Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain. ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes. The C. elegans pat-4 gene has been identified in genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments. pat-4 encodes the sole C. elegans homolog of ILK. In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked. Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments. Transgenically expressed 'kinase-dead' ILK fully rescues pat-4 loss-of-function mutants. UNC-112 has been identified as a new binding partner for ILK. These data strengthen the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner (Mackinnon, 2002).
The unc-52 gene of C. elegans encodes a homolog of the basement membrane heparan sulfate proteoglycan perlecan. Viable alleles reduce the abundance of UNC-52 in late larval stages and increase the frequency of distal tip cell (DTC) migration defects caused by mutations disrupting the UNC-6/netrin guidance system. These unc-52 alleles do not cause circumferential DTC migration defects in an otherwise wild-type genetic background. The effects of unc-52 mutations on DTC migrations are distinct from effects on myofilament organization and can be partially suppressed by mutations in several genes encoding growth factor-like molecules, including EGL-17/FGF, UNC-129/TGF-beta, DBL-1/TGF-beta, and EGL-20/WNT. It is proposed that UNC-52 serves dual roles in C. elegans larval development in the maintenance of muscle structure and the regulation of growth factor-like signaling pathways (Merz, 2003).
Mutations in the smu-1 gene of C. elegans suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. A presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. Reverse transcription-PCR and RNase protection were used to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. smu-1 has been identified molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. It is proposed that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes (Spike, 2001).
C. elegans MEC-8 is a putative RNA-binding protein that promotes specific alternative splices of unc-52 transcripts. MEC-8 is a nuclear protein found in hypodermis at most stages of development and not in most late embryonic or larval body-wall muscle. Overexpression of MEC-8 in hypodermis but not muscle can suppress certain unc-52 mutant phenotypes. These are unexpected results because it has been proposed that UNC-52 is produced exclusively by muscle. Various tissue-specific unc-52 minigenes were constructed and fused to a gene for green fluorescent protein. These minigenes have allowed monitoring of tissue-specific mec-8-dependent alternative splicing; mec-8 must be expressed in the same cell type as the unc-52 minigene in order to regulate the minigene's expression, supporting the view that MEC-8 acts directly on unc-52 transcripts and that UNC-52 must be synthesized primarily by the hypodermis. Indeed, this analysis of unc-52 genetic mosaics has shown that the focus of unc-52 action is not in body-wall muscle but most likely is in hypodermis (Spike, 2002).
A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor-binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator (Aviezer, 1994).
The internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) has been investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells has revealed saturable binding with a half-saturation constant of 76 nm and a maximal binding capacity of 183 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 is significantly inhibited by a cationic polypeptide poly-L-lysine, and compounds that contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B. Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells is significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis has revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrates that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes (Deguchi, 2002).
Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bind similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulate the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulates those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation is similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that is capable of binding FGF-2 is incapable of activating any BaF3 cell line unless the HS is removed from the protein core. The HS chains also exhibit greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity (Knox, 2002).
Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV-1 (IG2-9, 100 kDa) and IV-2 (IG10-15, 66 kDa). Both fragments bind to a heparin column, being eluted at ionic strengths either below (IV-2) or above (IV-1) physiological level, and can thus be readily purified. Electron microscopy demonstrated an elongated shape (20-25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid-phase and surface plasmon resonance assays demonstrated strong binding of fragment IV-1 to fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, with Kd values in the range 4-17 nM. The latter binding apparently occurs through nidogen-1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin-2 and collagen IV and none for fibulin-1 and BM-40. Fragment IV-2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin-2. None of these activities could be demonstrated for recombinant fragments corresponding to the N-terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein-protein interactions (Hopf, 1999).
Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. The crystal structure of the mouse nidogen-1 G2 fragment has been solved; this fragment contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding (Hopf, 2001a).
Perlecan, a major basement membrane proteoglycan, has a complex modular structure designed for the binding of many cellular and extracellular ligands. Its domain IV, which consists of a tandem of immunoglobulin-like modules (IG2-IG15), is rich in such binding sites, which have been mapped to different modules obtained by recombinant production. Heparin/sulfatide binding is restricted to IG5 and depends on four arginine residues that are close in space in beta strands B and E of the C-type IG fold. The nidogen-1 and nidogen-2 isoforms bind to IG3 with high affinity [K(d) approximately 10 nM]. This interaction depends on the globular nidogen domain G2 and is crucial for the formation of ternary complexes with laminins. Two loops of IG3 located between beta strands B/C and F/G, which are spatially close, make a major contribution to binding. Fibronectin binding is localized to IG4-5, and fibulin-2 binds to IG2 and IG13-15 with different affinities. This implicates a complex cluster of heterotypic interaction sites apparently important for the supramolecular organization of perlecan in tissues (Hopf, 2001b).
Perlecan, a widespread heparan sulfate proteoglycan, functions as a bioactive reservoir for growth factors by stabilizing them against misfolding or proteolysis. These factors, chiefly members of the fibroblast growth factor (FGF) gene family, are coupled to the N-terminal heparan sulfate chains, which augment high affinity binding and receptor activation. However, rather little is known about biological partners of the protein core. The major goal of this study was to identify novel proteins that interact with the protein core of perlecan. Using the yeast two-hybrid system and domain III of perlecan as bait, approximately 0.5 106 cDNA clones were screened from a keratinocyte library and a strongly interactive clone was identified. This cDNA corresponded to FGF-binding protein (FGF-BP), a secreted protein previously shown to bind acidic and basic FGF and to modulate their activities. Using a panel of deletion mutants, FGF-BP binding was localized to the second EGF repeat of domain III, a region very close to the binding site for FGF7. FGF-BP could be coimmunoprecipitated with an antibody against perlecan and bound in solution to recombinant domain III-alkaline phosphatase fusion protein. Immunohistochemical analyses revealed colocalization of FGF-BP and perlecan in the pericellular stroma of various squamous cell carcinomas suggesting a potential in vivo interaction. Thus, FGF-BP should be considered a novel biological ligand for perlecan, an interaction that could influence cancer growth and tissue remodeling (Mongiat, 2001).
PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, it was found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bind PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibits the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I does not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, does not bind perlecan. Confocal immunohistochemistry shows that PRELP outlines basement membranes with a location adjacent to perlecan. PRELP also binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualizes a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, a function is proposed for PRELP as a molecule anchoring basement membranes to the underlying connective tissue (Bengtsson, 2002).
The collagen-tailed form of acetylcholinesterase (AChE) is concentrated at the vertebrate neuromuscular junction (NMJ), where it is responsible for rapidly terminating neurotransmission. This unique oligomeric form of AChE, consisting of three tetramers covalently attached to a collagen-like tail, is more highly expressed in innervated regions of skeletal muscle fibers, where it is externalized and attached to the synaptic basal lamina interposed between the nerve terminal and the receptor-rich postsynaptic membrane. Although it is clear that the enzyme is preferentially synthesized in regions of muscle contacted by the motoneuron, the molecular events underlying its localization to the NMJ are not known. Perlecan, a multifunctional heparan sulfate proteoglycan concentrated at the NMJ, is shown to be the unique acceptor molecule for collagen-tailed AChE at sites of nerve-muscle contact and is the principal mechanism for localizing AChE to the synaptic basal lamina (Arikawa-Hirasawa, 2002)
The yeast two-hybrid system was used to find interactive partners of perlecan; perlecan domain V was used as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, a ~1.6-kb cDNA insert was isolated that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that was named ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C-terminus of ECM1 interacts specifically with the EGF-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression (Mongiat, 2003b).
Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme causes invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes develop a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation (Costell, 1999).
The gene encoding perlecan (Hspg2) was disrupted in mice. Approximately 40% of Hspg2-/- mice died at embryonic day (E) 10.5 with defective cephalic development. The remaining Hspg2-/- mice died just after birth with skeletal dysplasia characterized by micromelia with broad and bowed long bones, narrow thorax and craniofacial abnormalities. Only 6% of Hspg2-/- mice developed both exencephaly and chondrodysplasia. Hspg2-/- cartilage shows severe disorganization of the columnar structures of chondrocytes and defective endochondral ossification. Hspg2-/- cartilage matrix contained reduced and disorganized collagen fibrils and glycosaminoglycans, suggesting that perlecan has an important role in matrix structure. In Hspg2-/- cartilage, proliferation of chondrocytes is reduced and the prehypertrophic zone is diminished. The abnormal phenotypes of the Hspg2-/- skeleton are similar to those of thanatophoric dysplasia (TD) type I, which is caused by activating mutations in FGFR3, and to those of Fgfr3 gain-of-function mice. These findings suggest that these molecules affect similar signalling pathways (Arikawa-Hirasawa, 1999).
Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. The SJS1 locus has been localized to chromosome 1p34-p36.1; no evidence was found of genetic heterogeneity. Mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) are described in three SJS1 families. This study identifies the first human mutations in HSPG2, underscoring the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability (Nicole, 2000).
Mice lacking the perlecan gene (Hspg2) have a severe chondrodysplasia with dyssegmental ossification of the spine and show radiographic, clinical and chondro-osseous morphology similar to a lethal autosomal recessive disorder in humans termed dyssegmental dysplasia, Silverman-Handmaker type. This study reports a homozygous, 89-bp duplication in exon 34 of HSPG2 in a pair of siblings with DDSH born to consanguineous parents, and heterozygous point mutations in the 5' donor site of intron 52 and in the middle of exon 73 in a third, unrelated patient, causing skipping of the entire exons 52 and 73 of the HSPG2 transcript, respectively. These mutations are predicted to cause a frameshift, resulting in a truncated protein core. The cartilage matrix from these patients stained poorly with antibody specific for perlecan, but there was staining of intracellular inclusion bodies. Biochemically, truncated perlecan was not secreted by the patient fibroblasts, but was degraded to smaller fragments within the cells. Thus, DDSH is caused by a functional null mutation of HSPG2. These findings demonstrate the critical role of perlecan in cartilage development (Arikawa-Hirasawa, 2001).
Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2Delta 3/Delta 3 mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are both observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as protein uria, was detected in Hspg2Delta 3/Delta 3 mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2Delta 3/Delta 3 mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1-/- x Hspg2Delta 3/Delta 3 mice and degeneration proceeded faster than in Hspg2Delta 3/Delta 3 mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals (Rossi, 2003).
Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding (Hohenester, 1999).
Dystroglycan (DG) function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, roles for other molecules in this process have been defined. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell (Henry, 2001b).
Perlecan plays key roles in blood vessel growth and structural integrity. The C terminus of perlecan potently inhibits four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan is active at nanomolar concentrations and blocks endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth it has been named 'endorepellin.' Endothelial cells possess a significant number of high affinity [K(d) of 11 nm] binding sites for endorepellin and endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo (Mongiat, 2003a).
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