Recreation of the terminal events in physiological integrin activation
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ABSTRACT:
SUBMITTER: Feng Ye
PROVIDER: S-JCBD-200908045 | bioimages |
REPOSITORIES: bioimages
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Project description:Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.
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Project description:Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with beta1 and beta3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous alpha5beta1 or stably expressed alphaIIbbeta3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate alphaIIbbeta3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate beta1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.
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Project description:Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of "inside-out" signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals.
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Project description:Kindlin-2 (K2), a 4.1R-ezrin-radixin-moesin (FERM) domain adaptor protein, mediates numerous cellular responses, including integrin activation. The C-terminal 15-amino acid sequence of K2 is remarkably conserved across species but is absent in canonical FERM proteins, including talin. In CHO cells expressing integrin αIIbβ3, co-expression of K2 with talin head domain resulted in robust integrin activation, but this co-activation was lost after deletion of as few as seven amino acids from the K2 C terminus. This dependence on the C terminus was also observed in activation of endogenous αIIbβ3 in human erythroleukemia (HEL) cells and β1 integrin activation in macrophage-like RAW264.1 cells. Kindlin-1 (K1) exhibited a similar dependence on its C terminus for integrin activation. Expression of the K2 C terminus as an extension of membrane-anchored P-selectin glycoprotein ligand-1 (PSGL-1) inhibited integrin-dependent cell spreading. Deletion of the K2 C terminus did not affect its binding to the integrin β3 cytoplasmic tail, but combined biochemical and NMR analyses indicated that it can insert into the F2 subdomain. We suggest that this insertion determines the topology of the K2 FERM domain, and its deletion may affect the positioning of the membrane-binding functions of the F2 subdomain and the integrin-binding properties of its F3 subdomain. Free C-terminal peptide can still bind to K2 and displace the endogenous K2 C terminus but may not restore the conformation needed for integrin co-activation. Our findings indicate that the extreme C terminus of K2 is essential for integrin co-activation and highlight the importance of an atypical architecture of the K2 FERM domain in regulating integrin activation.
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Project description:Integrins contain two structurally homologous but distantly related domains: an I-like domain that is present in all beta-subunits and an I domain that is present in some alpha-subunits. Atomic resolution and mutagenesis studies of alpha I domains demonstrate a C-terminal, axial displacement of the alpha7-helix that allosterically regulates the shape and affinity of the ligand-binding site. Atomic resolution studies of beta I-like domains have thus far demonstrated no similar alpha7-helix displacement; however, other studies are consistent with the idea that alpha I and beta I-like domains undergo structurally analogous rearrangements. To test the hypothesis that C-terminal, axial displacement of the alpha7-helix, coupled with beta6-alpha7 loop reshaping, activates beta I-like domains, we have mimicked the effect of alpha7-helix displacement on the beta6-alpha7 loop by shortening the alpha7-helix by two independent, four-residue deletions of about one turn of alpha-helix. In the case of integrin alphaLbeta2, each mutant exhibits constitutively high affinity for the physiological ligand intercellular adhesion molecule 1 and full exposure of a beta I-like domain activation-dependent antibody epitope. In the case of analogous mutants in integrin alpha4beta7, each mutant shows the activated phenotype of firm adhesion, rather than rolling adhesion, in shear flow. The results show that integrins that contain or lack alpha I domains share a common pathway of beta I-like domain activation, and they suggest that beta I-like and alpha I domain activation involves structurally analogous alpha7-helix axial displacements.
| S-EPMC356951 | biostudies-literature
Project description:Integrins are integral membrane proteins that mediate cell-matrix and cell-cell adhesion. They are important for vascular development and hematopoiesis, immune and inflammatory responses, and hemostasis. Integrins are also signaling receptors that can transmit information bidirectionally across plasma membranes. Research in the past 2 decades has made progress in unraveling the mechanisms of integrin signaling and brings the field to the moment of attempting synthetic reconstruction of the signaling pathways in vitro. Reconstruction of biologic processes provides stringent tests of our understanding of the process, as evidenced by studies of other biologic machines, such as ATP synthase, lactose permease, and G-protein-coupled receptors. Here, we review recent progress in reconstructing integrin signaling and the insights that we have gained through these experiments.
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Project description:Integrins are heterodimeric adhesion receptors that link the extracellular matrix (ECM) to the cytoskeleton. Binding of the scaffold protein, talin, to the cytoplasmic tail of β-integrin causes a conformational change of the extracellular domains of the integrin heterodimer, thus allowing high-affinity binding of ECM ligands. This essential process is called integrin activation. Here we report that the Z-band alternatively spliced PDZ-motif-containing protein (Zasp) cooperates with talin to activate α5β1 integrins in mammalian tissue culture and αPS2βPS integrins in Drosophila. Zasp is a PDZ-LIM-domain-containing protein mutated in human cardiomyopathies previously thought to function primarily in assembly and maintenance of the muscle contractile machinery. Notably, Zasp is the first protein shown to co-activate α5β1 integrins with talin and appears to do so in a manner distinct from known αIIbβ3 integrin co-activators.
| S-EPMC3575701 | biostudies-literature
Project description:Background Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. Objective To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbβ3 activation. Methods Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbβ3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Results Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbβ3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > β-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbβ3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y 12 adenosine diphosphate (ADP) receptor, enhanced αIIbβ3 inactivation. Spreading assays showed that PKC or P2Y 12 inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. Conclusion PKC and autocrine ADP signaling contribute to persistent integrin αIIbβ3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.
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Project description:Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.
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Project description:Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation.
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