Kindlin-2 (Mig-2): a co-activator of β3 integrins

Integrin activation is essential for dynamically linking the extracellular environment and cytoskeletal/signaling networks. Activation is controlled by integrins' short cytoplasmic tails (CTs). It is widely accepted that the head domain of talin (talin-H) can mediate integrin activation by binding to two sites in integrin β's CT; in integrin β3 this is an NPLY747 motif and the membrane-proximal region. Here, we show that the C-terminal region of integrin β3 CT, composed of a conserved TS752T region and NITY759 motif, supports integrin activation by binding to a cytosolic binding partner, kindlin-2, a widely distributed PTB domain protein. Co-transfection of kindlin-2 with talin-H results in a synergistic enhancement of integrin αIIbβ3 activation. Furthermore, siRNA knockdown of endogenous kindlin-2 impairs talin-induced αIIbβ3 activation in transfected CHO cells and blunts αvβ3-mediated adhesion and migration of endothelial cells. Our results thus identify kindlin-2 as a novel regulator of integrin activation; it functions as a coactivator

So Many Plasminogen Receptors: Why?

Plasminogen and plasmin tether to cell surfaces through ubiquitously expressed and structurally quite dissimilar family of proteins, as well as some nonproteins, that are collectively referred to as plasminogen receptors. Of the more than one dozen plasminogen receptors that have been identified, many have been shown to facilitate plasminogen activation to plasmin and to protect bound plasmin from inactivation by inhibitors. The generation of such localized and sustained protease activity is utilized to facilitate numerous cellular responses, including responses that depend on cellular migration. However, many cells express multiple plasminogen receptors and numerous plasminogen receptors are expressed on many different cell types. Furthermore, several different plasminogen receptors can be used to support the same cellular response, such as inflammatory cell migration. Here, we discuss the perplexing issue: why are there so many different Plg-Rs

Combined functional and immunochemical analysis ofnormal and abnormal human factor

A B S T R A C T Human Factor X was isolated from Cohn fraction III and characterized by polyacrylamide gel electrophoresis, amino acid composition, and isoelectric focusing. Two molecular forms with biological activity were observed at isoelectric points of 4.8 and 5.0. Antisera generated to Factor X was monospecific and used to establish an equilibrium competitive inhibition radioimmunoassay. This assay was specific for human Factor X and did not cross-react with human prothrombin or bovine Factor X within the sensitivity range of 6-300 ng Factor X antigen/ml. The mean concentration of Factor X based on the antigen was 11.9 ,ug0ml, whereas concentration values based on coagulant activity was 7.8 ,ug/ml. This 30% difference in measurement appears to result from the presence of a subpopulation of Factor X molecules devoid of coagulant activity. The radioimmunoassay was used to qualitatively and quantitatively compare purified Factor X to plasmic Factor X obtained from normal, warfarintreated, acquired Factor X-deficient, and congenitaldeficient patients. In all but one case, the Factor X present in these plasmas was immunochemically identical to the purified Factor X and permitted precise quantitation of these abnormal Factor X molecules. Factor X procoagulant activity was analyzed relative to Factor X antigen and the specific activities were used to characterize normal and abnormal Factor X molecules. Reduced Factor X activity in plasmas from warfarin-treated and acquired Factor Xdeficient patients was attributed to both decreases in Factor X antigen and decreased function of the Factor X molecules. Congenitally deficient patients, in general, showed a reduction in Factor X antigen in parallel with Factor X procoagulant activities This is publicatio

Targeting Platelet–Leukocyte Interactions: Identification of the Integrin Mac-1 Binding Site for the Platelet Counter Receptor Glycoprotein Ibα

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibα. Previous studies have established a central role for the I domain, a stretch of ∼200 amino acids within the αM subunit, in the binding of GP Ibα. This study was undertaken to establish the molecular basis of GP Ibα recognition by αMβ2. The P201–K217 sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the αMI domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the αMI domain segments P201–G207 and R208–K217 were switched to the homologous, but non-GP Ibα binding, αL domain segments failed to support adhesion to GP Ibα. Mutation of amino acid residues within P201–K217, H210–A212, T213–I215, and R216–K217 resulted in the loss of the binding function of the recombinant αMI domains to GP Ibα. Synthetic peptides duplicating the P201–K217, but not scrambled versions, directly bound GP Ibα and inhibited αMβ2-dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P201–K217 sequence onto αL, converted αLβ2 into a GP Ibα binding integrin. Thus, the P201–K217 sequence within the αMI domain is necessary and sufficient for GP Ibα binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis

Myb-binding protein 1A (MYBBP1A) is essential for early embryonic development, controls cell cycle and mitosis, and acts as a tumor suppressor

MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.NHMRC: This work was supported by Associazione Italiana Ricerche sul Cancro (AIRC) grant 8929 and European Community FP7 201681 ‘‘Prepobedia’’ to FB, the Australian National Health and Medical Research Council to RK and TJG (project ID000115). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Function and Distribution of Apolipoprotein A1 in The Artery Wall Are Markedly Distinct From Those in Plasma

Background—Prior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall. Methods and Results—A monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaque–laden aorta showed \u3e100-fold enrichment of apoA1 compared with normal aorta (P\u3c0.001). Surprisingly, buoyant density fractionation revealed that only a minority (\u3c3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063≤d≤1.21). In contrast, the majority (\u3e90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d\u3e1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed ≈80% lower cholesterol efflux activity and ≈90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1. Conclusions—The function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired