Leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) is a modulator of FGFR1

Fibroblast growth factor receptors (FGFRs) play critical roles in craniofacial and skeletal development via multiple signaling pathways including MAPK, PI3K/AKT, and PLC-γ. FGFR-mediated signaling is modulated by several regulators. Proteins with leucine-rich repeat (LRR) and/or immunoglobulin (IG) superfamily domains have been suggested to interact with FGFRs. In addition, fibronectin leucine-rich repeat transmembrane protein 3 (FLRT3) has been shown to modulate the FGFR-mediated signaling via the fibronectin type III (FNIII) domain. Therefore proteins with LRR, IG, and FNIII are candidate regulators of the FGFRs. Here we identify leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) as a regulator of the FGFRs.     

Identification of the heparin-binding determinants within fibronectin repeat III1: role in cell spreading and growth

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III1 repeat (FNIII1). In this study, we localized the heparin-binding activity of FNIII1 to a cluster of basic amino acids, Arg613, Trp614, Arg615, and Lys617. Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin- and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII1.     

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.     

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.     

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.     

Existence of Tenascin-C Isoforms in Rat that Contain the Alternatively Spliced AD1 Domain are Developmentally Regulated During Hippocampal Development

Tenascin-C (TN-C) is a multimodular glycoprotein of the extracellular matrix which is important for the development of the nervous system and has a range of different functions which are mediated by the different protein domains present. TN-C contains eight constitutive fibronectin type III (FNIII) domains and a region of alternatively spliced FNIII domains. In the mouse and chick, six of these domains have been described and characterized, whereas in human there are nine of them. In this report, we show that seven alternatively spliced FNIII domains exist in rat and describe the differential expression pattern of the additional domain AD1 during embryonic and postnatal rat brain development. The AD1 domain of rat is homologous to the ones described in human and chick proteins but does not exist in mouse. Its expression can be located to the developing rat hippocampus and the lining of the lateral ventricle, regions where the TN-C protein may affect the behavior of stem and progenitor cells. During hippocampal development AD1 and the other alternatively spliced domains are differentially expressed as shown by RT-PCRs, immunocytochemistry and in situ hybridizations.     

Characterization of mouse serum tenascin-X

The interstitial extracellular matrix tenascin-X (iTNX), which has a molecular mass of roughly 450 kDa, is expressed at high levels in muscular tissues and skin. In this study, we identified the serum form of TNX (sTNX) with a molecular mass of 200 kDa in the mouse. Western blot analysis with specific antibodies against fibronectin type III-like (FNIII) repeats of TNX and N-terminal sequence analysis of 200-kDa sTNX revealed that the N-terminus of sTNX is located in the juncture between the 16th FNIII (M16) and 17th FNIII (M17) repeats of iTNX. The 200-kDa sTNX contains 15 FNIII repeats and a fibrinogen domain identical to the Cterminal portion of the iTNX. TNX-deficient mice lacked not only iTNX but also sTNX. Furthermore, 200-kDa sTNX was generated by cleavage of the spleen iTNX by spleen homogenate, and its generation was inhibited by protease inhibitors. These results suggest that sTNX is generated by proteolytic cleavage of iTNX.     

Fibronectin peptides derived from two distinct regions stimulate adipocyte differentiation by preventing fibronectin matrix assembly

Here, we show that fibronectin (FN) peptides derived from two distinct regions promote the insulin-induced adipocyte differentiation of ST-13 cells by preventing FN fibrillogenesis. ST-13 cells formed numerous FN fibrils under nonadipogenic conditions, whereas this FN fibrillogenesis was suppressed by adipose induction with insulin. The insulin-induced adipocyte differentiation was promoted by an amino-terminal 24-kDa fragment of FN, accompanied by further suppression of FN fibrillogenesis. The 24 K fragment prevented FN matrix assembly by direct incorporation into the FN matrix. Like the 24 K fragment, a peptide from the 14th type III repeat, termed FNIII14, which suppressed the integrin alpha 5 beta 1-mediated adhesion of ST-13 cells to FN, accelerated the adipocyte differentiation by preventing FN fibrillogenesis without direct incorporation into the FN matrix. FNIII14 induced the conformation change of beta1 integrins of K562 cells from active to resting, as judged by FACS analysis using a monoclonal antibody AG89 directed to an active beta1 integrin-dependent epitope. Binding of a (125)I-labeled FN fragment containing the RGD cell adhesive site to ST-13 cell surface was dissociated by FNIII14, with a concomitant binding of FNIII14 itself to the cell surface. The affinity labeling of ST-13 cells using biotinylated FNIII14 showed that FNIII14 specifically bound to a nonintegrin membrane protein with M(r) of around 50 kDa. Thus, the results indicated that prevention of FN fibrillogenesis by the 24 K Fib 1 fragment and FNIII14 caused the promotion of adipocyte differentiation of ST-13 cells and that the former was due to the direct incorporation into the FN matrix and that the latter might be interpreted by negative regulation of FN receptor alpha 5 beta 1 activity.     

Interdomain association in fibronectin: insight into cryptic sites and fibrillogenesis

The process by which fibronectin (FN), a soluble multidomain protein found in tissue fluids, forms insoluble fibrillar networks in the extracellular matrix is poorly understood. Cryptic sites found in FN type III domains have been hypothesized to function as nucleation points, thereby initiating fibrillogenesis. Exposure of these sites could occur upon tension-mediated mechanical rearrangement of type III domains. Here, we present the solution structures of the second type III domain of human FN ((2)FNIII), and that of an interaction complex between the first two type III domains ((1-2)FNIII). The two domains are connected through a long linker, flexible in solution. A weak but specific interdomain interaction maintains (1-2)FNIII in a closed conformation that associates weakly with the FN N-terminal 30 kDa fragment (FN30 kDa). Disruption of the interdomain interaction by amino-acid substitutions dramatically enhances association with FN30 kDa. Truncation analysis of (1-2)FNIII reveals that the interdomain linker is necessary for robust (1-2)FNIII-FN30 kDa interaction. We speculate on the importance of this interaction for FN function and present a possible mechanism by which tension could initiate fibrillogenesis.     

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.     

The mechanical hierarchies of fibronectin observed with single-molecule AFM

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.     

Ligation of the fibrin-binding domain by β-strand addition is sufficient for expansion of soluble fibronectin

How fibronectin (FN) converts from a compact plasma protein to a fibrillar component of extracellular matrix is not understood. "Functional upstream domain" (FUD), a polypeptide based on F1 adhesin of Streptococcus pyogenes, binds by anti-parallel β-strand addition to discontinuous sets of N-terminal FN type I modules, (2-5)FNI of the fibrin-binding domain and (8-9)FNI of the gelatin-binding domain. Such binding blocks assembly of FN. To learn whether ligation of (2-5)FNI, (8-9)FNI, or the two sets in combination is important for inhibition, we tested "high affinity downstream domain" (HADD), which binds by β-strand addition to the continuous set of FNI modules, (1-5)FNI, comprising the fibrin-binding domain. HADD and FUD were similarly active in blocking fibronectin assembly. Binding of HADD or FUD to soluble plasma FN exposed the epitope to monoclonal antibody mAbIII-10 in the tenth FN type III module ((10)FNIII) and caused expansion of FN as assessed by dynamic light scattering. Soluble N-terminal constructs truncated after (9)FNI or (3)FNIII competed better than soluble FN for binding of FUD or HADD to adsorbed FN, indicating that interactions involving type III modules more C-terminal than (3)FNIII limit β-strand addition to (1-5)FNI within intact soluble FN. Preincubation of FN with mAbIII-10 or heparin modestly increased binding to HADD or FUD. Thus, ligation of FNIII modules involved in binding of integrins and glycosaminoglycans, (10)FNIII and (12-14)FNIII, increases accessibility of (1-5)FNI. Allosteric loss of constraining interactions among (1-5)FNI, (10)FNIII, and (12-14)FNIII likely enables assembly of FN into extracellular fibrils.     

Engineering and expression of a recombinant fusion protein possessing fibroblast growth factor-2 and fibronectin fragment

There is a synergistic effect between fibronectin and fibroblast growth factor 2 (FGF-2) on osteoblast cell adhesion through an extracellular, signal-regulated kinase pathway. Here we describe the engineering of a fusion protein containing fibronectin fragment (FNIII9-10) connected to the COOH-terminus of FGF-2. Purified FGF2-FNIII9-10 fusion protein exhibited a significant increase of cell adhesion and proliferation of MG63 cells compared with FNIII9-10 alone (p < 0.05).     

Tenascin C Promiscuously Binds Growth Factors via Its Fifth Fibronectin Type III-Like Domain

Tenascin C (TNC) is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII) domains. The fifth TNCIII domain (TNCIII5) has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII), specifically FNIII12–14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1–5), as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF) family, the fibroblast growth factor (FGF) family, the transforming growth factor beta (TGF-β) superfamily, the insulin-like growth factor binding proteins (IGF-BPs), and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1–5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.     

Cancer-Derived Mutations in the Fibronectin III Repeats of PTPRT/PTPρ Inhibit Cell-Cell Aggregation

Abstract

The receptor protein tyrosine phosphatase T PTPρ is the most frequently mutated tyrosine phosphatase in human cancer. PTPρ mediates homophilic cell-cell aggregation. In its extracellular region, PTPρ has cell adhesion molecule–like motifs, including a MAM domain, an immunoglobulin domain, and four fibronectin type III (FNIII) repeats. Tumor-derived mutations have been identified in all of these extracellular domains. Previously, the authors determined that tumor-derived mutations in the MAM and immunoglobulin domains of PTPρ reduce homophilic cell-cell aggregation. In this paper, the authors describe experiments in which the contribution of the FNIII repeats to PTPρ-mediated cell-cell adhesion was evaluated. The results demonstrate that deletion of the FNIII repeats of PTPρ result in defective cell-cell aggregation. Furthermore, all of the tumor-derived mutations in the FNIII repeats of PTPρ also disrupt cell-cell aggregation. These results further support the hypothesis that mutational inactivation of PTPρ may lead to cancer progression by disrupting cell-cell adhesion.     

Effect of transfection with a gene coding for the fibronectin FNIII/10 fragment upon contact inhibition of C3H10T1/2 fibroblasts

The density-dependent growth inhibition of non-transformed cells may be associated with inefficient transduction of the proliferative signal from cell adhesion molecules. To verify this concept, the C3H10T1/2 fibroblasts were stably transfected with the gene coding for the fibronectin fragment III/10 (FNIII/10). This resulted in differences in gene's expression between original C3H10T1/2 cells and their FNIII/10 transfectants. No significant differences in growth properties were observed in the original or in the transfected cells. C3H10T1/2 cells and their transfectants, when co-cultured, displayed more cells at confluence than the cells cultured alone. Moreover, co-cultured C3H10T1/2 cells and their transfectants showed elevated levels of phospho-ERK1/2 compared to homogenous cultures. Results obtained indicate that cellular homogeneity is responsible for density-dependent growth inhibition.     

Protein H--a bacterial surface protein with affinity for both immunoglobulin and fibronectin type III domains.

Several bacterial species express surface proteins with affinity for the constant region (Fc) of immunoglobulin (Ig) G. The biological consequences of the interaction with IgG are poorly understood but it has been demonstrated that genes encoding different IgG Fc-binding proteins have undergone convergent evolution, suggesting that these surface molecules are connected with essential microbial functions. One of the molecules, protein H, is present in some strains of Streptococcus pyogenes, the most significant streptococcal species in clinical medicine. In contrast to other Ig-binding bacterial proteins tested, protein H was found to interact also with the neural cell adhesion molecule (N-CAM), a eukaryotic cell surface glycoprotein mediating homo- and heterophilic cell-cell interactions. The affinity for the interaction between protein H and N-CAM was 1.6 x 10(8)/M and the binding site on protein H was mapped to the NH2-terminal 80 amino acid residues. N-CAM and IgG are both members of the Ig superfamily and analogous to N-CAM, IgG binds to the NH2-terminal part of protein H. However, the binding sites for the two proteins were found to be separate, an unexpected result which was explained by the observation that the fibronectin type III (FNIII) domains and not the Ig-like domains of N-CAM are responsible for the interaction with protein H. Thus, the binding of N-CAM to protein H was blocked with fibronectin but not with IgG. Moreover, apart from fibronectin itself and N-CAM, fragments of fibronectin and the matrix protein cytotactin/tenascin containing FNIII domains also showed affinity for protein H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical shift assignments of the fibronectin III like domains 7–8 of type VII collagen

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain. The NC1 domain has subdomains with homology to adhesion molecules and mediates important interactions within the extracellular matrix. An 185 amino acid long part of the NC1-subdomain termed fibronectin III like domains 7 and 8 (FNIII7-8) was investigated. Antibodies against this region are pathogenic in a mouse model of EBA and one reported missense mutations of Col7 lies within these domains. The nearly complete NMR resonance assignment of recombinant FNIII7-8 of Col7 is reported.     

The structure and function of tenascins in the nervous system.

The tenascins are a family of large extracellular matrix glycoproteins that comprise five known members. Three of these, tenascin-C (TN-C) tenascin-R (TN-R) and tenascin-Y (TN-Y) are expressed in specific patterns during nervous system development and are down-regulated after maturation. The expression of TN-C, the best studied member of the family, persists in restricted areas of the nervous system that exhibit neuronal plasticity and is reexpressed after lesion. Numerous studies in vitro suggest specific roles for tenascins in the nervous system involving precursor cell migration, axon growth and guidance. TN-C has been shown to occur in a large number of isoform variants generated by combinatorial variation of alternatively spliced fibronectin type III (FNIII) repeats. This finding indicates that TN-C might specify neural microenvironments, a hypothesis supported by recent analysis of TN-C knockout animals, which has begun to reveal subtle nervous system dysfunctions.