plasma membrane. The extracellular EGF-like domain acts as a mitogen and motogen upon ectodomain shedding, but the functional roles of the transmembrane and cytoplasmic domains are largely unknown. We demonstrate here that cytoplasmic domain of HB-EGF is phosphorylated by external stimuli, and that the phosphorylation site is involved in HB-EGF-dependent tumorigenesis. Treatment of Vero cells overexpressing human HB-EGF with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused ectodomain shedding of HB-EGF and generated two carboxyl (C)-terminal fragments with distinct electrophoretic mobilities. Mutation analysis showed that Ser207 in the cytoplasmic domain of HB-EGF is phosphorylated upon TPA stimulation, generating two C-terminal fragments with distinct phosphorylation states. Treatment of cells with lysophosphatidic acid, anisomycin, and calcium ionophore, all of which are known to induce ectodomain shedding, also caused phosphorylation of HB-EGF. Although ectodomain shedding and phosphorylation of HB-EGF occurred coordinately, Ala substitution of Ser207 had no effect on TPA-induced or constitutive ectodomain shedding. Injection of cells overexpressing HB-EGF into nude mice showed that Ala substitution of Ser207 reduced the tumorigenic activity of HB-EGF, even though the cell surface level and ectodomain shedding of HB-EGF were not affected by the mutation. Moreover, we found that the cytoplasmic domain of another EGFR ligand, transforming growth factor-alpha, is phosphorylated upon TPA stimulation. Thus, the present results suggest a novel role for the cytoplasmic domain of HB-EGF and other EGF family growth factors that is regulated by phosphorylation.     

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF). EGF receptor phosphorylation and cell proliferation assays demonstrated that the biological activity of the coil-tagged EGF versions was comparable to that of non-tagged EGF. Additionally, analysis of the binding of E5/K5-coil-EGF to cell surface EGFR or to soluble EGFR ectodomain, as measured by cell-based binding competition assays and by SPR-based biosensor experiments, indicated that the coil-tagged EGF versions bound to EGFR with affinities similar to that of non-tagged EGF. Finally, we show that E-coil-tagged EGF, but not non-tagged EGF, can retarget a K-coil containing adenovirus to EGF receptor expressing glioblastoma tumor cells. Overall these results indicate that E. coli expression offers a practical platform for the reproducible production of fully biologically active E5/K5-coil-tagged EGF, and support applications of heterodimerizing coil-tagged ligands, e.g. the targeting of viruses or other entities such as nanoparticles to tumor cells, or growth factor immobilization on cell culture scaffolds for tissue engineering.