Cryptic domains of tenascin-C differentially control fibronectin fibrillogenesis

The three-dimensional organization of the ubiquitous extracellular matrix glycoprotein fibronectin regulates cell fate and morphogenesis during development; in particular tubule formation that constitutes the vasculature, lung and kidney. Tenascin-C is a matrix protein with a restricted expression pattern; it is specifically up-regulated at sites of fibronectin fibril assembly during development and in remodeling adult tissues. Here we demonstrate that specific domains of tenascin-C inhibit fibronectin matrix assembly whereas full-length tenascin-C does not. These domains act via distinct mechanisms: TNfn1-8 blocks fibrillogenesis by binding to fibronectin fibrils and preventing intermolecular fibronectin interactions whilst FBG acts independently of binding to fibronectin and instead is internalized and causes cytoskeletal re-organization. We also show that TNfn1-8 disrupts epithelial cell tubulogenesis. Our data demonstrate that tenascin-C contains cryptic sites which can control tissue levels of fibrillar fibronectin either by preventing de novo fibril assembly or reducing levels of deposited fibronectin. Exposure of these domains during tissue remodeling may provide a novel means of controlling fibronectin assembly and tubulogenic processes dependent on the assembly of this matrix.     

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.     

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.     

Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix

Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.     

Mechanical Design of the Third FnIII Domain of Tenascin-C

By combining single-molecule atomic force microscopy (AFM), proline mutagenesis and steered molecular dynamics (SMD) simulations, we investigated the mechanical unfolding dynamics and mechanical design of the third fibronectin type III domain of tenascin-C (TNfn3) in detail. We found that the mechanical stability of TNfn3 is similar to that of other constituting FnIII domains of tenascin-C, and the unfolding process of TNfn3 is an apparent two-state process. By employing proline mutagenesis to block the formation of backbone hydrogen bonds and introduce structural disruption in β sheet, we revealed that in addition to the important roles played by hydrophobic core packing, backbone hydrogen bonds in β hairpins are also responsible for the overall mechanical stability of TNfn3. Furthermore, proline mutagenesis revealed that the mechanical design of TNfn3 is robust and the mechanical stability of TNfn3 is very resistant to structural disruptions caused by proline substitutions in β sheets. Proline mutant F88P is one exception, as the proline mutation at position 88 reduced the mechanical stability of TNfn3 significantly and led to unfolding forces of < 20 pN. This result suggests that Phe88 is a weak point of the mechanical resistance for TNfn3. We used SMD simulations to understand the molecular details underlying the mechanical unfolding of TNfn3. The comparison between the AFM results and SMD simulations revealed similarities and discrepancies between the two. We compared the mechanical unfolding and design of TNfn3 and its structural homologue, the tenth FnIII domain from fibronectin. These results revealed the complexity underlying the mechanical design of FnIII domains and will serve as a starting point for systematically analyzing the mechanical architecture of other FnIII domains in tenascins-C, and will help to gain a better understanding of some of the complex features observed for the stretching of native tenascin-C.     

Modulating the Mechanical Stability of Extracellular Matrix Protein Tenascin-C in a Controlled and Reversible Fashion

Stretching force can induce conformational changes of proteins and is believed to be an important biological signal in the mechanotransduction network. Tenascin-C is a large extracellular matrix protein and is subject to stretching force under its physiological condition. Regulating the mechanical properties of the fibronectin type III domains of tenascin-C will alter its response to mechanical stretching force and thus may provide the possibility of regulating the biological activities of tenascin-C in living cells. However, tuning the mechanical stability of proteins in a rational and systematic fashion remains challenging. Using the third fibronectin type III domain (TNfn3) of tenascin-C as a model system, here we report a successful engineering of a mechanically stronger extracellular matrix protein via engineered metal chelation. Combining steered molecular dynamics simulations, protein engineering and single-molecule atomic force microscopy, we have rationally engineered a bihistidine-based metal chelation site into TNfn3. We used its metal chelation capability to selectively increase the unfolding energy barrier for the rate-limiting step during the mechanical unfolding of TNfn3. The resultant TNfn3 mutant exhibits enhanced mechanical stability. Using a stronger metal chelator, one can convert TNfn3 back to a state of lower mechanical stability. This is the first step toward engineering extracellular matrix proteins with defined mechanical properties, which can be modulated reversibly by external stimuli, and will provide the possibility of using external stimuli to regulate the biological functions of extracellular matrix proteins.     

Cell surface molecules that bind fibronectin's matrix assembly domain

The assembly of fibronectin into disulfide cross-linked extracellular matrices requires the interaction of mesenchymal cells with two distinct sites on fibronectin, the Arg-Gly-Asp cell adhesive site and an amino-terminal site contained within the first five type I homologous repeats (Quade, B. J., and McDonald, J. A. (1988) J. Biol. Chem. 263, 19602-19609). Proteolytically derived 29-kDa fragments of fibronectin (29kDa) containing these repeats bind to monolayers of cultured fibroblasts and inhibit fibronectin matrix assembly. The cell surface molecules interacting with fibronectin's 29-kDa matrix assembly domain have resisted purification using conventional methods such as affinity chromatography. Accordingly, in order to identify molecules which bind this fragment, 125I-labeled 29kDa was allowed to bind to fibroblast monolayers and chemically cross-linked to the cell surface with bis(sulfosuccinimidyl) suberate. Extraction of the cross-linked cell layer yielded radiolabeled complexes of 56, 150, and 280 kDa. Formation of these cross-linked complexes was specifically inhibited by the addition of excess unlabeled 29kDa but was unaffected by the presence of fibronectin fragments containing other type I repeats outside of the 29kDa matrix assembly domain. The cross-linked complexes were insoluble in nondenaturing detergents but soluble when denatured and reduced, suggesting that 29kDa may be cross-linked to components of the pericellular matrix. Immunoprecipitation of cross-linked cell extracts with a polyclonal antibody to fibronectin that does not recognize the amino terminus demonstrate that the 280-kDa band contains 29kDa cross-linked to fibronectin present on the cell surface. Formation of the 150-kDa complex was inhibited by EDTA, suggesting that divalent cations are required for its formation. Although the molecular mass and divalent cation requirement suggest that the 150-kDa complex may be related to an integrin, this complex was not immunoprecipitated by polyclonal antibodies generated to the alpha 5 beta 1 integrin fibronectin receptor.     

Structure of the prion Ure2p in protein fibrils assembled in vitro

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers. Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Fibronectin's amino-terminal matrix assembly site is located within the 29-kDa amino-terminal domain containing five type I repeats

Fibronectin is organized into disulfide cross-linked, insoluble pericellular matrix fibrils by fibroblasts in vitro. Two sites, the Arg-Gly-Asp-Ser-containing cell attachment domain and a site located in the first 70 kDa of fibronectin, are required for matrix assembly. The first 70 kDa of fibronectin contain two structural motifs termed type I and type II homologies, which are repeated nine and two times, respectively. Previous work has implicated the amino-terminal region and the carboxyl terminus containing three type I repeats in matrix assembly, suggesting that type I repeats possess binding activity essential for fibronectin matrix assembly. To test this hypothesis, we developed a sensitive capture immunoassay to quantify insoluble matrix fibronectin and tested a panel of fibronectin fragments, containing all of the type I repeats found in the intact protein, for their ability to inhibit matrix assembly. Only fragments containing the first five type I repeats inhibited fibronectin matrix assembly, although sequences carboxyl-terminal to this domain enhanced this activity. Additional evidence for the specific recognition of the amino-terminal type I repeats by matrix assembling cells was found when the reversible, detergent-sensitive binding of a 125I-labeled fragment containing the first five type I repeats (29 kDa) to cell monolayers was studied. Only monolayers of cell lines that incorporate fibronectin into a fibrillar matrix specifically bound 125I-labeled 29 kDa. Binding of the radiolabeled amino-terminal fragment to matrix-forming cells was inhibited by unlabeled fragments containing the first five type I repeats but not by unlabeled fragments containing the remaining seven type I repeats. Matrix assembly is therefore not a generalized property of type I repeats. Rather, a critical site is located within the first 29 kDa of fibronectin.     

Structural characterization of the fibrillar form of the yeast Saccharomyces cerevisiae prion Ure2p

The protein Ure2 from the yeast Saccharomyces cerevisiae has prion properties. It assembles in vitro into long, straight, insoluble fibrils that are similar to amyloids in that they bind Congo Red and show green-yellow birefringence and have an increased resistance to proteolysis. We recently showed that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta-core. A model for fibril formation, where assembly is driven by non-native inter- and/or intramolecular interaction between Ure2p monomers following subtle conformational changes was proposed [Bousset et al. (2002) EMBO J. 21, 2903-2911]. An alternative model for the assembly of Ure2p into fibrils where assembly is driven by the stacking of 40-70 N-terminal amino acid residues of Ure2p into a central beta-core running along the fibrils from which the C-terminal domains protrude was proposed [Baxa et al. (2003) J. Biol. Chem. 278, 43717-43727]. We show here that Ure2p fibril congophilia and the associated yellow-green birefringence in polarized light are not indicative that the fibrils are of amyloid nature. We map the structures of the fibrillar and soluble forms of Ure2p using limited proteolysis and identify the reaction products by microsequencing and mass spectrometry. Finally, we demonstrate that the C-terminal domain of Ure2p is tightly involved in the fibrillar scaffold using a sedimentation assay and a variant Ure2p where a highly specific cleavage site between the N- and C-terminal domains of the protein was engineered. Our results are inconsistent with the cross-beta-core model and support the model for Ure2p assembly driven by subtle conformational changes and underline the influence of the natural context of the N-terminal domain on the assembly of Ure2p.     

A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly

F1 is an adhesin of Streptococcus pyogenes which binds the N-terminal 70-kDa region of fibronectin with high affinity. The fibronectin binding region of F1 is comprised of a 43-residue upstream domain and a repeat domain comprised of five tandem 37-residue sequences. We investigated the effects of these domains on the assembly of fibronectin matrix by human dermal fibroblasts, MG63 osteosarcoma cells, or fibroblasts derived from fibronectin-null stem cells. Subequimolar or equimolar concentrations of recombinant proteins containing both the upstream and repeat domains or just the repeat domain enhanced binding of fibronectin or its N-terminal 70-kDa fragment to cell layers; higher concentrations of these recombinant proteins inhibited binding. The enhanced binding did not result in greater matrix assembly and was caused by increased ligand binding to substratum. In contrast, recombinant or synthetic protein containing the 43 residues of the upstream domain and the first 6 residues from the repeat domain exhibited monophasic inhibition with an IC(50) of approximately 10 nm. Truncation of the 49-residue sequence at its N or C terminus caused loss of inhibitory activity. The 49-residue upstream sequence blocked incorporation of both endogenous cellular fibronectin and exogenous plasma fibronectin into extracellular matrix and inhibited binding of 70-kDa fragment to fibronectin-null cells in a fibronectin-free system. Inhibition of matrix assembly by the 49-mer had no effect on cell adhesion to substratum, cell growth, formation of focal contacts, or formation of stress fibers. These results indicate that the 49-residue upstream sequence of F1 binds in an inhibitory mode to N-terminal parts of exogenous and endogenous fibronectin which are critical for fibronectin fibrillogenesis.     

The heparin III-binding domain of fibronectin (III4–5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly

Fibronectin matrix assembly involves interactions among various regions of the molecule, which contribute to elongation and stabilization of the fibrils. In this study, we examined the possible role of the heparin III domain of fibronectin (repeats III4-5) in fibronectin fibrillogenesis. We show that a recombinant fragment comprising these repeats (FNIII4-5 fragment) blocked fibronectin fibril formation and the incorporation of 125I-fibronectin into cell layers. Binding assays using a biosensor revealed that FNIII4-5 bound fibronectin and the amino-terminal 70 kDa and 29 kDa fragments. It also bound to itself, indicating a previously unidentified self-association site in repeats III4-5. These interactions were specific since FNIII4-5 did not bind to the FNIII7-10 fragment, representing a central region in fibronectin. The fibronectin-binding property of the III4-5 domain, but not its matrix assembly inhibitory function, was apparently cryptic in larger fragments. By mutating the arginine residues in the WTPPRAQITGYRLTVGLTRR proteoglycan-binding sequence (HBP/III5 site) of FNIII4-5 [Moyano, J.V., Carnemolla, B., Albar, J.P., Leprini, A., Gaggero, B., Zardi, L., Garcia-Pardo, A., 1999. Cooperative role for activated alpha4beta1 integrin and chondroitin sulfate proteoglycans in cell adhesion to the heparin III domain of fibronectin. Identification of a novel heparin and cell binding sequence in repeat III5. J. Biol. Chem. 274, 135-142.], we found that the first two arginine residues in HBP/III5 were involved in the fibronectin-binding property of FNIII4-5, while the last two arginine residues in HBP/III5 were required for inhibition of matrix assembly and the binding of 125I-fibronectin to cell layers. Both properties appear to function independently from each other, depending on the conformation of the fibronectin dimer.     

Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α₅β₁ integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α₅β₁ integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α₅β₁ integrin, suggesting that these two domains of fibronectin cannot bind to the α₅β₁ integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α₅β₁ integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.     

Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.     

Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif.     

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.     

Identification of the fibronectin sequences required for assembly of a fibrillar matrix

During extracellular matrix assembly, fibronectin (FN) binds to cell surface receptors and initiates fibrillogenesis. As described in this report, matrix assembly has been dissected using recombinant FN polypeptides (recFNs) expressed in mammalian cells via retroviral vectors. RecFNs were assayed for incorporation into the detergent-insoluble cell matrix fraction and for formation of fibrils at the cell surface as detected by indirect immunofluorescence. Biochemical and immunocytochemical data are presented defining the minimum domain requirements for FN fibrillogenesis. The smallest functional recFN is half the size of native FN and contains intact amino- and carboxy-terminal regions with a large internal deletion spanning the collagen binding domain and the first seven type III repeats. Five type I repeats at the amino terminus are required for assembly and have FN binding activity. The dimer structure mediated by the carboxy-terminal interchain disulfide bonds is also essential. Surprisingly, recFNs lacking the RGDS cell binding site formed a significant fibrillar matrix. Therefore, FN-FN interactions and dimeric structure appear to be the major determinants of fibrillogenesis.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.