The cysteine-rich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting

Snake venom metalloproteinases (SVMPs) are members of the Reprolysin family of metalloproteinases to which the ADAM (a disintegrin and metalloproteinase) proteins also belong. The disintegrin-like/cysteine-rich domains of the ADAMs have been implicated in their function. In the case of the SVMPs, we hypothesized that these domains could function to target the metalloproteinases to key extracellular matrix proteins or cell surface proteins. Initially we detected interaction of collagen XIV, a fibril-associated collagen with interrupted triple helices containing von Willebrand factor A (VWA) domains, with the PIII SVMP catrocollastatin. Next we investigated whether other VWA domain-containing matrix proteins could support the binding of PIII SVMPs. Using surface plasmon resonance, the PIII SVMP jararhagin and a recombinant cysteine-rich domain from a PIII SVMP were demonstrated to bind to collagen XIV, collagen XII, and matrilins 1, 3, and 4. Jararhagin was shown to cleave these proteins predominantly at sites localized at or near the VWA domains suggesting that it is the VWA domains to which the PIII SVMPs are binding via their cysteine-rich domain. In light of the fact that these extracellular matrix proteins function to stabilize matrix, targeting the SVMPs to these proteins followed by their specific cleavage could promote the destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries could contribute to their disruption and hemorrhage. Although there is only limited structural homology shared by the cysteine-rich domains of the PIII SVMPs and the ADAMs our results suggest an analogous function for the cysteine-rich domains in certain members of the expanded ADAM family of proteins to target them to VWA domain-containing proteins.     

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.     

O-glucosylation and O-fucosylation occur together in close proximity on the first epidermal growth factor repeat of AMACO (VWA2 protein)

AMACO (VWA2 protein) is an extracellular matrix protein of unknown function associated with certain basement membranes in skin, lung, and kidney. AMACO is a member of the von Willebrand factor A-like (VWA) domain containing protein superfamily and in addition to three VWA domains it also contains two epidermal growth factor-like domains. One of these contains the rare, overlapping consensus sequences for both O-glucosylation and O-fucosylation. In earlier studies of other proteins the attachment of either core glucose and fucose moieties or of the respective elongated glycans starting with these monosaccharides has been described. By a detailed mass spectrometric analysis we show that both elongated O-glucosylated (Xyl1-3Xyl1-3Glc) and elongated O-fucosylated glycan chains (NeuAc2-3Gal1-4GlcNAc1-3Fuc) can be attached to AMACO in close proximity on the same epidermal growth factor-like domain. It has been reported that the lack of O-fucosylation can markedly decrease secretion of proteins. However, the secretion of AMACO is not significantly affected when the glycosylation sites are mutated. The number of extracellular matrix proteins carrying the overlapping consensus sequence is very limited and it could be that these modifications have a new, yet unknown function.     

An interaction between integrin and the talin FERM domain mediates integrin activation but not linkage to the cytoskeleton

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is thought to have two roles: mediating inside-out activation of integrins, and connecting extracellular matrix (ECM)-bound integrins to the cytoskeleton. Talin's amino-terminal head, which consists of a FERM domain, binds an NPxY motif within the cytoplasmic tail of most integrin beta subunits. This is consistent with the role of FERM domains in recruiting other proteins to the plasma membrane. We tested the role of the talin-head-NPxY interaction in integrin function in Drosophila. We found that introduction of a mutation that perturbs this binding in vitro into the isolated talin head disrupts its recruitment by integrins in vivo. Surprisingly, when engineered into the full-length talin, this mutation did not disrupt talin recruitment by integrins nor its ability to connect integrins to the cytoskeleton. However, it reduced the ability of talin to strengthen integrin adhesion to the ECM, indicating that the function of the talin-head-NPxY interaction is solely to regulate integrin adhesion.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

The human leukocyte adhesion glycoprotein Mac-1 (complement receptor type 3, CD11b) alpha subunit. Cloning, primary structure, and relation to the integrins, von Willebrand factor and factor B

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or "L" domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.     

Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.     

A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.     

The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis

CLCA (chloride channel, calcium-activated) proteins are novel pulmonary vascular addresses for blood-borne, lung-metastatic cancer cells. They facilitate vascular arrest of cancer cells via adhesion to beta4 integrin and promote early, intravascular, metastatic growth. Here we identify the interacting binding domains of endothelial CLCA proteins (e.g. hCLCA2, mCLCA5, mCLCA1, and bCLCA2) and beta4 integrin. Endothelial CLCAs share a common beta4-binding motif (beta4BM) in their 90- and 35-kDa subunits of the sequence F(S/N)R(I/L/V)(S/T)S, which is located in the second extracellular domain of the 90-kDa CLCA and near the N terminus of the 35-kDa CLCA, respectively. Using enzyme-linked immunosorbent, pull-down, and adhesion assays, we showed that glutathione S-transferase fusion proteins of beta4BMs from the 90- and 35-kDa CLCA subunits bind to the beta4 integrin in a metal ion-dependent manner. Fusion proteins from fibronectin and the integrins beta1 and beta3 served as negative controls. beta4BM fusion proteins competitively blocked the beta4/CLCA adhesion and prevented lung colonization of MDA-MB-231 breast cancer cells. A disrupted beta4BM in hCLCA1, which is not expressed in endothelia, failed to interact with beta4 integrin. The corresponding CLCA-binding domain of the beta4 integrin is localized to the specific determining loop (SDL). Again enzyme-linked immunosorbent, pull-down, and adhesion assays were used to confirm the interaction with CLCA proteins using a glutathione S-transferase fusion protein representing the C-terminal two-thirds of beta4 SDL (amino acids 184-203). A chimeric beta4 integrin in which the indicated SDL sequence had been replaced with the corresponding sequence from the beta1 integrin failed to bind hCLCA2. The dominance of the CLCA ligand in beta4 activation and outside-in signaling is discussed in reference to our previous report that beta4/CLCA ligation elicits selective signaling via focal adhesion kinase to promote metastatic growth.     

Copine A,a calcium-dependent membrane-binding protein,transiently localizes to the plasma membrane and intracellular vacuoles in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Background  

Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways.     

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.     

Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions (review)

Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the alpha and beta subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the alpha/beta interface. Better atomic-level resolution structures of the alpha/beta transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the beta-tails. The concept of the beta integrin tail as a focal adhesion interaction 'hub' for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localization, are described.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

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.     

Transmembrane and cytoplasmic domains in integrin activation and protein-protein interactions (Review)

Integrins are heterodimeric membrane-spanning adhesion receptors that are essential for a wide range of biological functions. Control of integrin conformational states is required for bidirectional signalling across the membrane. Key components of this control mechanism are the transmembrane and cytoplasmic domains of the α and β subunits. These domains are believed to interact, holding the integrin in the inactive state, while inside-out integrin activation is accompanied by domain separation. Although there are strong indications for domain interactions, the majority of evidence is insufficient to precisely define the interaction interface. The current best model of the complex, derived from computational calculations with experimental restraints, suggests that integrin activation by the cytoplasmic protein talin is accomplished by steric disruption of the α/β interface. Better atomic-level resolution structures of the α/β transmembrane/cytoplasmic domain complex are still required for the resting state integrin to corroborate this. Integrin activation is also controlled by competitive interactions involving the cytoplasmic domains, particularly the β-tails. The concept of the β integrin tail as a focal adhesion interaction ‘hub’ for interactions and regulation is discussed. Current efforts to define the structure and affinity of the various complexes formed by integrin tails, and how these interactions are controlled, e.g. by phosphorylation and localization, are described.     

Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation     

The collagens of hydra provide insight into the evolution of metazoan extracellular matrices

A collagen-based extracellular matrix is one defining feature of all Metazoa. The thick sheet-like extracellular matrix (mesoglia) of the diploblast, hydra, has characteristics of both a basement membrane and an interstitial matrix. Several genes associated with mesoglea have been cloned including a basement membrane and fibrillar collagen and an A and B chain of laminin. Here we report the characterization of a further three fibrillar collagen genes (Hcol2, Hcol3, and Hcol5) and the partial sequence of a collagen gene with a unique structural organization consisting of multiple von Willebrand factor A domains interspersed with interrupted collagenous triple helices (Hcol6) from Hydra vulgaris. Hcol2 and -5 have major collagenous domains of classical length ( approximately 1020 amino acid residues), whereas the equivalent domain in Hcol3 is shorter (969 residues). The N-propeptide of Hcol2 contains a whey acid protein four-cysteine repeat (WAP) domain, and the equivalent domain of Hcol3 contains two WAP and two von Willebrand factor A domains. Phylogenetic analyses reveal that the hydra fibrillar collagen genes form a distinct clade that appears related to the protostome/deuterostome A clade of fibrillar collagens. Data base searches reveal Hcol2, -5, and -6 are highly conserved in Hydra magnipapillata, which also provided preliminary evidence for the expression of a B-clade fibrillar collagen. All four of the H. vulgaris collagens are expressed specifically by the ectoderm. The expression pattern for Hcol2 is similar to that previously reported for Hcol1 (Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S., Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000) Development 127, 4669-4680) but distinct from the pattern shared by Hcol3 and Hcol5. The characterization of multiple collagen genes in relatively simple diploblastic organisms provides new insights into the molecular evolution of collagens and the origins of the collagen-based extracellular matrix found throughout the multicellular animal kingdom.     

Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor.

The vitronectin receptor mediates cell adhesion to the extracellular matrix proteins vitronectin, fibrinogen, von Willebrand factor, and thrombospondin in an RGD-dependent manner. We previously demonstrated the direct interaction between the vitronectin receptor and an RGD-containing peptide by photoaffinity labeling the receptor with 125I-sulfosuccinimidyl-2-(p-azido-salicylamido)-1,3'-dithioprop ion ate (SASD)-GRGDSPK (Smith, J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263, 18726-18731). In that report, we identified amino acid residues 61-203 of the beta-subunit as proximal to the ligand binding site. Here we demonstrate that 125I-SASD-GRGDSPK affinity labels the alpha-subunit of the receptor at least two distinct sites within the region encompassing residues 139-349. Both of these regions are within the putative divalent cation binding region of the alpha-subunit. Collectively, our results suggest that discrete amino-terminal domains of both subunits of the receptor contribute to the structure of the ligand binding domain and furthermore that the ligand and divalent cation binding domains are spatially and functionally linked.     

A Disintegrin-like and Metalloprotease (Reprolysin-type) with Thrombospondin Type 1 Motif (ADAMTS) Superfamily: Functions and Mechanisms

Together with seven ADAMTS-like proteins, the 19 mammalian ADAMTS proteases constitute a superfamily. ADAMTS proteases are secreted zinc metalloproteases whose hallmark is an ancillary domain containing one or more thrombospondin type 1 repeats. ADAMTS-like proteins resemble ADAMTS ancillary domains and lack proteolytic activity. Vertebrate expansion of the superfamily reflects emergence of new substrates, duplication of proteolytic activities in new contexts, and cooperative functions of the duplicated genes. ADAMTS proteases are involved in maturation of procollagen and von Willebrand factor, as well as in extracellular matrix proteolysis relating to morphogenesis, angiogenesis, ovulation, cancer, and arthritis. New insights into ADAMTS mechanisms indicate significant regulatory roles for ADAMTS ancillary domains, propeptide processing, and glycosylation. ADAMTS-like proteins appear to have regulatory roles in the extracellular matrix.