Integrin Cytoplasmic Tail Research Articles

Integrin bi‐directional signaling across the plasma membrane

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi-directional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bi-directional signals through transmembrane/cytoplasmic domains.

Cell Adhesion: A FERM Grasp of the Tail Sorts Out Integrins

As well as modulating integrin activation, a conserved NPxY motif in integrin cytoplasmic tails that binds the FERM-domain-containing proteins kindlin and sorting nexin 17 plays pivotal roles in integrin recycling and degradation.

Proteomic analysis of α4β1 integrin adhesion complexes reveals α‐subunit‐dependent protein recruitment

Integrin adhesion receptors mediate cell–cell and cell–extracellular matrix interactions, which control cell morphology and migration, differentiation, and tissue integrity. Integrins recruit multimolecular adhesion complexes to their cytoplasmic domains, which provide structural and mechanosensitive signaling connections between the extracellular and intracellular milieux. The different functions of specific integrin heterodimers, such as α4β1 and α5β1, have been attributed to distinct signal transduction mechanisms that are initiated by selective recruitment of adhesion complex components to integrin cytoplasmic tails. Here, we report the isolation of ligand-induced adhesion complexes associated with wild-type α4β1 integrin, an activated α4β1 variant in the absence of the α cytoplasmic domain (X4C0), and a chimeric α4β1 variant with α5 leg and cytoplasmic domains (α4Pα5L), and the cataloguing of their proteomes by MS. Using hierarchical clustering and interaction network analyses, we detail the differential recruitment of proteins and highlight enrichment patterns of proteins to distinct adhesion complexes. We identify previously unreported components of integrin adhesion complexes and observe receptor-specific enrichment of molecules with previously reported links to cell migration and cell signaling processes. Furthermore, we demonstrate colocalization of MYO18A with active integrin in migrating cells. These datasets provide a resource for future studies of integrin receptor-specific signaling events.

Abstract 496: Competition Between Filamin and Kindlin-2 Regulates Integrin Activation

Cell migration and adhesion during hemostasis, angiogenesis and inflammation are dynamically regulated by integrin heterodimeric adhesion receptors. Their interactions with cytosolic proteins, filamin (FLN), talin (TLN) and Kindlin (Kn2) enable them to convey intracellular signals (inside-out-signaling) to the external environment by engaging extracellular matrix ligands. While TLN and Kn2 activate integrins, FLN inhibits cell migration. TLN and Kn2 bind to membrane-proximal and -distal NPxY motifs of β integrin cytoplasmic tails (CTs), respectively, and an integrin binding site for FLN resides in between these two sequences. Competition between TLN and FLN regulates integrin activation, but it is unknown if FLN and Kn2 compete and regulate integrin inside-out signaling. This competition was tested using αIIbβ3 (platelet-specific) and β7 (lymphocyte-specific; strong FLN binder) integrins in multiple cell types. siRNA depletion of FLNA in K562 cells stably expressing αIIbβ3 integrin (K562-αIIbβ3) significantly enhanced PAC-1 (specific for activated αIIbβ3) binding compared to control siRNA, demonstrating its effect on β3 activation. In pulldown assays using GST-β3 CT, Kn2 bound β3 in CHO lysates transfected with Kn2, either alone or with FLN repeat 21; however, FLN binding to β3 CT was observed only when FLN repeat 21 was expressed alone. Under similar conditions using GST-β7 CT, FLN-β7 interaction was not perturbed by Kn2. This was more pronounced in endothelial cell lysates where GST-β7 CT bound endogenous FLNA but not Kn2. Weak talin-β7 CT binding in this assay was noted. Moreover, in K562-αIIbβ3 cells, exogenous Kn2 overcame the suppressive effect of FLN on αIIbβ3 activation. Overall, our data shows that FLN inhibits β3 integrin function, and competition between FLN and Kn2 can indeed regulate integrin activation.

Abstract 375: Regulation of Integrin Activation by Kindlin-3

The contributions of integrins to cellular adhesive responses depend upon the dynamic regulation of their activation states, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin activation. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin activation in hematopoietic cells, where kindlin-3 deficiency leads to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Here, we used human erythroleukemia HEL cells to characterize integrin-activating kindlin-3 function and the kindlin-3-β3 integrin interaction in greater detail. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen readily. Thrombopoetin or PMA stimulation activated β3-containing integrins, the cells adhere and spread on fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. We expressed various β3 CT as chimeric constructs containing the extracellular domain of PSGL-1 and evaluated their effects on β3-mediated cell spreading on fibrinogen. Expression of the wild-type β3 CT chimera impaired β3-mediated cell spreading, and introduction of S752P and Y759A mutations within the kindlin binding core abolished this inhibitory activity. Thus, our data emphasizes a role of direct kindlin-3 and β3 integrin cytoplasmic tail interaction in kindlin-3 induced integrin activation in hematopoietic cells.

Abstract 369: Localization and Specification of a Kindlin-2 Binding Core Within Integrin Beta Cytoplasmic Tails

Integrins are widely expressed cell adhesion receptors. One of the key regulatory mechanisms of integrins is their ability to undergo activation. Based on in vivo studies kindlins and talin are know to play indispensable roles in integrin activation, and this function depends on their binding to the cytoplasmic tail (CT) of integrin β subunits, However, the requisite sequences within the integrin β subunit that specify kindlin is as of yet unresolved. Using overlapping synthetic peptides corresponding to the highly conserved C-terminal region of integrin β CT as inhibitors of β3 CT: kindlin-2 interaction in pull-down assays, we identified the kindlin-2 binding core to a twelve amino-acid sequence. This sequence includes the membrane-distal NxxY region and S/T cluster, which are conserved across integrin β CT, but also variable residues flanking these two motifs. We postulated that the contribution of these flanking residues in β subunits might impose selectivity upon the interaction of kindlin-2 with various β subunits. Indeed, we found that kindlin-2 interacts with β1and β3, but poorly with the β2 CT, in both pull-down and surface plasmon resonance experiments. This selectivity was determined primarily by the non-conserved residues at the extreme C-termini of the β CT. By swapping amino acids in this region, reactivity with kindlin-2 could either be enhanced or blunted. By surface plasmon resonance, we also demonstrated that the β3 subunits can accommodate both kindlin-2 and talin head, even though their binding sites are very close in the β3 subunit. Kindlin-2 and talin head do not interact with each other but can bind simultaneously to the integrin β3 CT without mutual disturbance. This work specifies a binding site within β CT for kindlin-2, provides the first evidence for selectivity in the interaction of kindlin-2 with integrin β subunits, and excludes a spatial influence of talin and kindlin-2 on their binding to integrin CT in supporting integrin activation.

Integrin-linked kinase at a glance

Integrins are a large family of cell–extracellular matrix (ECM) and cell–cell adhesion molecules that regulate development and tissue homeostasis by controlling cell migration, survival, proliferation and differentiation ([Hynes, 2002][1]). They are non-covalently associated heterodimers

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.

Distinct roles for talin‐1 and kindlin‐3 in LFA‐1‐dependent neutrophil rolling and arrest

β2 integrins, including LFA‐1 (αLβ2), play critical roles in leukocyte recruitment during inflammation by binding to ICAM‐1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA‐1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA‐1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin activating proteins, talin‐1 and kindlin‐3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post‐capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin‐1 is necessary for both LFA‐1‐dependent neutrophil rolling and arrest. In contrast, kindlin‐3 was required only for neutrophil arrest and was dispensable for rolling mediated by intermediate affinity LFA‐1. We further corroborate our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. These data indicate that talin‐1 is sufficient to induce LFA‐1 extension to an intermediate affinity state whereas kindlin‐3 is critical for the transition of LFA‐1 to a high affinity state.

Distinct Roles for Talin-1 and Kindlin-3 in Regulating LFA-1 Conformation during Neutrophil Rolling and Arrest

β2 integrins, including LFA-1 (αLβ2), play critical roles in leukocyte trafficking. LFA-1 mediates the recruitment of neutrophils during inflammation through interaction with ICAM-1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA-1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA-1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin tail-binding activating proteins, talin-1 and kindlin-3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post-capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin-1 is necessary for both LFA-1-dependent neutrophil rolling and arrest. In contrast, kindlin-3 is required only for neutrophil arrest and is dispensable for rolling mediated by intermediate affinity LFA-1. These data indicate that talin-1 is sufficient to induce LFA-1 extension to an intermediate affinity state whereas kindlin-3 is critical for the transition of LFA-1 to a high affinity state. We further corroborated our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. Our results are the first to demonstrate that members of the talin and kindlin protein families play distinct roles in regulating integrin conformation.

Tyrosine Phosphorylation as a Conformational Switch: A CASE STUDY OF INTEGRIN β3 CYTOPLASMIC TAIL

Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, α(IIb)β(3) integrin. We have analyzed phosphorylated (Tyr(747) and Tyr(759)) β(3) integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr(747) phosphorylation prevents β(3)CT from binding to its heterodimer partner α(IIb)CT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of β(3)CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.

Monophosphorylated (747pY) beta3 integrin cytoplasmic tail under membrane mimetic conditions

monophosphorylated (747pY) beta3 integrin cytoplasmic tail under membrane mimetic conditions

Association between α4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration

α4β1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail. We found that substituting Glu982 in the α4 tail with alanine (E982A) disrupts the α4-MIIA association without interfering with the paxillin binding or Ser988 phosphorylation. By comparing stably transfected CHO cells, we show that the E982A mutation reduces the ability of α4β1 integrin to mediate cell spreading and to promote front-back polarization. In addition, we show that E982A impairs shear-flow-induced migration of the α4-integrin-expressing CHO cells by reducing their migration speed and directional persistence. The E982A mutation also leads to defects in the organization of MIIA filament bundles. Furthermore, when cells are plated on fibronectin and simulated with shear flow, α4β1 integrin forms filament-like patterns that co-align with MIIA filament bundles. These results provide a new mechanism for linking integrins to the actomyosin cytoskeleton and for regulating cell migration by integrins and non-muscle myosin II.

A potential role for α-actinin in inside-out αIIbβ3 signaling

AbstractMany different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y12-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.

Nanoscale architecture of integrin-based cell adhesions

Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a ∼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

Integrin β3 Phosphorylation Dictates Its Complex with the Shc Phosphotyrosine-binding (PTB) Domain

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β(3), Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β(3)integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr(759), which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr(747) of the highly conserved NPXY motif of β(3). The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.

Abstract 3841: An integrin αvβ3/c-Src oncogenic unit promotes anchorage independence and tumor progression

Abstract Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin αvβ3 is associated with progression of a variety of human tumors. We recently described an adhesion-independent role for integrin αvβ3 in pancreatic cancer and other carcinomas resulting in increased anchorage-independent tumor growth in vitro and lymph node metastases in vivo. These effects required recruitment of c-Src to the β3 integrin cytoplasmic tail, leading to c-Src activation. Pharmacological blockade of c-Src kinase activity with Dasatinib or genetic suppression of endogenous c-Src not only inhibited anchorage-independent growth, but also suppressed metastasis in vivo. We now show that αvβ3 exerts these effects through ligand-independent clustering of the integrin on the surface of non-adherent tumor cells. Self-assembly/clustering appears to be a unique property of αvβ3 relative to other integrins and is dependent on c-Src recruitment to αvβ3. These data describe a c-Src-dependent self-assembly property of αvβ3 leading to increased anchorage-independence and malignancy of tumor cells expressing this integrin. These findings also underscore the potential benefits of using Src inhibitors, such as Dasatinib, for treating patients with αvβ3-expressing solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3841.

Analysis of conserved residues in the βpat-3cytoplasmic tail reveals important functions of integrin in multiple tissues

Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans betapat-3 cytoplasmic tail. The beta1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the beta1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a beta1C-like variant, which results in a frameshift, was constructed. The betapat-3(beta1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin.

Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface β1 integrins

Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell's extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to beta1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the beta1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.

Integrin αIIbβ3 Activation in Chinese Hamster Ovary Cells and Platelets Increases Clustering Rather than Affinity

Integrin alphaIIbbeta3 affinity regulation by talin binding to the cytoplasmic tail of beta3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of alphaIIbbeta3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn(2+), a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of beta3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets.