Integrin ␣8␤1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of ␣8␤1 integrin toward a panel of RGD-containing ligands. Integrin ␣8␤1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ؎ 0.01 nM, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to ␣8␤1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central "linker" segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to ␣8␤1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrinligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of ␣8␤1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIF-EIER sequence was strongly inhibitory, and was ϳ2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to ␣8␤1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to ␣8␤1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).2 They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed ␣ and ␤. In mammals, 18 ␣ and 8 ␤ subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers.Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen-and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include ␣5␤1, ␣8␤1, ␣IIb␤3, and ␣V-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities. The ␣8 integrin subunit was originally identified in chick nerves (4). Integrin ␣8␤1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis.

Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system.

Background: Polydom/SVEP1 is a putative extracellular matrix protein of unknown function. Results: Polydom/SVEP1 is a potent ligand for integrin ␣9␤1 and colocalizes with the integrin in vivo. Conclusion: Polydom/SVEP1 is a hitherto unknown high affinity ligand for integrin ␣9␤1. Significance: The identification of this high affinity ligand offers important clues toward better understanding of the consequences of integrin ␣9␤1-mediated cell-extracellular matrix interactions.

Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (␣, ␤, and ␥), in which three laminin globular modules in the ␣ chain and the Glu residue in the C-terminal tail of the ␥ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the ␤ chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the ␤1 or ␤2 chain toward a panel of laminin-binding integrins, and we found that ␤2 chain-containing laminins (␤2-laminins) bound more avidly to ␣3␤1 and ␣7X2␤1 integrins than ␤1 chain-containing laminins (␤1-laminins), whereas ␣6␤1, ␣6␤4, and ␣7X1␤1 integrins did not show any preference toward ␤2-laminins. Because ␣3␤1 contains the "X2-type" variable region in the ␣3 subunit and ␣6␤1 and ␣6␤4 contain the "X1-type" region in the ␣6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between ␤1-laminins and ␤2-laminins. In support of this possibility, a putative X2-type variant of ␣6␤1 was produced and found to bind preferentially to ␤2-laminins. Production of a series of swap mutants between the ␤1 and ␤2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by ␤2-laminins. Taken together, the results provide evidence that the C-terminal region of ␤ chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.Laminins are large glycoproteins exclusively localized in basement membranes, which represent thin sheets of extracellular matrix bound by a variety of cell types, including epithelial, endothelial, muscle, and glial cells. Laminins are composed of three polypeptide chains (␣, ␤, and ␥), which assemble into a disulfide-bonded heterotrimer with a cross-shaped structure.There are five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) in mammals (1, 2), combinations of which give rise to at least 12 distinct isoforms expressed in tissue-specific and developmentally regulated manners (1, 3).Laminins play pivotal roles in embryonic development. Mice deficient in expression of the ␥1 chain, which is present in most laminin isoforms except for laminin-332 2 and some ␥3 chaincontaining isoforms, fail to deposit basement membranes and die at the peri-implantation stage of embryonic development (4). Gene knockouts of other laminin chains also result in severe phenotypes. Mice deficient in the ␣5 chain die around embryonic day 17 because of multiple developmental abnormalities, including failure of neural tube closure and digit separation and abnormal placental, kidney, and lung morphogenesis (5-7). Mice lacking the ␣2 chain show adult lethality because of severe and progressive skeletal muscle degeneration (8, 9). These phenotypes can be accounted for by defects in the physical strength of basement membra...

Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed ␣, ␤, and ␥. Recently, we found that the Glu residue at the third position from the C termini of the ␥1 and ␥2 chains is critically involved in integrin binding by laminins. However, the ␥3 chain lacks this Glu residue, suggesting that laminin isoforms containing the ␥3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of ␥3 chain-containing isoforms and those containing the ␥1 or ␥2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the ␥3 chain with the C-terminal nine amino acids of the ␥1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the ␥1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gainof-function evidence that laminin isoforms containing the ␥3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins.Laminins are heterotrimeric glycoproteins found in basement membranes and consist of three covalently linked chains termed ␣, ␤, and ␥. There are five ␣ chains (␣1-␣5), three ␤ chains (␤1-␤3), and three ␥ chains (␥1-␥3) that can give rise to at least 15 different functional laminin isoforms (1-3). These isoforms have been implicated in a wide variety of biological processes involving cell-basement membrane interactions through binding to cell surface receptors including integrins, syndecans, and dystroglycan (1, 4 -9).Integrins are ␣␤ transmembrane receptors that play critical roles in cell matrix adhesion in multicellular organisms. Several members of the integrin family proteins, including ␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1, serve as laminin receptors on a variety of cell types (10). The putative binding sites for these integrins have been mapped to the globular (G) 3 domain of the laminin ␣ chains (11-16), although trimerization with ␤ and ␥ chains is necessary for the G domain to exert its integrin binding activity (17)(18)(19). Recently, we found that the C-terminal regions of the ␥ chains are critically involved in integrin binding by laminins (20). Briefly, deletion of the C-terminal three but not two amino acids of the ␥1 chain completely abrogated the integrin binding activity of laminin-511 (␣5␤1␥1), while substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity; thereby underscoring a critical role of Glu-1607 in i...

CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.

The niche is a specialized microenvironment for tissue stem cells in vivo. It has long been emphasized that niche ECM molecules act on tissue stem cells to regulate their behavior, but the molecular entities of these interactions remain to be fully elucidated. Here, we report that laminin forms the in vivo ECM niche for trophoblast stem cells (TSCs), the tissue stem cells of the placenta. TSCs expressed fibronectin-binding, vitronectin-binding, and laminin-binding integrins, whereas the integrin ligands present in the TSC niche were collagen and laminin. Therefore, the only niche integrin ligand available for TSCs in vivo was laminin. Laminin promoted TSC adhesion and proliferation in vitro in an integrin binding–dependent manner. Importantly, when the integrin-binding ability of laminin was genetically ablated in mice, the size of the TSC population was significantly reduced compared with that in control mice. The present findings underscore an ECM niche function of laminin to support tissue stem cell maintenance in vivo.

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