Ligand binding initiates single-molecule integrin conformational activation.

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

A Structural Change in Butyrophilin upon Phosphoantigen Binding Underlies Phosphoantigen-Mediated Vγ9Vδ2 T Cell Activation.

Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.

Atypical structure and function of integrin αV ß8.

Integrins are heterodimeric transmembrane proteins that play important roles in various biological processes. Most integrins serve as adhesion molecules and transmit bidirectional signaling across the cell membrane through global conformational changes from the bent closed to the extended open conformation. However, integrin ß8 is distinctive in structure and function. Its cytoplasmic domain lacks the conserved protein-binding sequence, which is important in transmitting inside-out signals, suggesting that integrin ß8 may have a different activation mechanism or lack such signaling. In addition, the ligand-binding or activating metal ion Mn2+ does not induce a global conformational change in integrin ß8 . It may have only one conformation, that is, an extended, closed conformation, but with high affinity for ligands under physiological conditions, and is, therefore, considered an atypical integrin member. The extended structure and high ligand-binding affinity of integrin αv ß8 make it ideal for encountering and binding ligands expressed on an opposing cell or in the extracellular matrix. In this review, we summarize the progress in integrin ß8 research with a focus on its distinctive function and structure among integrin members.

Fc receptor inside-out signaling and possible impact on antibody therapy.

Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR-mediated signaling and triggering of cellular effector functions. FcR activation is tightly regulated to prevent immune responses by non-antigen bound antibodies or in the absence of 'danger signals'. FcR activity may be modulated at the plasma membrane via cross-talk with integrins. In addition, cytokines at the site of infection/inflammation can increase FcR avidity, a process referred to as inside-out signaling. This regulatory mechanism has been described for FcγRI (CD64), FcγRIIa (CD32a), and FcαRI (CD89) and is also well-known for integrins. Key cellular events during inside-out signaling are (de)phosphorylation, clustering, cytoskeleton rearrangements, and conformational changes. The latter can be studied with antibodies that specifically recognize epitopes exposed by the active (high affinity) or inactive (low affinity) state of the FcR. These antibodies are important tools to investigate the role of FcR activation in disease settings. Research on FcR has gained momentum with the rise of monoclonal antibodies (mAb) entering the clinic for the treatment of cancer and other diseases. The clinical outcome of mAb therapy may be improved by increasing FcR avidity by cytokine stimulation.

SLAT promotes TCR-mediated, Rap1-dependent LFA-1 activation and adhesion through interaction of its PH domain with Rap1.

SLAT (also known as DEF6) promotes T cell activation and differentiation by regulating NFAT-Ca(2+) signaling. However, its role in TCR-mediated inside-out signaling, which induces integrin activation and T cell adhesion, a central process in T cell immunity and inflammation, has not been explored. Here, we show that SLAT is crucial for TCR-induced adhesion to ICAM-1 and affinity maturation of LFA-1 in CD4(+) T cells. Mechanistic studies revealed that SLAT interacts, through its PH domain, with a key component of inside-out signaling, namely the active form of the small GTPase Rap1 (which has two isoforms, Rap1A and Rap1B). This interaction has been further shown to facilitate the interdependent recruitment of Rap1 and SLAT to the T cell immunological synapse upon TCR engagement. Furthermore, a SLAT mutant lacking its PH domain drastically inhibited LFA-1 activation and CD4(+) T cell adhesion. Finally, we established that a constitutively active form of Rap1, which is present at the plasma membrane, rescues the defective LFA-1 activation and ICAM-1 adhesion in SLAT-deficient (Def6(-/-)) T cells. These findings ascribe a new function to SLAT, and identify Rap1 as a target of SLAT function in TCR-mediated inside-out signaling.

PAI-1 uncouples integrin-ß1 from restrain by membrane-bound ß-catenin to promote collagen fibril remodeling in obesity-related neoplasms.

The paracrine actions of adipokine plasminogen activator inhibitor-1 (PAI-1) are implicated in obesity-associated tumorigenesis. Here, we show that PAI-1 mediates extracellular matrix (ECM) signaling via epigenetic repression of DKK1 in endometrial epithelial cells (EECs). While the loss of DKK1 is known to increase ß-catenin accumulation for WNT signaling activation, this epigenetic repression causes ß-catenin release from transmembrane integrins. Furthermore, PAI-1 elicits the disengagement of TIMP2 and SPARC from integrin-ß1 on the cell surface, lifting an integrin-ß1-ECM signaling constraint. The heightened interaction of integrin-ß1 with type 1 collagen (COL1) remodels extracellular fibrillar structures in the ECM. Consequently, the enhanced nanomechanical stiffness of this microenvironment is conducive to EEC motility and neoplastic transformation. The formation of extensively branched COL1 fibrils is also observed in endometrial tumors of patients with obesity. The findings highlight PAI-1 as a contributor to enhanced integrin-COL1 engagement and extensive ECM remodeling during obesity-associated neoplastic development.

Garcinol acts as a novel integrin αIIbß3 inhibitor in human platelets.

AIMS: Platelet activation plays a central role in arterial thrombosis. Platelets are activated by adhesive proteins (i.e., collagen) or soluble agonists (i.e., thrombin), the respective receptor-specific signaling cause inside-out signaling, leading to the binding of fibrinogen to integrin αIIbß3. This binding triggers outside-in signaling, resulting in platelet aggregation. Garcinol, a polyisoprenylated benzophenone, is extracted from the fruit rind of Garcinia indica. Although garcinol exhibits considerable bioactivities, few studies have investigated the effect of garcinol on platelet activation. MAIN METHODS: Aggregometry, immunoblotting, flow cytometer, confocal microscopic analysis, fibrin clot retraction, animal studies such as fluorescein-induced platelet plug formation in mesenteric microvessels, acute pulmonary thromboembolism, and tail bleeding time were performed in this study. KEY FINDINGS: This study indicates that garcinol inhibited platelet aggregation stimulated by collagen, thrombin, arachidonic acid, and U46619. Garcinol reduced integrin αIIbß3 inside-out signaling, including ATP release; cytosolic Ca2+ mobilization; P-selectin expression; and Syk, PLCγ2/PKC, PI3K/Akt/GSK3ß, MAPKs, and NF-κB activation stimulated by collagen. Garcinol directly inhibited integrin αIIbß3 activation by interfering with FITC-PAC-1 and FITC-triflavin by collagen. Additionally, garcinol affected integrin αIIbß3-mediated outside-in signaling, such as decreasing platelet adhesion and the single-platelet spreading area; suppressing integrin ß3, Src, FAK, and Syk phosphorylation on immobilized fibrinogen; and inhibiting thrombin-stimulated fibrin clot retraction. Garcinol substantially reduced mortality caused by pulmonary thromboembolism and prolonged the occlusion time of thrombotic platelet plug formation without extending bleeding time in mice. SIGNIFICANCE: This study identified that garcinol, a novel antithrombotic agent, acts as a naturally occurring integrin αIIbß3 inhibitor.

Targeting the high affinity receptor, FcγRI, in autoimmune disease, neuropathy, and cancer.

The Fc gamma receptor I (FcγRI or CD64) is the only human Fc receptor with a high affinity for monomeric IgG. It plays a crucial role in immunity, as it mediates cellular effector functions of antibodies including phagocytosis, antigen presentation, and cytokine production. FcγRI is constitutively saturated with monomeric IgG and this feeds the dogma that it has no role in immune responses. However, recent findings have implicated a role for FcγRI in various autoimmune disorders, neuropathies, and antibody therapy in tumor models. By a process known as 'inside-out' signaling, stimulation of myeloid cells with cytokines such as tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) enhances FcγRI binding to immune complexes (ICs), including antibody-opsonized pathogens or tumor cells. This review focuses on the current knowledge on interaction of FcγRI with IgG and ICs and the effect of inside-out signaling on FcγRI functioning. Additionally, this review will address potential clinical applications of targeting FcγRI, and the tools that can be used to overcome IC-mediated autoimmune diseases on the one hand, and to enhance antibody-based anti-cancer therapy on the other.

P120 catenin potentiates constitutive E-cadherin dimerization at the plasma membrane and regulates trans binding.

Cadherins are essential adhesion proteins that regulate tissue cohesion and paracellular permeability by assembling dense adhesion plaques at cell-to-cell contacts. Adherens junctions are central to a wide range of tissue functions; identifying protein interactions that potentiate their assembly and regulation has been the focus of research for over 2 decades. Here, we present evidence for a new, unexpected mechanism of cadherin oligomerization on cells. Fully quantified spectral imaging fluorescence resonance energy transfer (FSI-FRET) and fluorescence intensity fluctuation (FIF) measurements directly demonstrate that E-cadherin forms constitutive lateral (cis) dimers at the plasma membrane. Results further show that binding of the cytosolic protein p120ctn binding to the intracellular region is required for constitutive E-cadherin dimerization. This finding differs from a model that attributes lateral (cis) cadherin oligomerization solely to extracellular domain interactions. The present, novel findings are further supported by studies of E-cadherin mutants that uncouple p120ctn binding or with cells in which p120ctn was knocked out using CRISPR-Cas9. Quantitative affinity measurements further demonstrate that uncoupling p120ctn binding reduces the cadherin trans binding affinity and cell adhesion. These findings transform the current model of cadherin assembly at cell surfaces and identify the core building blocks of cadherin-mediated intercellular adhesions. They also identify a new role for p120ctn and reconcile findings that implicate both the extracellular and intracellular cadherin domains in cadherin clustering and intercellular cohesion.

Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells.

The integrin LFA-1 (CD11a/CD18) plays a critical role in the interaction of T cells with antigen presenting cells (APCs) to promote lymphocyte differentiation and proliferation. This integrin can be present either in a closed or in an open active conformation and its activation upon T-cell receptor (TCR) stimulation is a critical step to allow interaction with APCs. In this study we demonstrate that the serine/threonine kinase Ndr2 is critically involved in the initiation of TCR-mediated LFA-1 activation (open conformation) in T cells. Ndr2 itself becomes activated upon TCR stimulation and phosphorylates the intracellular integrin binding partner Filamin A (FLNa) at serine 2152. This phosphorylation promotes the dissociation of FLNa from LFA-1, allowing for a subsequent association of Talin and Kindlin-3 which both stabilize the open conformation of LFA-1. Our data suggest that Ndr2 activation is a crucial step to initiate TCR-mediated LFA-1 activation in T cells.

Ndr2 Kinase Controls Neurite Outgrowth and Dendritic Branching Through α1 Integrin Expression.

The serine/threonine kinase Ndr2 has been shown to control the inside-out activation of the ß1subunit of integrins and the formation of neurites in both primary neurons and neurally differentiated pheochromacytoma (PC12) cells. In this study, we demonstrate that Ndr2 kinase furthermore determines the substrate specificity of neurite extension in PC12 cells via expression of α1ß1 integrins. We show that stable overexpression of Ndr2 in PC12 cells increases neurite growth and extension on poly-D-lysine substrate, likely involving an increased expression of active ß1 integrin in the growth tips of these cells. By contrast, the Ndr2 overexpressing cells do not show the α1ß1 integrin-mediated enhancement of neurite growth on collagen IV substrate that can be seen in control cells. Moreover, they entirely fail to increase in response to activation of α1ß1 integrins via a soluble KTS ligand and show a diminished accumulation of α1 integrin in neurite tips, although the expression of this subunit is induced during differentiation to comparable levels as in control cells. Finally, we demonstrate that Ndr2 overexpression similarly inhibits the α1ß1 integrin-dependent dendritic growth of primary hippocampal neurons on laminin 111 substrate. By contrast, lack of Ndr2 impairs the dendritic growth regardless of the substrate. Together, these results suggest that Ndr2 regulates α1 integrin trafficking in addition to ß1 integrin subunit activation and thereby controls the neurite growth on different extracellular matrix (ECM) substrates.

B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk.

Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10-20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation.DOI: http://dx.doi.org/10.7554/eLife.02069.001.

The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism.

The small GTPase Rap1 induces integrin activation via an inside-out signaling pathway mediated by the Rap1-interacting adaptor molecule (RIAM). Blocking this pathway may suppress tumor metastasis and other diseases that are related to hyperactive integrins. However, the molecular basis for the specific recognition of RIAM by Rap1 remains largely unknown. Herein we present the crystal structure of an active, GTP-bound GTPase domain of Rap1 in complex with the Ras association (RA)-pleckstrin homology (PH) structural module of RIAM at 1.65 Å. The structure reveals that the recognition of RIAM by Rap1 is governed by side-chain interactions. Several side chains are critical in determining specificity of this recognition, particularly the Lys31 residue in Rap1 that is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases. Lys31 forms a salt bridge with RIAM residue Glu212, making it the key specificity determinant of the interaction. We also show that disruption of these interactions results in reduction of Rap1:RIAM association, leading to a loss of co-clustering and cell adhesion. Our findings elucidate the molecular mechanism by which RIAM mediates Rap1-induced integrin activation. The crystal structure also offers new insight into the structural basis for the specific recruitment of RA-PH module-containing effector proteins by their small GTPase partners.

Why integrin as a primary target for imaging and therapy.

Integrin-mediated cell adhesion is involved in many essential normal cellular and pathological functions including cell survival, growth, differentiation, migration, inflammatory responses, platelet aggregation, tissue repair and tumor invasion. 24 different heterodimerized transmembrane integrin receptors are combined from 18 different α and 8 different ß subunits. Each integrin subunit contains a large extracellular domain, a single transmembrane domain and a usually short cytoplasmic domain. Integrins bind extracellular matrix (ECM) proteins through their large extracellular domain, and engage the cytoskeleton via their short cytoplasmic tails. These integrin-mediated linkages on either side of the plasma membrane are dynamically linked. Thus, integrins communicate over the plasma membrane in both directions, i.e., outside-in and inside-out signaling. In outside-in signaling through integrins, conformational changes of integrin induced by ligand binding on the extracellular domain altered the cytoplasmic domain structures to elicit various intracellular signaling pathways. Inside-out signaling originates from non-integrin cell surface receptors or cytoplasmic molecules and it activates signaling pathways inside the cells, ultimately resulting in the activation/deactivation of integrins. Integrins are one of key family proteins for cell adhesion regulation through binding to a large number of ECM molecules and cell membrane proteins. Lack of expression of integrins may result in a wide variety of effects ranging from blockage in pre-implantation to embryonic or perinatal lethality and developmental defects. Based on both the key role they played in angiogenesis, leukocytes function and tumor development and easy accessibility as cell surface receptors interacting with extracellular ligands, the integrin superfamily represents the best opportunity of targeting both antibodies and small-molecule antagonists for both therapeutic and diagnostic utility in various key diseases so far.

FcγRIIIB stimulation promotes ß1 integrin activation in human neutrophils.

The molecular stimuli involved in receptor-induced integrin activation are still poorly defined. We have investigated the role of receptors for the Fc portion of immunoglobulin G molecules (FcγR) on activation of integrins in human neutrophils. Cross-linking of FcγRIIA induced an increase in surface expression of ß2 integrins but had no effect on ß1 integrins. In contrast, cross-linking of FcγRIIIB not only increased ß2 integrins on the cell surface but also induced ß1 integrin activation, as indicated by an increase in binding to fibronectin and the appearance of an activation epitope detected by the monoclonal antibody 15/7. The FcγRIIIB-induced increase of ß2 integrins required Src-family tyrosine kinases, Syk kinase, and phosphatidylinositol-3 kinase (PI-3K), as the corresponding, specific inhibitors, PP2, Piceatannol, and LY294002, completely blocked it. Contrary to this, FcγRIIIB-indued ß1 integrin activation was not blocked by PP2 or LY294002. It was, however, enhanced by Piceatannol. After FcγRIIIB cross-linking, colocalization of FcγRIIIB and active ß1 integrins was detected on the neutrophil membrane. These data show, for the first time, that cross-linking of FcγRIIIB induces an inside-out signaling pathway that leads to ß1 integrin activation. This activation is independent of Src-family kinases, and PI-3K and may be induced in part by the interaction of FcγRIIIB with ß1 integrins.