Yoshikazu Takada

VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.

Cytokine-activated human endothelial cells express vascular cell adhesion molecule-1 (VCAM-1), which binds lymphocytes. We now identify the integrin VLA-4 as a receptor for VCAM-1 because VLA-4 surface expression on K-562 cells (following transfection of the VLA alpha 4 subunit cDNA) resulted in specific cell adhesion to VCAM-1, and anti-VLA-4 antibodies completely inhibited VCAM-1-dependent cell-cell attachment. In addition, VLA-4 expression allowed K-562 cells to attach to the heparin II binding region (FN-40) of fibronectin. However, VLA-4/VCAM-1 and VLA-4/FN-40 interactions are readily distinguishable: only the former was inhibited by the anti-VLA-4 monoclonal antibody HP1/3, and only the latter was inhibited by soluble FN-40. The VCAM-1/VLA-4 ligand-receptor pair may play a major role in the recruitment of mononuclear leukocytes to inflammatory sites in vivo.

Fibulin-5/DANCE is essential for elastogenesis in vivo.

The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-β-binding proteins) and polymerized elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins αvβ3, αvβ5 and α9β1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.

Structure of the integrin VLA-4 and its cell-cell and cell-matrix adhesion functions.

Article synthese sur la variabilite de la structure moleculaire et sur la diversite fonctionnelle de la proteine VLA-4 appartenant a la famille des integrines

RGD-independent Binding of Integrin α9β1 to the ADAM-12 and -15 Disintegrin Domains Mediates Cell-Cell Interaction

ADAMs (a disintegrinand metalloproteases) mediate several important processes (e.g. tumor necrosis factor-α release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin α9β1specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and α9β1supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for α9β1/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/α6β1 interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that α9β1 may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and α9β1, this interaction may be of potential biological and pathological significance.

The primary structure of the alpha 4 subunit of VLA-4: homology to other integrins and a possible cell-cell adhesion function.

VLA‐4 is a cell surface heterodimer in the integrin superfamily of adhesion receptors. Anti‐VLA‐4 antibodies inhibited cytolytic T cell activity, with inhibitory activity directed against the effector T cells rather than their targets. Thus, whereas other VLA receptors appear to mediate cell–matrix interactions, VLA‐4 may have a cell–cell adhesion function. To facilitate comparative studies of VLA‐4 and other integrins, cDNA clones for the human alpha 4 subunit of VLA‐4 were selected and then sequenced. The 3805 bp sequence encoded for 999 amino acids, with an N‐terminus identical to that previously obtained from direct sequencing of purified alpha 4 protein. The alpha 4 amino acid sequence was 17‐24% similar to other integrin alpha chains with known sequences. Parts of the alpha 4 sequence most conserved in other alpha chains include (i) the positions of 19/24 cysteine residues, (ii) three potential divalent cation binding sites of the general structure DXDXDGXXD and (iii) the transmembrane region. However, alpha 4 stands apart from all other known integrin alpha subunit sequences because (i) alpha 4 has neither an inserted I‐domain, nor a disulfide‐linked C‐terminal fragment, (ii) its sequence is the most unique and (iii) only alpha 4 has a potential protease cleavage site, near the middle of the coding region, which appears responsible for the characteristic 80,000 and 70,000 Mr fragments of alpha 4.

Defining the Topology of Integrin α5β1-Fibronectin Interactions Using Inhibitory Anti-α5 and Anti-β1 Monoclonal Antibodies EVIDENCE THAT THE SYNERGY SEQUENCE OF FIBRONECTIN IS RECOGNIZED BY THE AMINO-TERMINAL REPEATS OF THE α5 SUBUNIT

The high affinity interaction of integrin α5β1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on α5β1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-α5 mAbs (P1D6 and JBS5) to α5β1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to α5β1. In contrast, binding of the anti-β1 mAb P4C10 to α5β1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with α5β1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the α5 subunit. Our results indicate that the synergy region is recognized primarily by the α5 subunit (in particular by its NH2-terminal repeats) but that the β1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.

Integrins α2β1 and α4β1 Can Mediate SA11 Rotavirus Attachment and Entry into Cells

ABSTRACT Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins α2β1 and α4β1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing α2β1 or α4β1 integrins via transfection is increased over virus binding to and infection of cells transfected with α3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of α2β1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing α2β1 was elevated over binding to control cells and was specifically blocked by the anti-α2 monoclonal antibody AK7. Virus growth in α4-transfected K562 cells which had also been induced to express α2β1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, α2β1 and α4β1, are capable of acting as cellular receptors for SA11 rotavirus.

Identification of putative ligand binding sites within I domain of integrin α2β1(VLA-2, CD49b/CD29)

Integrin alpha 2 beta 1 is a cell surface adhesion receptor for collagen and echovirus 1. Here we localized the epitopes for anti-alpha 2 monoclonal antibodies using interspecies (human/bovine) alpha 2 chimeras with different lengths of human alpha 2 sequence on the amino-terminal side and site-directed mutagenesis. The antibodies that block the collagen and/or echovirus 1 binding to human alpha 2 beta 1 (6F1, RMAC11, 12F1, and AA10) recognizes a small region (residues 173-259) within the I domain. Asp-160 and Arg-242 are critical for binding of the two other function-inhibiting antibodies, P1H5 and 5E8, respectively. Notably, mutations of Asp-151 and Asp-254 block the binding of alpha 2 beta 1 to collagen. These data suggest that the I domain (residues 140-359) is critically involved in the ligand/receptor interactions, and collagen and echovirus 1 binding sites are adjacent or overlapping within the I domain. The sequence of the residues 173-259 of alpha 2 overlap with the peptide sequences (M11 and M20) that derive from von Willebrand factor A1 and A3 domains (homologous to the alpha 2 I domain) and block von Willebrand factor/collagen interaction, suggesting that the epitope region of alpha 2 (residues 173-259) may really be involved in ligand recognition.

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

ABSTRACT Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection.

Changing ligand specificities of αvβ1 and αvβ3 integrins by swapping a short diverse sequence of the β subunit

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin αvβ3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while αvβ1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the β1 and β3 subunits. When the sequence CTSEQNC (residues 187–193) of β1 is replaced with the corresponding CYDMKTTC sequence of β3, the ligand specificity of αvβ1 is altered. The mutant (αvβ1–3-1), like αvβ3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The αvβ1–3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal β3–1-3 mutation blocks binding of αvβ3 to these multiple ligands and to LM609, a function-blocking anti-αvβ3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of β, in which the sequence is located in the ligand binding site.