Arjan van der Flier

Function and interactions of integrins

Abstract. Integrins are heterodimeric cell adhesion molecules that link the extracellular matrix to the cytoskeleton. The integrin family in man comprises 24 members, which are the result of different combinations of 1 of 18 α- and 1 of 8 β-subunits. Alternative splicing of mRNA of some α- and β-subunits and postranslational modifications of integrin subunits further increase the diversity of the integrin family. In their capacity as adhesion receptors that organize the cytoskeleton, integrins play an important role in controlling various steps in the signaling pathways that regulate processes as diverse as proliferation, differentiation, apoptosis, and cell migration. The intracellular signals that lead to these effects may be transduced via cytoplasmic components, which have been identified as integrin-binding proteins in yeast two-hybrid screens and which could mediate the coupling of integrins to intracellular signaling pathways. In this review an overview is given of the function and ligand-binding properties of integrins as well as of proteins that associate with integrins and may play a role in their signaling function.

Structural and functional aspects of filamins.

Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.

Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin β subunits

Integrins connect the extracellular matrix with the cell interior, and transduce signals through interactions of their cytoplasmic tails with cytoskeletal and signaling proteins. Using the yeast two-hybrid system, we isolated a novel splice variant (filamin-Bvar-1) of the filamentous actin cross-linking protein, filamin-B, that interacts with the cytoplasmic domain of the integrin β1A and β1D subunits. RT-PCR analysis showed weak, but wide, expression of filamin-Bvar-1 and a similar splice variant of filamin-A (filamin-Avar-1) in human tissues. Furthermore, alternative splice variants of filamin-B and filamin-C, from which the flexible hinge-1 region is deleted (ΔH1), were induced during in vitro differentiation of C2C12 mouse myoblasts. We show that both filamin-Avar-1 and filamin-Bvar-1 bind more strongly than their wild-type isoforms to different integrin β subunits. The mere presence of the high-affinity binding site for β1A is not sufficient for targeting the filamin-Bvar-1 construct to focal contacts. Interestingly, the simultaneous deletion of the H1 region is required for the localization of filamin-B at the tips of actin stress fibers. When expressed in C2C12 cells, filamin-Bvar-1(ΔH1) accelerates their differentiation into myotubes. Furthermore, filamin-B variants lacking the H1 region induce the formation of thinner myotubes than those in cells containing variants with this region. These findings suggest that specific combinations of filamin mRNA splicing events modulate the organization of the actin cytoskeleton and the binding affinity for integrins.

Endothelial α5 and αv integrins cooperate in remodeling of the vasculature during development

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the α5 and αv integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin α5-floxed mouse line and ablated α5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin α5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin α5 and αv and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both α5 and αv integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial α5 and αv integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin α5 knockout endothelial cells and markedly reduced in integrin α5/αv double-knockout endothelial cell lines. Therefore, neither α5 nor αv integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.

A novel β1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal muscle

The mRNAs of several integrin subunits are alternatively spliced in the region encoding cytoplasmic domains, that may potentially provide alternative integrin‐cytoskeleton interactions and transmembrane signaling pathways. We identified a novel cytoplasmic tail variant of the human β1 subunit by reverse transcriptase polymerase chain reaction. This fourth β1 variant, named β1D, is specific for skeletal and cardica muscle. The determined genomic organization of the 3′‐region of the human β1 gene reveals that β1D is produced by alternative splicing of mRNA. In addition, we show that the expression of β1D is developmentally regulated during murine myoblast differentiation, suggesting a role for β1D in myogenesis.

The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins

Myotilin and the calsarcin family member FATZ-1 (also called calsarcin-2 or myozenin-1) are recently discovered sarcomeric proteins implicated in the assembly and stabilization of the Z-discs in skeletal muscle. The essential role of myotilin in skeletal muscle is attested by the observation that certain forms of myofibrillar myopathy and limb girdle muscular dystrophy are caused by mutations in the human myotilin gene. Here we show by transfection, biochemical and/or yeast two-hybrid assay that: (1) myotilin is able to interact with the C-terminal region of FATZ-1 and that the N- or C-terminal truncations of myotilin abrogate binding; (2) myotilin can also interact with another calsarcin member, FATZ-2 (calsarcin-1, myozenin-2); (3) myotilin and FATZ-1 bind not only to the C-terminal region of filamin-C containing the Ig repeats 19-24, but also to the other two filamins, filamin-A and filamin-B, as well as the newly identified filamin-Bvar-1variant; (4) the binding of myotilin to filamin-C involves binding sites in its N-terminal region, whereas FATZ-1 associates with filamin-C via sequences within either its N- or C-terminal region; and finally, (5) the C-terminal region of filamin-C like filamin-B and filamin-Bvar-1, shows binding activity with the β1A integrin subunit. Our findings further dissect the molecular interactions within the Z-disc that are essential for its organization, and provide evidence for a novel connection between Z-disc proteins and the sarcolemma via filamins and β1 integrins. These data shed new light on the complex organization of the Z-disc that is highly relevant to understanding muscular dystrophies.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Spatial and temporal expression of the β1D integrin during mouse development

The β1D protein is a recently characterized isoform of the integrin β1 subunit that is present in cardiac and skeletal muscles. In this study, we have examined the expression of β1D in different types of skeletal muscle and in cardiac muscle and studied its distribution during mouse development, using new monoclonal antibodies specific for β1D. Immunoprecipitation studies revealed that, while β1A is strongly expressed in proliferating C2C12 myoblasts, β1D is only expressed after their differentiation to myotubes. In these myotubes, β1D is associated with different α subunits, namely α3A, α5, α7A, or α7B. Initially, during embryogenesis, the β1A subunit is the only β1 variant expressed in skeletal and cardiac muscle. The β1D subunit is first detected in skeletal muscle at E17.5, whereas in cardiac muscle its expression begins around the time of birth. Later the expression of β1A in skeletal and cardiac muscle becomes restricted to capillary cells, whereas β1D eventually becomes the only variant expressed in adult cardiac and skeletal muscle cells. The switch from the β1A to the β1D subunit in cardiac muscle cells coincides with the expression of α7. In adults there is a distinct concentration of β1D at the myotendinous junctions of muscle fibers and at costameres in both cardiac and skeletal muscle. In addition, β1D is present at intercalated discs in cardiac muscle and at neuromuscular junctions in skeletal muscle cells. The amount of β1D in different types of skeletal muscle (fast, slow, and mixed‐type) was similar, but cardiac muscle expressed almost five times as much of this protein. We suggest that β1D plays a role in the maintenance of the cytoarchitecture of mature muscle and in the functional integrity of the muscle cells. Dev. Dyn. 1997;210:472–486.

Secondary reduction of α7B integrin in laminin α2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The α7β1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the α7 integrin are laminin-1 (α1-β1-γ1), laminin-2 (α2-β1-γ1) and laminin-4 (α2-β2-γ1). Targeted deletion of the gene for the α7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the α7B and β1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the α7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the β1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of α7B integrin was significantly reduced at the sarcolemma in six patients with laminin α2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin α2 chain expressed. In contrast, the expression of the laminin α2 chain was not altered in the skeletal muscle of the α7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the α7 integrin subunit and that of the laminin α2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of α7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the β1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of β1D integrin was significantly reduced in the α7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of α7B in laminin α2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in α7 deficient mice, implies that the α7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

Interaction of filamin A with the integrin β7 cytoplasmic domain: Role of alternative splicing and phosphorylation

Integrin–filamin binding plays an important role in adhesion‐mediated control of the actin cytoskeleton. Here, using the interaction between recombinant fragments from the C‐terminus of filamin A and the cytoplasmic tail of integrin β7 as a model, we report a negative regulatory role for filamin alternative splicing. Splice variant forms of filamin A lacking a 41‐amino acid segment interacted more strongly than full‐length fragments. In addition, we provide evidence that phosphorylation of the splice variant region is unlikely to represent the mechanism by which binding is reduced.