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.

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Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by β3 integrin

The Journal of Cell Biology ◽

10.1083/jcb.200212082 ◽

2003 ◽

Vol 162 (3) ◽

pp. 499-509 ◽

Cited By ~ 127

Author(s):

Roberta Faccio ◽

Deborah V. Novack ◽

Alberta Zallone ◽

F. Patrick Ross ◽

Steven L. Teitelbaum

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Cell Attachment ◽

Cytoplasmic Tail ◽

Cytoplasmic Domain ◽

Small Gtpases ◽

Cytoskeletal Reorganization ◽

Integrin Activation ◽

Intracellular Signals ◽

Β3 Integrin

The β3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony–stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the β integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various β3 integrins with cytoplasmic tail mutations in β3-deficient OC precursors. We find that S752 in the β3 cytoplasmic tail is required for growth factor–induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional β3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on β3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional β3. Instead, its activation is dependent upon intracellular calcium, and on the β2 integrin. Thus, the β3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

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A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets

Journal of Experimental Medicine ◽

10.1084/jem.20070058 ◽

2007 ◽

Vol 204 (7) ◽

pp. 1571-1582 ◽

Cited By ~ 117

Author(s):

Ronit Pasvolsky ◽

Sara W. Feigelson ◽

Sara Sebnem Kilic ◽

Amos J. Simon ◽

Guy Tal-Lapidot ◽

...

Keyword(s):

T Lymphocytes ◽

Leukocyte Adhesion ◽

Splice Junction ◽

Integrin Activation ◽

Leukocyte Adhesion Deficiency ◽

Protein Levels ◽

Guanine Exchange Factor ◽

Β3 Integrin ◽

Inside Out

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

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Nov/CCN3 Enhances Long-Term Repopulating Activity of Mouse Hematopoietic Stem Cells Via Intergin β3 Signaling Collaborating with Thrombopoietin

Blood ◽

10.1182/blood.v118.21.862.862 ◽

2011 ◽

Vol 118 (21) ◽

pp. 862-862

Author(s):

Jun Ishihara ◽

Terumasa Umemoto ◽

Masayuki Yamato ◽

Yoshiko Shiratsuchi ◽

Brian G. Petrich ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Integrin Αvβ3 ◽

Αvβ3 Integrin ◽

Integrin Activation ◽

Hematopoietic Stem ◽

Β3 Integrin ◽

Inside Out ◽

Positive Effect

Abstract Abstract 862 Hematopoietic stem cells (HSCs) govern hematopoiesis by giving rise to lymphoid, myeloid, and erythroid cells throughout adult life. HSCs reside in a specialized microenvironment termed “niche,” where their functions are regulated by several factors such as cytokines and extracellular matrix (ECM). Nov (CCN3), a member of the CCN family, is a well-known soluble factor that regulates several biological events by binding to integrin receptors, growth factors, and ECM. Recently, Nov has been reported to function as a positive regulator of HSCs, as evidenced by enhanced HSC activity in response to enforced Nov expression in HSCs (Gupta et al., Science, 2007). Furthermore, Nov has been identified as a target gene for Hoxb4, a transcription factor that governs the self-renewal capacity of HSCs (Ohshima et al., Blood, 2011). In addition, Nov expression in HSCs is upregulated by IL-3 via STAT5 activation (Kimura et al., J. Biol. Chem., 2010). Thus, models for the regulation of Nov expression in HSCs have been proposed; however, the mechanisms underlying the regulation of HSC functions by Nov remain unclear. Here, we present a novel mechanism for the enhancement of HSC activity by Nov. We first suspected that thrombopoietin (TPO), an essential cytokine for HSC maintenance, may promote Nov expression, given that TPO not only induces STAT5 activation in HSCs (Seita et al., PNAS, 2007) but also stimulates Hoxb4 expression (Kirito et al., Blood, 2003). Therefore, we examined the expression of Nov by real-time quantitative RT-PCR in CD150+ CD34− c-kit+ Sca-1+ lineage− (CD150+ CD34− KSL) HSCs that were treated with TPO. Similar to IL-3, TPO significantly enhanced Nov expression, compared to that in fresh unstimulated HSCs (p < 0.05). In contrast, stem cell factor (SCF), a critical cytokine for the maintenance of HSC functions, completely lost Nov expression. This strong link between Nov and TPO in HSCs suggests that TPO may play a key role in the regulation of HSCs by Nov. Therefore, we examined the long-term repopulating (LTR) activity of HSCs in transplantation assays following treatment with exogenous Nov in the presence of TPO or SCF. Interestingly, TPO stimulation supported the Nov-induced enhancement of HSC LTR activity (p < 0.05), whereas this positive effect was completely abolished in the presence of SCF. Furthermore, treatment with TPO, but not with SCF, increased the capture of Nov by HSCs, as measured by flow cytometry analyses using Alexa647-labeled Nov (p < 0.001), which strongly suggests that the positive effect of exogenous Nov on the LTR activity of HSCs is specifically dependent on TPO. More importantly, this TPO-mediated promotion of Nov binding to HSCs was blocked by antibodies against integrin αv or β3, indicating that integrin avβ3 is the primary receptor for Nov on HSCs. Previously, we demonstrated that outside-in signaling via phosphorylated Tyr747 of integrin 3 (β3PY747) is indispensable for the TPO-dependent maintenance of mouse HSCs, which requires the activation (conformational change for high affinity ligand binding) of αvβ3 integrin via TPO-induced inside-out signaling (Umemoto et al., Blood ASH abstract, 2009). Given our previous data, the results from the present study suggest that Nov regulates the LTR activity of HSCs through outside-in signaling especially via β3PY747, following its ligation to integrin αvβ3 that has been activated by TPO-induced inside-out signaling. Finally, we confirmed our hypothesis by using β3 integrin mutant mice that harbor an alanine substitution of tyrosine 747 in the cytoplasmic tail of β3 integrin (Y747A), which impairs integrin inside-out and outside-in signaling. Transplantation assays using Y747A-expressing HSCs revealed that inhibition of bidirectional integrin signaling by the Y747A mutation completely abolished the positive TPO-dependent effect of Nov, even when αvβ3 integrin activation was rescued by Mn2+, an external inducer of integrin activation that acts independently of inside-out signaling. Taken together, our findings demonstrate that Nov positively regulates HSC activity through outside-in signaling via β3PY747, following its TPO-dependent ligation to integrin αvβ3. Thus, we present a novel mechanistic link between Nov, β3 integrin, and TPO in HSCs. Disclosures: No relevant conflicts of interest to declare.

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The Dual Structural Roles of the Membrane Distal Region of α Integrin Cytoplasmic Tail in Integrin inside-out Activation

Blood ◽

10.1182/blood.v124.21.1431.1431 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1431-1431

Author(s):

Jieqing Zhu ◽

Jiafu Liu ◽

Yan-Qing Ma ◽

Zhengli Wang

Keyword(s):

Cytoplasmic Membrane ◽

Cytoplasmic Tail ◽

K562 Cells ◽

Distal Region ◽

Proximal Region ◽

Integrin Activation ◽

Membrane Proximal Region ◽

Inside Out ◽

Complete Deletion

Abstract Integrin inside-out activation is essential for platelet aggregation mediated by αIIbβ3 and leukocytes migration and arresting mediated by αLβ2. How integrin is activated by the inside-out stimulation is not completely understood. Integrin activation from inside the cell is regulated through the transmembrane and cytoplasmic domains. Mutagenesis and structural studies revealed that the inactive integrin conformation is maintained by the specific interactions at the transmembrane and cytoplasmic domains. Inside-out signals impinging on integrin cytoplasmic domain disturb the transmembrane and cytoplasmic associations, resulting in conformational change of extracellular domain that is required for binding ligands. Studies on the mechanism of integrin inside-out activation have been focused on β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators including talin and kindlin. The integrin α cytoplasmic tails only share a conserved GFFKR motif at the membrane-proximal region that forms specific interface with the membrane-proximal region of β cytoplasmic tail. The membrane-distal regions after the GFFKR motif are diverse significantly both in length and sequence. Their roles in integrin activation have not been well characterized. In this study, by comprehensive mutagenesis, we defined the role of the membrane-distal region of α integrin cytoplasmic tail in maintaining integrin in the resting state and in integrin inside-out activation. We found that complete deletion of the αIIb cytoplasmic membrane-distal region greatly enhances αIIbβ3 activation induced by the active mutations such as β3-K716A and β3-G708L, indicating that the missing of membrane-distal region facilitates integrin activation, i.e. the αIIb membrane-distal region contributes to the inactive integrin conformation. On the other hand, complete deletion of the αIIb membrane-distal region abolished integrin activation induced by the active mutations of αIIb-R995 and β3-D723, indicating that the αIIb membrane-distal region also contributes to integrin inside-out activation. We demonstrated that deletion of the membrane-distal region of αIIb, αV, or αL integrin greatly diminished ligand binding induced by overexpression of talin-1 head and/or kindlin-2 or -3 in 293FT cells. We further confirmed the effect of α cytoplasmic membrane-distal region on integrin inside-out activation in K562 cells. In the absence of αIIb cytoplasmic membrane-distal region, PMA failed to induce ligand binding to αIIbβ3 integrin expressed in K562 cells. This effect was due to the lack of talin-1-head and kindlin-induced integrin conformational change (ectodomain extension and headpiece opening) in the absence of α cytoplasmic membrane-distal region as reported by the conformation-dependent monoclonal antibodies. Structural superposition of αIIbβ3 transmembrane-cytoplasmic heterodimer and talin-1-head/β-tail complex reveals steric clashes between talin-1 head and the αIIb membrane-distal residues (NR997) immediately follow the GFFKR motif, which has been suggested to play a role in talin-mediated integrin activation. To test this possibility, we retained two native residues, NR997 for the αIIb membrane-distal region and found that it partially restores talin-1-head-induced integrin activation. Replacing the NR997 with small amino acids, GG997 or AA997 has little effect, while with the bulky residues YY997 significantly reduced talin-1-head-induced αIIbβ3 activation. Interestingly, retaining two native residues for the membrane-distal region of αV or αL integrin failed to restore talin-1-head-induced αVβ3 or αLβ2 activation. Retaining as long as 8 native residues for the αL membrane-distal region is not sufficient to restore talin-1-head-induced αLβ2 activation to the level of intact αL. These data demonstrate that a steric clash might play a role but is not the sole mechanism by which the α cytoplasmic membrane-distal region participates in integrin inside-out activation. A proper length and amino acids of the membrane-distal region is required for talin-induced integrin activation. Our data established an essential role of the α integrin cytoplasmic membrane-distal region in integrin activation and provide new insight of how talin and kindlin induce the high affinity integrin conformation that is required for fully functional integrins. Disclosures No relevant conflicts of interest to declare.

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Regulation of αIIbβ3-Signaling by Myomesin Family of Proteins.

Blood ◽

10.1182/blood.v110.11.3893.3893 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3893-3893

Author(s):

Kumar B. Reddy

Keyword(s):

Cell Model ◽

Cell Types ◽

Positive Control ◽

Variable Number ◽

Model System ◽

Type I ◽

Integrin Receptors ◽

Β3 Integrin ◽

Cell Model System ◽

Inside Out

Abstract Integrins are the type-I transmembrane proteins. They function as heterodimers of α and β subunits. The platelet integrin αIIbβ3 (GPIIb-IIIa) served as the model system in understanding the signaling across integrin receptors. αIIbβ3 exists in a resting state on circulating platelets and can be activated via several inside-out signaling pathways resulting in binding to its ligands, such as fibrinogen (Fg). The inside-out signaling appears to be regulated by several cytoskeletal and signaling proteins, which directly associate with the cytoplasmic tails of αIIbβ3. We have previously identified skelemin/myomesin-I as the cytoskeletal protein that interacted with the cytoplasmic tail of β3-integrins. Skelemin/myomesin-I is very closely related to two other members: myomesins-II and III. Myomesins I-III, along with other proteins such as titin and myosin light chain kinases, are members of a much larger superfamily of proteins. The hallmark of this family of proteins is the presence of a variable number of fibronectin (Fn)-like and immunoglobulin (Ig)-like motifs. Myomesin-I contains five Fn-motifs flanked by a total of seven Ig-like motifs. The interaction between β3-integrin and myomesin-I occurs via the C-terminal Ig motifs 3–7 of myomsin-I. In order to understand the functional consequence of this interaction between myomesin-I and β3-integrin, we measured the ligand binding properties of αIIbβ3 in a CHO cell model system. We found that expression of Ig motifs 3–7 increased Fg binding to the αIIbβ3, comparable to talin-head domain, used as the positive control. However, the expression of smaller and individual Ig motifs 4 and 5 resulted in still significantly higher Fg-binding to αIIbβ3. Ig motifs from myomesin-II also behaved in a similar fashion. We also found that myomesin-II is ubiquitously expressed in all the cell types tested, including platelets, suggesting myomesin-II is the likely candidate among various myomesin family members for the regulation of signaling across αIIbβ3. Our results suggest that the Ig motifs of the myomesin represent novel domains, which can regulate inside-out signaling across αIIbβ3.

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Semaphorin 4D/Plexin-B1–mediated R-Ras GAP activity inhibits cell migration by regulating β1 integrin activity

The Journal of Cell Biology ◽

10.1083/jcb.200508204 ◽

2006 ◽

Vol 173 (4) ◽

pp. 601-613 ◽

Cited By ~ 69

Author(s):

Izumi Oinuma ◽

Hironori Katoh ◽

Manabu Negishi

Keyword(s):

Cell Migration ◽

Cell Types ◽

Β1 Integrin ◽

Integrin Activation ◽

Kinase Activation ◽

Surface Receptors ◽

Semaphorin 4D ◽

Ras Activation ◽

Ras Family

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration (Oinuma, I., Y. Ishikawa, H. Katoh, and M. Negishi. 2004. Science. 305:862–865). We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras–mediated phosphatydylinositol 3-kinase activation, and β1 integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras–specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing β1 integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated β1 integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1–mediated R-Ras GAP activity controls cell migration by modulating the activity of β1 integrins.

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Talin controls integrin activation

Biochemical Society Transactions ◽

10.1042/bst0320434 ◽

2004 ◽

Vol 32 (3) ◽

pp. 434-437 ◽

Cited By ~ 110

Author(s):

D.A. Calderwood

Keyword(s):

Conformational Changes ◽

Critical Role ◽

Dynamic Control ◽

Common Element ◽

Actin Binding ◽

Cytoplasmic Domains ◽

Integrin Activation ◽

Intracellular Signals ◽

Integrin Binding Site ◽

Activation Pathways

Tight, dynamic control of the affinity of integrin adhesion receptors for their extracellular ligands (integrin activation) is essential for the development and functioning of multicellular organisms. Integrin activation is controlled by intracellular signals that, through their action on integrin cytoplasmic domains, induce conformational changes in integrin extracellular domains, resulting in increased affinity for the ligand. Recent results indicate that the binding of talin, a major actin-binding protein, to integrin β tails represents a final common step in integrin activation pathways. The major integrin-binding site lies within the talin FERM (four-point-one, ezrin, radixin, moesin) domain, and binding occurs via a variant of the classical PTB domain (phosphotyrosine-binding domain)–NPxY interaction. Formation of this talin–integrin complex plays a critical role in integrin activation, since mutations, in either talin or integrin β tails, which disrupt complex formation, inhibit integrin activation. Furthermore, use of RNA interference to knockdown talin expression selectively reveals that talin is essential for integrin activation in response to physiological agonists. Thus the association of the cytoskeletal protein talin with integrin β cytoplasmic domains is a critical step during integrin activation, and regulation of this step may be a final common element in the signalling pathways that control integrin activation.

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