ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.     

Cooperation of the metalloprotease, disintegrin, and cysteine-rich domains of ADAM12 during inhibition of myogenic differentiation

The extracellular domain of the mature form of ADAM12 consists of the metalloprotease, disintegrin, cysteine-rich, and epidermal growth factor (EGF)-like domains. The disintegrin, cysteine-rich, and EGF-like fragments have been shown previously to support cell adhesion via activated integrins or proteoglycans. In this study, we report that the entire extracellular domain of mouse ADAM12 produced in Drosophila S2 cells supported efficient adhesion and spreading of C2C12 myoblasts even in the absence of exogenous integrin activators. This adhesion was not mediated by beta1 integrins or proteoglycans, was myoblast-specific, and required the presence of both the metalloprotease and disintegrin/cysteine-rich domains of ADAM12. Analysis of the recombinant proteins by far-UV circular dichroism suggested that the secondary structures of the autonomously expressed metalloprotease domain and the disintegrin/cysteine-rich/EGF-like domains differ from the structures present in the intact extracellular domain. Furthermore, the intact extracellular domain (but not the metalloprotease domain or the disintegrin/cysteine-rich/EGF-like fragment alone) decreased the expression of the cell cycle inhibitor p21 and myogenin, two markers of differentiation, and inhibited C2C12 myoblast fusion. Thus, the novel protein-protein interaction reported here involving the extracellular domain of ADAM12 may have important biological consequences during myoblast differentiation.     

Putative function of ADAM9, ADAM10, and ADAM17 as APP alpha-secretase

The putative alpha-secretase cleaves the amyloid precursor protein (APP) of Alzheimer's disease in the middle of the amyloid beta peptide (Abeta) domain. It is generally thought that the alpha-secretase pathway mitigates Abeta formation in the normal brain. Several studies have suggested that ADAM9, ADAM10, and ADAM17 are candidate alpha-secretases belonging to the ADAM (a disintegrin and metalloprotease) family, which are membrane-anchored cell surface proteins. In this comparative study of ADAM9, ADAM10, and ADAM17, we examined the physiological role of ADAMs by expressing these ADAMs in COS-7 cells, and both "constitutive" and "regulated" alpha-secretase activities of these ADAMs were determined. We tried to suppress the expression of these ADAMs in human glioblastoma A172 cells, which contain large amounts of endogenous alpha-secretase, by lipofection of the double-stranded RNA (dsRNA) encoding each of these ADAMs. The results indicate that ADAM9, ADAM10, and ADAM17 catalyze alpha-secretory cleavage and therefore act as alpha-secretases in A172 cells. This is the first report that to suggest the endogenous alpha-secretase is composed of several ADAM enzymes.     

Multiple non-catalytic ADAMs are novel integrin α4 ligands

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM–integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these “dead” enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.     

Selective modulation of integrin-mediated cell migration by distinct ADAM family members

A disintegrin and a metalloprotease (ADAM) family members have been implicated in many biological processes. Although it is recognized that recombinant ADAM disintegrin domains can interact with integrins, little is known about ADAM-integrin interactions in cellular context. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (alpha4beta1, alpha5beta1, or both), and cell migration on full-length fibronectin or on its alpha4beta1 or alpha5beta1 binding fragments was studied. We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAM12 inhibited cell migration mediated by the alpha4beta1 but not the alpha5beta1 integrin. ADAM17 had the reciprocal effect; it inhibited alpha5beta1- but not alpha4beta1-mediated cell migration. ADAM19 and ADAM33 inhibited migration mediated by both alpha4beta1 and alpha5beta1 integrins. A point mutation in the ADAM12 disintegrin loop partially reduced the inhibitory effect of ADAM12 on cell migration on the alpha4beta1 binding fragment of fibronectin, whereas mutations that block metalloprotease activity had no effect. Our results indicate that distinct ADAMs can modulate cell migration mediated by specific integrins in a pattern dictated, at least in part, by their disintegrin domains.     

Crystal structures of human ADAMTS-1 reveal a conserved catalytic domain and a disintegrin-like domain with a fold homologous to cysteine-rich domains

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type I motifs) family of proteases plays a role in pathological conditions including arthritis, cancer, thrombotic thrombocytopenic purpura and the Ehlers-Danlos type VIIC and Weill-Marchesani genetic syndromes. Here, we report the first crystal structures for a member of the ADAMTS family, ADAMTS-1. Originally cloned as an inflammation-associated gene, ADAMTS-1 has been shown to be involved in tissue remodelling, wound healing and angiogenesis. The crystal structures contain catalytic and disintegrin-like domains, both in the inhibitor-free form and in complex with the inhibitor marimastat. The overall fold of the catalytic domain is similar to related zinc metalloproteinases such as matrix metalloproteinases and ADAMs (a disintegrin and metalloproteinases). The active site contains the expected organisation of residues to coordinate zinc but has a much larger S1' selectivity pocket than ADAM33. The structure also unexpectedly reveals a double calcium-binding site. Also surprisingly, the previously named disintegrin-like domain showed no structural homology to the disintegrin domains of other metalloproteinases such as ADAM10 but is instead very similar in structure to the cysteine-rich domains of other metalloproteinases. Thus, this study suggests that the D (for disintegrin-like) in the nomenclature of ADAMTS enzymes is likely to be a misnomer. The ADAMTS-1 cysteine-rich domain stacks against the active site, suggesting a possible regulatory role.     

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     

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.     

ADAMs: modulators of cell-cell and cell-matrix interactions

ADAMs contain adhesive and metalloprotease domains. As major ectodomain sheddases, they release a variety of cell-surface proteins, including growth factors, cytokines, cell adhesion molecules and receptors. ADAMs can also cleave and remodel components of the extracellular matrix. Hence, ADAMs are emerging as key modulators of cell-cell and cell-matrix interactions. Important questions, including if and how ADAM adhesive domains promote ADAM protease function, are currently being addressed.     

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.     

The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion

ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.     

Expression of ADAMs and their inhibitors in sputum from patients with asthma

ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV(1)) (r = -0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV(1) (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.     

Cloning and expression in Pichia pastoris of metalloprotease domain of ADAM 9 catalytically active against fibronectin

ADAM 9 is a member of the cellular metalloprotease/disintegrin/cysteine-rich (MDC) gene family, related to soluble snake venom metalloproteases (SVMP). ADAMs may play important roles in cell-cell fusion, cell-matrix interaction, and other cellular functions. To investigate catalytic activity of human ADAM 9 we have cloned and expressed the metalloprotease domain of human ADAM 9 in Pichia pastoris. The recombinant protein was purified in a three-step purification procedure and activity was detected against gelatin, beta-casein, and fibronectin. In addition we identified five normal and cancer cell lines expressing mRNA of human ADAM 9.     

Crystal structure of the catalytic domain of human ADAM33

Adam33 is a putative asthma susceptibility gene encoding for a membrane-anchored metalloprotease belonging to the ADAM family. The ADAMs (a disintegrin and metalloprotease) are a family of glycoproteins implicated in cell-cell interactions, cell fusion, and cell signaling. We have determined the crystal structure of the Adam33 catalytic domain in complex with the inhibitor marimastat and the inhibitor-free form. The structures reveal the polypeptide fold and active site environment resembling that of other metalloproteases. The substrate-binding site contains unique features that allow the structure-based design of specific inhibitors of this enzyme.     

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.     

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.     

Catalytic activity of ADAM28

ADAMs are membrane-anchored glycoproteins containing a disintegrin and metalloprotease domain that have important roles in fertilization, development, and diseases such as Alzheimer's dementia. Here we present the first evidence for catalytic activity of ADAM28, a protein that is highly expressed in the epididymis and lymphocytes. Recombinant ADAM28 cleaves myelin basic protein at two sites. The catalytic activity of ADAM28 is not sensitive to tissue inhibitors of metalloproteases 1 and 2, but can be abolished by a mutation in the catalytic site. Catalytically active ADAM28 will be valuable for further studies of its role in sperm maturation and lymphocyte function.     

The lymphocyte metalloprotease MDC-L (ADAM 28) is a ligand for the integrin alpha4beta1.

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.     

Positive selection at reproductive ADAM genes with potential intercellular binding activity

Many genes with a role in reproduction, including those implicated in fertilization and spermatogenesis, have been shown to evolve at a faster rate relative to genes associated with other functions and tissues. These survey studies usually group a wide variety of genes with different characteristics and evolutionary histories as reproductive genes based on their site of expression or function. We have examined the molecular evolution of the ADAM (a disintegrin and metalloprotease) gene family, a structurally and functionally diverse group of genes expressed in reproductive and somatic tissue to test whether a variety of protein characteristics such as phylogenetic clusters, tissue of expression, and proteolytic and adhesive function can group fast evolving ADAM genes. We found that all genes were evolving under purifying selection (d(N)/d(S) < 1), although reproductive ADAMs, including those implicated in fertilization and spermatogenesis, evolved at the fastest rate. Genes with a role in binding to cell receptors in endogenous tissue appear to be evolving under purifying selection, regardless of the tissue of expression. In contrast, positive selection of codon sites in the disintegrin/cysteine-rich adhesion domains was detected exclusively in ADAMs 2 and 32, two genes expressed in the testis with a potential role in sperm-egg adhesion. Positive selection was detected in the transmembrane/cytosolic tail region of ADAM genes expressed in a variety of tissues.