The Requirement of the Glutamic Acid Residue at the Third Position from the Carboxyl Termini of the Laminin γ Chains in Integrin Binding by Laminins

Ido, Hiroyuki; Nakamura, Aya; Kobayashi, Reiko; Ito, Shunsuke; Li, Shaoliang; Futaki, Sugiko; Sekiguchi, Kiyotoshi

doi:10.1074/jbc.m609402200

Cited by 94 publications

(115 citation statements)

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“…K1606 is adjacent to a conserved glutamate (γ E1605) (Fig. 6), shown previously to be crucial for integrin binding (45). The cross-linking results support a model in which the γ chain tail is in the vicinity of the LG1/LG2 interface and may assist in forming the cloverleaf association, explaining why the crystal structure of LG1 through LG3, which lacked the coiled coil and γ chain tail, did not form the cloverleaf and why removal of γ E1605 undermined integrin binding (45).…”

Section: Resultsmentioning

confidence: 95%

“…6), shown previously to be crucial for integrin binding (45). The cross-linking results support a model in which the γ chain tail is in the vicinity of the LG1/LG2 interface and may assist in forming the cloverleaf association, explaining why the crystal structure of LG1 through LG3, which lacked the coiled coil and γ chain tail, did not form the cloverleaf and why removal of γ E1605 undermined integrin binding (45). The LG docking models and observed interactions with residues near the coiled-coil terminus, while yielding only approximate orientations, nevertheless provide molecular insight into self-assembly of cell-binding regions of laminin.…”

Section: Resultsmentioning

confidence: 98%

“…We present here a definitive experimental determination of the subunit order and a prediction of the subunit register for the laminin coiled coil. In addition, we obtained insights into the location of the Lβ knob with respect to the other coiled-coil subunits and into the association of LG domains with the carboxyl terminus of the coiled coil, a requirement for integrin binding (45). The functional importance of the laminin protein family in interaction and signaling between cells and the ECM warrants continued efforts to explore the structure and dynamics of this remarkable macromolecule.…”

Section: Discussionmentioning

confidence: 99%

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Cross-linking reveals laminin coiled-coil architecture

Armony

Jacob

Moran

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.L aminins are network-forming constituents of the extracellular matrix (ECM) (1, 2). They interact with the cell surface and other ECM components to generate a physical and functional framework affecting cell viability, identity, and activity. The laminin family appears to have arisen during the evolution of multicellularity in animals (3), and laminins contribute to a diversity of basement membrane and connective tissue structures in mammals (2). Laminins are studied in the context of development (4), stem cell biology (5), tissue engineering (6), cancer (7), and aging (8). The remarkable structural organization of laminins underlies their important physiological functions.Laminins are composed of three subunits, α, β, and γ, that assemble into a roughly humanoid form as visualized using rotary shadowing electron microscopy (9). The individual subunits separately form the "head" and two "arms," which are composed of epidermal growth factor (EGF)-like cysteine-rich repeats with globular domains embedded (10) (Fig. 1). Following the head and arms, the three subunits come together to form a long coiled coil, constituting the "body." Additional globular domains unique to the α subunit are the "feet." The laminin head and arms may splay to form a tripod (2), a feature not captured in rotary shadowing images or in diagrams of domain composition. Due to the centrality of laminin in cell-ECM interactions, efforts have been made to analyze the functional regions of the trimer, to determine which α, β, and γ paralogs associate into physiological heterotrimers and to understand how trimers self-assemble into higher-order networks. Laminin fragments have been generated to assess their binding properties (11, 12) and as targets for structure determination by X-ray crystallography (13)(14)(15)(16)(17)(18)(19)(20).Despite this progress, few insights into the overall 3D architecture of laminin have been made in the past few decades, and certain regions of the complex have been neglected as targets of structural techniques. In particular, no structure has been determined for any segment of th...

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Cross-linking reveals laminin coiled-coil architecture

Armony

Jacob

Moran

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The PCR-amplified cDNA was digested with BamHI/ PmeI and inserted into the corresponding restriction sites of the pEF expression vector in-frame to the sequence encoding the "BASE" peptide and a His 6 tag at the 3Ј end of the integrin ␤8 cDNA as described previously (35). Another pEF vector encoding the extracellular region of integrin ␤8 lacking a C-terminal His 6 tag was also constructed by overlap extension PCR for expression of recombinant ␣v␤8 integrin lacking the His 6 tag (designated as ␣v␤8(⌬His)).…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis of the Ligand Binding Specificity of αvβ8 Integrin

Ozawa

Sato

Imabayashi

et al. 2016

Journal of Biological Chemistry

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␣v␤8 is an integrin that recognizes an Arg-Gly-Asp (RGD) motif and interacts with fibronectin, vitronectin, and latent TGF-␤1. We comprehensively determined the binding activity of the ␣v␤8 integrin toward 25 secreted proteins having an RGD motif. The ␣v␤8 integrin strongly bound to latent TGF-␤1 but showed marginal activity for other RGD-containing proteins, including fibronectin and vitronectin. Site-directed mutagenesis of latent TGF-␤1 demonstrated that the high affinity binding of ␣v␤8 integrin to latent TGF-␤1 was defined by Leu-218 immediately following the RGD motif within the latency-associated peptide of TGF-␤1. Consistent with the critical role of Leu-218 in latent TGF-␤1 recognition by ␣v␤8 integrin, a 9-mer synthetic peptide containing an RGDL sequence strongly inhibited interactions of latent TGF-␤1 with ␣v␤8 integrin, whereas a 9-mer peptide with an RGDA sequence was ϳ60-fold less inhibitory. Because ␣v␤3 integrin did not exhibit strong binding to latent TGF-␤1 or distinguish between RGDL-and RGDA-containing peptides, we explored the mechanism by which the integrin ␤8 subunit defines the high affinity binding of latent TGF-␤1 by ␣v␤8 integrin. Production of a series of swap mutants of integrin ␤8 and ␤3 subunits indicated that the high affinity binding of ␣v␤8 integrin with latent TGF-␤1 was ensured by interactions between the Leu-218 residue and the ␤8 I-like domain, with the former serving as an auxiliary recognition residue defining the restricted ligand specificity of ␣v␤8 integrin toward latent TGF-␤1. In support of this conclusion, high affinity binding toward the ␣v␤8 integrin was conferred on fibronectin by substitution of its RGDS motif with an RGDL sequence.Integrins are a family of adhesion receptors that bind to a variety of extracellular ligands, typically cell adhesion proteins in the extracellular matrix (ECM).2 Integrins play mandatory roles in embryonic development and the maintenance of tissue architecture 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, among which 18 isoforms function as ECM receptors. Based on their ligand binding specificities, ECM-binding integrins are classified into three major groups as follows: laminin-, collagen-, and Arg-Gly-Asp (RGD)-binding integrins (1, 2), 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, which interact with a variety of ECM ligands containing RGD motifs with distinct binding specificities.The integrin ␣v subunit was originally identified as a receptor for vitronectin (3). The ␣v-containing integrins are widely expressed on many cell types, including neural crest cells, glial cells, muscle cells, osteoclasts, epithelial cells, and vascular endothelial cells during embryonic development (4...

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“…Genetic analysis with laminin-class integrin subunit-deficient mice has revealed the importance of the LG domain integrin subunits for tissue and organ morphogenesis, including epidermal/ dermal attachment, brain development, and glomerular and lung development (GeorgesLabouesse et al 1996;Kreidberg et al 1996;Di Persio et al 1997;Mayer et al 1997;GeorgesLabouesse et al 1998). Binding of these integrins depends on contributions arising from g1 and b1 subunit coiled-coil domain sequences located in the vicinity of the LG domains (Ido et al 2007;Taniguchi et al 2009). A g1-subunit glutamic acid residue (E1607) important for integrin-binding is absent in the g3 laminin subunit (Ido et al 2008).…”

Section: Integrin Interactionsmentioning

confidence: 99%