Genetic analysis of β1 integrin “activation motifs” in mice

Akey feature of integrins is their ability to regulate the affinity for ligands, a process termed integrin activation. The final step in integrin activation is talin binding to the NPXY motif of the integrin β cytoplasmic domains. Talin binding disrupts the salt bridge between the α/β tails, leading to tail separation and integrin activation. We analyzed mice in which we mutated the tyrosines of the β1 tail and the membrane-proximal aspartic acid required for the salt bridge. Tyrosine-to-alanine substitutions abolished β1 integrin functions and led to a β1 integrin–null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the β1 integrin tail are essential for β1 integrin function, whereas tyrosine phosphorylation and the membrane-proximal salt bridge between α and β1 tails have no apparent function under physiological conditions in vivo

What mouse mutants teach us about extracellular matrix function. Annu Rev Cell Dev Biol 22

Abstract For many years the extracellular matrix was viewed as a benign scaffold for arranging cells within connective tissues, but it is now being redefined as a dynamic, mobile, and flexible key player in defining cellular behavior. Gene targeting, transgene expression, and spontaneous mutations of extracellular matrix proteins in mice have greatly accelerated our mechanistic view of the structural and instructive functions of the extracellular matrix in developmental and regenerative processes. This review summarizes the phenotypes of genetic mouse models carrying mutations in extracellular matrix proteins, with specific emphasis on recent advances. The application of reverse genetics has demonstrated the multifunctionality of matrix proteins in a biological context and, in addition, has brought a novel perspective to the understanding of human pathologies

Extracellular matrix

(ECM). A network of secreted proteins and polysaccharides that surrounds all the connective tissues and underlines all the epithelial and the endothelial sheets. It provides a physical support for tissues, as well as a sink for the storage, release and presentation of growth factors. Focal adhesion A highly specialized celladhesion structure that connects actin filaments to the ECM through integrins. Immature focal adhesions are known as focal complexes, and those that are formed through interactions with fibronectin mature into structures known as fibrillar adhesions. The extracellular matrix (ECM) provides the structural framework for the formation of tissues and organs. The ECM binds to substrate-adhesion molecules on the surface of cells and influences various intracellular signalling pathways that regulate survival, proliferation, polarity and differentiation. An important family of adhesion molecules that bind to the ECM are the integrins. Integrins are heterodimeric transmembrane molecules that consist of α and β subunits, and they are composed of large extracellular domains and relatively small cytoplasmic domains 1 . Integrins can switch between active and inactive conformations. In the inactive state, integrins have a low affinity for ligands. Intracellular signalling events such as protein-kinase-C stimulation can prime the integrins, which results in a conformational change that exposes the ligand-binding site Among the integrins, β1 integrin contributes to a large number of integrin heterodimers whereas β3 integrin has an important adhesion role in platelets -an excellent system in which to study cell-ECM adhesion 1 . β1 and β3 integrins are widely expressed, and studies on the function of β1 and β3 integrins have provided many general insights into integrin-mediated adhesion. Deletion of the highly conserved β1 integrin gene in different organisms has been associated with defects in adhesion, proliferation, survival and polarity Three proteins that have emerged from these studies as important regulators of integrin-mediated signalling are the integrin-linked kinase (ILK), and the adaptor proteins PINCH (particularly interesting Cys-His-rich protein) and parvin. These molecules form a heterotrimeric complex we refer to as the IPP complex, which is named after its components in order of their discovery. Recent reports have provided a wealth of data to expand the known functions of the IPP complex into almost every aspect of cell behaviour and fate. This review will provide an overview of our current knowledge regarding the function of the IPP complex. Interactions between the IPP components and numerous binding partners will be discussed to explain how the IPP complex functions both as an adaptor between integrins and the actin cytoskeleton, and as a hub that regulates several signalling pathways. Furthermore, this review will address the latest results in the ongoing controversy regarding the function of the putative kinase activity of ILK. We will conclude by describing the results of in vivo studies in model organisms, which provide insights into the role of the IPP-mediated integrin-signalling functions during development. Identification, architecture and assembly of IPP ILK was identified in 1996 in a yeast two-hybrid screen for proteins that could bind to the cytoplasmic tail of β1 integrin 7 . The protein that was cloned contained three domain

PIP5KIγ90 generated phosphatidylinositol-4,5-bisphosphate promotes uptake of Staphylococcus aureus by host cells

Staphylococcus aureus, a gram-positive pathogen, invades cells mainly in an integrin-dependent manner. As the activity or conformation of several integrin-associated proteins can be regulated by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ), we investigated the roles of PI-4,5-P2 and PI-4,5-P2 -producing enzymes in cellular invasion by S. aureus. PI-4,5-P2 accumulated upon contact of S. aureus with the host cell and targeting of an active PI-4,5-P2 phosphatase to the plasma membrane reduced bacterial invasion. Knockdown of individual phosphatidylinositol-4-phosphate 5-kinases revealed that phosphatidylinositol-4-phosphate 5-kinase γ (PIP5KIγ) plays an important role in bacterial internalization. Specific ablation of the talin and FAK binding motif in PIP5KIγ90 reduced bacterial invasion, which could be rescued by re-expression of an active, but not inactive PIP5KIγ90. Furthermore, PIP5KIγ90-deficient cells showed normal basal PI-4,5-P2 levels in the plasma membrane but reduced accumulation of PI-4,5-P2 and talin at sites of S. aureus attachment, and overall lower levels of FAK phosphorylation. These results highlight the importance of local synthesis of PI-4,5-P2 by a focal adhesion-associated lipid kinase for integrin-mediated internalization of S. aureus.publishe

Sphingosine kinase 2 (Sphk2) regulates platelet biogenesis by providing intracellular sphingosine 1-phosphate (S1P)

Key Points Sphk2 provides a source of intracellular S1P that tightly controls thrombopoiesis by regulating SFK expression and activity in MKs. Modulation of intracellular S1P by regulating Sphk2 may provide a new strategy to enhance platelet production in patients with thrombocytopenia.</jats:p

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis