Abbreviations used in this paper: CT, cytoplasmic tail; HUVEC, human umbilical vein endothelial cell; talin-H, talin head domain.The online version of this paper contains supplemental material. IntroductionIntegrin activation, the rapid transition from a low to a high affi nity state for ligand, regulates the numerous cellular responses consequent to integrin engagement by extracellular matrix proteins or counter-receptors on other cells ( Hynes, 2002 ). This transformation is tightly controlled by the integrin cytoplasmic tails (CTs) ( Qin et al., 2004 ;Ma et al., 2007 ). Mutational and structural analyses suggest that the ␤ 3 CT can be divided two regions, and both infl uence integrin activation. The membraneproximal region of the ␤ 3 CT is primarily ␣ -helix, which interacts with the membrane-proximal helix of the ␣ subunit through several electrostatic and hydrophobic bonds ( Vinogradova et al., 2002 ). Unclasping of the complex is a critical event in integrin activation ( Hughes et al., 1996 ;Kim et al., 2003 ;Ma et al., 2006 ). The membrane-distal region of the ␤ 3 CT contains two NXXY turn motifs, NPLY 747 and NITY 759 , which are separated by a short helix containing a T/S cluster, the TS 752 T region ( Fig. 1 A ). The head domain of talin (talin-H) docks at the NPLY 747 motif through its F 3 domain and also interacts with the membrane-proximal region, perturbing the membrane clasp and leading to at least partial integrin activation ( Vinogradova et al., 2002 ;Tadokoro et al., 2003 ;Wegener et al., 2007 ). The T/S cluster and the NITY motif are also critical for integrin activation ( Chen et al., 1994 ;O ' Toole et al., 1995 ;Xi et al., 2003 ;Ma et al., 2006 ). However, the mechanisms underlying their effects remain unresolved. In this study, we found that kindlin-2, a widely distributed PTB domain protein, interacts with the C terminus of ␤ 3 CT at the TS 752 T and NITY 759 motifs and markedly enhances talin-induced integrin activation. Thus, kindlin-2 is identifi ed as a coactivator of integrins. Results and discussionTo address the functional signifi cance of the membrane-distal region of the ␤ 3 CT, we considered whether it might interact with intracellular regulator(s). A CHO cell line stably expressing ␣ IIb ␤ 3 was transfected with cDNAs encoding for wild-type or mutated ␤ 3 CT based on the rationale that these expressed constructs would compete for integrin binding partners. A similar strategy had been used previously to screen the ␤ CT binding partners essential for integrin activation ( Fenczik et al., 1997 ). In our studies, these ␤ 3 CT were expressed as chimeric constructs containing the extracellular domain of PSGL-1 so that expression levels of the various ␤ 3 CT could be verifi ed. As assessed by fl ow cytometry (FACS), PSGL-1 expression differed by less than 10%. The effects of the various ␤ 3 CT on ␣ IIb ␤ 3 -mediated cell spreading on immobilized fi brinogen were evaluated. Compared with cells expressing PSGL-1 alone, expression of the wild-type ␤ 3 CT chimera totally abolished ␣ IIb ␤...

Monogenic deficiency diseases provide unique opportunities to define the contributions of individual molecules to human physiology and to identify pathologies arising from their dysfunction. Here we describe a deficiency disease in two human siblings that presented with severe bleeding, frequent infections and osteopetrosis at an early age. These symptoms are consistent with but more severe than those reported for people with leukocyte adhesion deficiency III (LAD-III). Mechanistically, these symptoms arose from an inability to activate the integrins expressed on hematopoietic cells, including platelets and leukocytes. Immortalized lymphocyte cell lines isolated from the two individuals showed integrin activation defects. Several proteins previously implicated in integrin activation, including Ras-associated protein-1 (RAP1) 1 and calcium and diacylglycerol-regulated guanine nucleotide exchange factor-1 (CALDAG-GEF1) 2 , were present and functional in these cell lines. The genetic basis for this disease was traced to a point mutation in the coding region of the KINDLIN3 (official gene symbol FERMT3) gene 3 . When wild-type KINDLIN-3 was expressed in the immortalized lymphocytes, their integrins Correspondence should be addressed to T.V.B. (byzovat@ccf.org). 6 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Medicine website. AUTHOR CONTRIBUTIONS N.L.M. identified the Kindlin-3 mutation, performed molecular biology and protein biochemistry studies and wrote the manuscript; L.Z. contributed to study design and experiments on primary leukocytes from subjects; J.C. performed assays with EGFP-Kindlin-3 rescue and siRNA-mediated KINDLIN3 knockdown and western blotting; A.C. performed microscopy studies and FACS analysis; O.R. performed cell culture work and molecular biology; Y.-Q.M. performed molecular biology and Kindlin-3-specific antibody preparation; E.A.P. performed platelet studies; M.T. performed neutrophil analysis; D.P.L. and A.I.C. performed osteogenesis assays; S.B.S. originally described the subjects, designed clinical studies and wrote the manuscript; E.F.P. designed the studies, interpreted the results and wrote the manuscript; T.V.B. performed experiments with platelets and leukocytes, designed the general strategy, interpreted data and wrote the manuscript. Kindlin-3 is one of the three-member kindlin family of intracellular proteins that are linked to the actin cytoskeleton 3 . The family is evolutionarily conserved with an ortholog, UNC-112, found in Caenorhabditis elegans 4 . Each kindlin contains a C-terminal FERM domain that is most similar to that of talin, another cytoskeletal protein involved in integrin regulation. Kindlins and talin bind to nonoverlapping sites in the cytoplasmic tails of integrins 5 . Kindler disease, associated with a deficiency of Kindlin-1, has multiple symptoms, including skin blistering and poikiloderma 6 . Kindlin-2 deficiency is embryonically lethal in zebrafish and mice but has not been described in hu...

Summary Activation of heterodimeric (α/β) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin β membrane-proximal cytoplasmic tails (β-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin β-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.

Heterodimeric integrin adhesion receptors regulate diverse biological processes including angiogenesis, thrombosis and wound healing. The transmembrane-cytoplasmic domains (TMCDs) of integrins play a critical role in controlling activation of these receptors via an inside-out signaling mechanism, but the precise structural basis remains elusive. Here, we present the solution structure of integrin ␣IIb␤3 TMCD heterodimer, which reveals a righthanded coiled-coil conformation with 2 helices intertwined throughout the transmembrane region. The helices extend into the cytoplasm and form a clasp that differs significantly from a recently published ␣IIb␤3 TMCD structure. We show that while a point mutation in the clasp interface modestly activates ␣IIb␤3, additional mutations in the transmembrane interface have a synergistic effect, leading to extensive integrin activation. Detailed analyses and structural comparison with previous studies suggest that extensive integrin activation is a highly concerted conformational transition process, which involves transmembrane coiled-coil unwinding that is triggered by the membrane-mediated alteration and disengagement of the membrane-proximal clasp. Our results provide atomic insight into a type I transmembrane receptor heterocomplex and the mechanism of integrin inside-out transmembrane signaling.NMR ͉ protein structure ͉ transmembrane domain I ntegrins are a major class of cell adhesion receptors that are found in almost every living organism (1). They are obligate heterodimers (␣,␤) in which each subunit is composed of a large extracellular domain, a single pass transmembrane (TM) segment, and a small cytoplasmic tail (CT). Integrins interact with extracellular matrix (ECM) proteins via their extracellular domains and with intracellular proteins via their CTs. This interconnection allows integrins to regulate diverse cellular adhesive processes. A central and unresolved issue in integrin biology is the molecular basis for signal transmission across the cell membrane. A large body of genetic, cell biological, and biochemical data indicate that the conformational states of integrin ␣/␤ transmembrane-cytoplasmic domains (TMCDs) control the ability of integrins to bind extracellular ligands (inside-out signaling) and to cluster and form focal adhesions (outside-in signaling) (for reviews see refs. 1 and 2). Biochemical and structural evidence suggests that the ␣/␤ TMCDs associate via both their TMs (3-6) and their CTs (7-9) to maintain integrins in a resting state. Dissociation of the TMs or CTs triggers receptor activation and signaling (7,(10)(11)(12)(13)(14). However, many different computational models for the TM association exist (3-5, 11, 13, 15-18) and the structural analyses of the CT interaction are inconsistent (6)(7)(8)(9)19). Earlier studies failed to observe heterodimeric TMCD interaction in micelles (20), which reflects the technical difficulty in structurally characterizing this kind of transmembrane heterodimers (21). Here, we have successfully determined the NMR ...

Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with ␤1 and ␤3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using ␣IIb␤3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells.Cell-extracellular matrix (ECM) 3 adhesion is a fundamental process that is mediated by transmembrane receptors such as integrins (1-6). The interactions of integrins with ECM ligands can be controlled by integrin activation via "inside-out" signaling. Talin, a FERM (Band 4.1 (four point one)/ezrin/radixin/ moesin) domain-containing focal adhesion (FA) protein, can play a key role in this process (for recent reviews, see Refs. 7-10). Binding of the talin FERM domain to the ␤ integrin cytoplasmic domains results in separation of the ␣ and ␤ integrin cytoplasmic tails and consequently in an increase in integrin extracellular ligand-binding affinity (i.e. integrin activation) (11-13). Integrin extracellular ligand-binding affinity plays an important role in control of initial cell-ECM adhesion. Additionally, integrin-mediated cell-ECM adhesion can be enhanced through interactions with cytoskeletal proteins, a process that has been termed cytoskeletal strengthening (14 -16). The physical basis underlying the cytoskeletal strengthening of cell-ECM adhesion has been well described (16). However, the molecular interactions that mediate this process remain to be defined.MIG-2 (mitogen-inducible gene-2, also known as kindlin-2) is a widely expressed and evolutionarily conserved cytoplasmic protein (17-21). Genetic studies have shown that Caenorhabditis elegans UNC-112, a homolog of MIG-2, is required for attachment of body-wall muscle cells to the hypodermis (17,19). Loss of UNC-112 in C. elegans results in an embryonic lethal Pat (paralyzed, arrested elongation at two-fold) phenotype resembling that of ␣ or ␤ integrin loss (17, 19). In mammalian organisms, MIG-2 has been detected in many cell types, including fibroblasts, muscle cells, endothelial cells, and epithelial cells (20,22). In these cells, it concentrates at FAs. MIG-2 interacts with migfilin (20), a filamin-and VASP (vasodilatorstimulated p...

Background: Kindlin-2 is a key regulator of integrin activation. Results: Kindlin-2 contains a PH domain with a distinct binding pocket for phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) that promotes talin-mediated integrin activation. Conclusion: PIP3-mediated membrane binding of kindlin-2 is crucial for the cooperation of kindlin-2 with talin in activating integrin. Significance: Learning how kindlin-2 functions is crucial for understanding the integrin-mediated cell adhesion.

P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and integrin ␣M␤2 (Mac-1, CD11bCD18) are leukocyte adhesion molecules essential for innate immunity and inflammation. The interaction of PSGL-1 with P-selectin (CD62P) mediates tethering, rolling, and weak adhesion of leukocytes, during which they become sufficiently activated in situ by locally released or displayed cytokines and chemoattractants for integrin-mediated firm adhesion. However, communication between P-selectin and the integrin, whether P-selectin can trigger ␤2-integrin activation, remains controversial. We found that Pselectin immunoglobulin chimera and PSGL-1 monoclonal antibodies (mAbs) increased adhesion of human neutrophils to immobilized, but not soluble, fibrinogen. This intermediate state of neutrophil adhesion was defined by moderate clustering of integrin ␣M␤2, no increase in CBRM1/5 (a mAb specific for the activation epitope on the ␣M subunit) recognition, and no increase in surface expression of ␣M␤2, whereas phorbol myristate acetate (PMA) induced extensive changes in these 3 parameters. Furthermore, platelet-activating factor or interleukin 8 acted in concert with P-selectin for further enhancing the activation of ␣M␤2. We thus propose a model in which P-selectin induces an intermediate state of integrin activation and then cooperates with other extracellular stimuli to support maximal adhesion of human neutrophils. IntroductionThe recruitment of leukocytes to the site of inflammation entails a cascade of cellular adhesive events, which include tethering (initial attachment), rolling, firm adhesion, and transendothelial migration of the responding cells. 1,2 Members of the selectin family of cell adhesion molecules expressed on the endothelial surface interact with their cognate glycoprotein ligands on leukocytes to mediate the tethering and rolling phase of leukocyte recruitment; that is, the weak adhesive interactions. 3,4 Members of the ␤2 subfamily of leukocyte integrins, as well as other integrin subfamilies, recognize their cognate ligands to then mediate the firm adhesion (arrest) of leukocytes. The initial weak adhesion brings leukocytes into proximity of cytokines/chemoattractants displayed on or released from the activated endothelium, such as interleukin 8 (IL-8) and platelet-activating factor (PAF). These transduce signals through their G protein-coupled receptors that activate the integrins to induce the firm leukocyte adhesion to the endothelium. 1,5 Such integrin-mediated firm adhesion can occur through direct ligand engagement or indirect, bridging mechanisms. For example, activated ␤2 integrins can bind various members of intercellular cell adhesion molecule (ICAM) family on endothelial cells to support leukocyte adhesion and transmigration. 6,7 As an example of the bridging mechanism, integrin ␣M␤2 can recognize fibrinogen or fibrin deposited on the endothelial surface or at the sites of inflammation to promote the accumulation of leukocytes. [8][9][10][11] Fibrinogen engagement by activated ␣M␤2 is also one of the s...

Background:The talin and kindlin play indispensable roles in integrin activation. Results: The C-terminal 12 amino acids of ␤ 1 and ␤ 3 integrins mediate kindlin-2 binding. Conclusion: Kindlin-2 binding to the extreme C terminus allows ␤ subunits to accommodate both kindlin-2 and talin. Significance: Binding of talin and kindlin-2 to distinct sites in integrins regulates receptor activation, a pivotal event in cellular responses.