Trans-dominant inhibition of integrin function.

Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.     

A novel gain-of-function mutation of the integrin alpha2 VWFA domain.

Integrin alpha2beta1 is the major receptor for collagens in human tissues, being involved in cell adhesion and the control of collagen and collagenase gene expression. The collagen binding site of alpha2beta1 has been localized to the alpha2 von Willebrand Factor type A (VWFA) domain (A-domain or I-domain) and the residues responsible for the interaction with collagen have been mapped. We report a study of alpha2 VWFA domain in which residue E318, which lies outside the collagen binding site, is mutated to tryptophan, showing that this is a gain-of-function mutation. Recombinant alpha2-E318W VWFA domain showed elevated and specific binding to collagen I compared with the wild-type. Side chain hydrophobicity was important for the gain-of-function as elevated binding was seen with E318I and E318Y, but not with E318R. The E318W mutation had additional effects on VWFA domain properties as alpha2-E318W VWFA domain differed from the wild-type in its cation preferences for ligand binding and in binding to monoclonal antibody JA203, which bound at a site distal to E318. The gain-of-function effect was not restricted to binding to collagen I as alpha2-E318W also showed elevated binding to collagen IV, collagen I C-propeptide, laminin and E-cadherin. Binding to these ligands was inhibited by collagen peptide containing the GFOGER motif, indicating that these bound to the VWFA domain by a similar mechanism to collagen I. These data indicate that residue E318 plays a novel and important role in modulating alpha2 VWFA domain--ligand binding and may be involved in the conformational changes associated with its regulation.     

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated. The integrin alpha 6 beta 4 is expressed along the basal surface of various epithelial cells. We show here that this integrin localizes to hemidesmosomes as determined by immunoelectron microscopy using antibodies directed against both the extra- and intracytoplasmic domains of alpha 6 beta 4. This result, which agrees with a recent study, suggests a functional role for the alpha 6 beta 4 integrin in the hemidesmosomes. We therefore investigated such a potential role for this integrin using the cultured rat bladder carcinoma cell line 804G, which has the uncommon ability to form hemidesmosomes in vitro when maintained on uncoated glass substrates. By immunoprecipitation and immunofluorescence, we show that 804G cells express alpha 6 beta 4 along their basal surface in a punctate pattern that overlaps with the distribution of hemidesmosomal plaque antigens. However, this pattern is altered when cells are plated in the presence of an antiserum directed against alpha 6 beta 4. Furthermore, no hemidesmosomes are detectable at the ultrastructural level in the alpha 6 beta 4 antibody-treated cells compared with control cells. These results indicate that integrins may play a critical role in assembly and adhesive functions of the hemidesmosome.     

Laminin‐332–β1 integrin interactions negatively regulate invadopodia

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin‐associated focal contacts and keratin‐associated hemidesmosomes, both of which form on laminin‐332 (Ln‐332). In epithelial‐derived cancer cells, additional actin‐linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln‐332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln‐332 and forms all three types of adhesions. Expression of shRNA to Ln‐332 γ2 chain (γ2‐kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating γ2‐kd cells on Ln‐332 or collagen‐I fully recovered cell spreading and inhibition of invadopodia. Inhibition of α3 or β1, but not α6 or β4, phenocopied the effect of γ2‐kd, suggesting that α3β1‐mediated focal contacts, rather than α6β4‐mediated hemidesmosome pathways, intersect with invadopodia regulation. γ2‐kd cells exhibited alterations in focal contact‐type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin‐based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln‐332‐β1 interactions as a potent upstream regulator that limits cell invasion. J. Cell. Physiol. 223: 134–142, 2010. © 2009 Wiley‐Liss, Inc.     

A novel integrin (alpha E beta 4) from human epithelial cells suggests a fourth family of integrin adhesion receptors.

A new member of the integrin superfamily of adhesion receptors was isolated from human epithelial cells. Analogously to other integrins, this molecule is a heterodimer comprised of structurally unrelated subunits, both glycosylated. Unequivocal amino-acid sequence homologies were observed between these subunits and integrin alpha and beta chain sequences, indicating that this epithelial heterodimer is a novel integrin. No obvious serologic cross-reactivities were detected with other integrins. The beta chain of the epithelial integrin displayed a mol. wt significantly higher than other integrin beta chains, possibly due to a large sialic acid content. Integrin heterodimers are grouped into three families, based on which of three beta chains (beta 1, beta 2 and beta 3) they contain. Therefore, the epithelial integrin may represent the prototype of a fourth integrin family, because it contains a structurally distinct beta chain. The designation alpha E beta 4 is proposed for this novel human integrin.     

Regulation of leukocyte integrin function: Affinity vs. avidity

Leukocytes circulate freely in the bloodstream until receiving signals which activate adhesive mechanisms essential for immune responsiveness. Key mediators of these adhesion events are heterodimeric cell surface receptors called integrins. It is now apparent that several components may contribute to successful integrin-mediated adhesion: alterations in individual receptors lead to enhanced affinity for ligand; integrin clustering causes an increase in avidity; by spreading, the adhering cell is less susceptible to shear force. Model systems have allowed us to examine the contribution of each of these factors in generating adhesion. In more physiologically relevant situations, it can now be questioned whether integrin-mediated adhesion is regulated via alterations in receptor affinity or avidity, or whether both these mechanisms are involved. © 1996 Wiley-Liss, Inc.     

Chondrocyte integrin expression and function

The extracellular matrix (ECM) is an "information rich" environment and interactions between the chondrocyte and ECM regulate many biological processes important to cartilage homeostasis and repair including cell attachment, growth, differentiation, and survival. The integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these processes. Chondrocytes have been found to express several members of the integrin family which can serve as receptors for fibronectin (alpha 5 beta 1), types II and VI collagen (alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1), laminin (alpha 6 beta 1), and vitronectin and osteopontin (alpha V beta 3). Integrin expression can be regulated by growth factors including IGF-I and TGF-beta. By providing a link between the ECM and the cytoskeleton, integrins may be important transducers of mechanical stimuli. Integrin binding stimulates intracellular signaling which can affect gene expression and regulate chondrocyte function. Further studies are needed to more clearly define the role of integrins in cartilage.     

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.     

The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function

Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to - and degradation of - the extracellular matrix. The key proteins in these structures include the actin regulators cortactin and neural Wiskott-Aldrich syndrome protein (N-WASP), the adaptor proteins Tyr kinase substrate with four SH3 domains (TKS4) and Tyr kinase substrate with five SH3 domains (TKS5), and the metalloprotease membrane type 1 matrix metalloprotease (MT1MMP; also known as MMP14). Many cell types can produce these structures, including invasive cancer cells, vascular smooth muscle and endothelial cells, and immune cells such as macrophages and dendritic cells. Recently, progress has been made in our understanding of the regulatory and functional aspects of podosome and invadopodium biology and their role in human disease.     

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.     

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.     

A novel structural model for regulation of clathrin function.

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.     

整合素在细胞黏附中的生物学功能

整合素是一种跨膜蛋白,属于黏附分子家族.其主要功能是参与细胞和细胞、细胞和细胞外基质(ECM)的黏附和信号转导.整合素是含有α和β两条肽链的异源二聚体,来源不同的α、β亚基所形成的整合素具有不同的ECM结合能力.阐述了整合素的结构、生物学功能以及生理、病理学意义,并概述了其研究进展.     

Hydroxyl-radical production in physiological reactions. A novel function of peroxidase.

Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.     

The αMβ2 integrin and its role in neutrophil function

Neutrophils are the first cell type to arrive at the injury sites and play a critical role in host defense,by virtue of its ability to adhere and transmigrate through endothelium,to phagocytose foreign pathogens,and to produce free oxygen radicals and proteolytic enzymes.Yet,inappropriate neutrophil activation causes tissue damage and various inflammatory diseases.These physiological and pathological functions of neutrophils depend on the engagement of certain surface receptors,especially αMβ2,the major β2 integrin receptor present on neutrophil surface.Understanding of the molecular mechanisms underlying ligand binding by αMβ2,as well as the rolea of αMβ2ligand interactions in neutrophil functions will enable us to regulate more precisely neutrophil activities:that is,to promote their host defense functions,and at the same time to minimize their deleterious effects of normal cells.     

Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia

Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.