scholarly journals The Talin Head Domain Binds to Integrin β Subunit Cytoplasmic Tails and Regulates Integrin Activation

1999 ◽  
Vol 274 (40) ◽  
pp. 28071-28074 ◽  
Author(s):  
David A. Calderwood ◽  
Roy Zent ◽  
Richard Grant ◽  
D. Jasper G. Rees ◽  
Richard O. Hynes ◽  
...  
Keyword(s):  
Β Subunit ◽  
Integrin Activation ◽  
Head Domain ◽  
Cytoplasmic Tails

scholarly journals Integrin Activation Involves a Conformational Change in the α1 Helix of the β Subunit A-domain

2002 ◽  
Vol 277 (22) ◽  
pp. 19800-19805 ◽  
Author(s):  
A. Paul Mould ◽  
Janet A. Askari ◽  
Stephanie Barton ◽  
Adam D. Kline ◽  
Paul A. McEwan ◽  
...  
Keyword(s):  
Conformational Change ◽  
Β Subunit ◽  
Integrin Activation ◽  
Subunit A

scholarly journals Spatial Coordination of Kindlin-2 with Talin Head Domain in Interaction with Integrin β Cytoplasmic Tails

2012 ◽  
Vol 287 (29) ◽  
pp. 24585-24594 ◽  
Author(s):  
Kamila Bledzka ◽  
Jianmin Liu ◽  
Zhen Xu ◽  
H. Dhanuja Perera ◽  
Satya P. Yadav ◽  
...  
Keyword(s):  
Spatial Coordination ◽  
Head Domain ◽  
Cytoplasmic Tails

A Role for α-Actinin in Inside-out αIIbβ3 Signaling.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1849-1849 ◽  
Author(s):  
Seiji Tadokoro ◽  
Masamichi Shiraga ◽  
Hirokazu Kashiwagi ◽  
Tsuyoshi Kamae ◽  
Masao Akiyama ◽  
...  
Keyword(s):  
Initial Velocity ◽  
Late Phase ◽  
Initial Step ◽  
Cytoskeletal Proteins ◽  
Velocity Analysis ◽  
Integrin Activation ◽  
Time Points ◽  
Cytoplasmic Tails ◽  
Inside Out

Abstract Integrin activation is regulated by many different biochemical signaling pathways through the integrin cytoplasmic tails. Multiprotein complexes assembled around the integrin cytoplasmic tail are linked to the actin cytoskeleton. Binding of the cytoskeletal proteins to integrin cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that modulate their affinity. Talin-1 or Kindlin-3 has been identified as integrin activator complex proteins. α-Actinin also links the cytoplasmic domains of integrin β tails to actin filaments. We report here a new role for α-actinin in inside-out integrin activation. To explore the role of α-actinin in inside-out signaling, platelets were stimulated with protease-activated receptor (PAR) - activating peptides (AP) under non-stirring condition for up to 20 min. Immunoprecipitation with anti-αIIbβ3 followed by immnoblotting with anti-α-actinin revealed that in resting platelets α-actinin was constitutively associated with αIIbβ3. When platelets were stimulated by PAR1-AP, α-actinin was dissociated from αIIbβ3 as an initial step. Interestingly α-actinin re-bound to αIIbβ3 at 20 min after PAR1-AP stimulation. In contrast to PAR1-AP stimulation, the α-actinin dissociation from αIIbβ3 induced by PAR4-AP was long-lasting. To reveal the dynamic changes in αIIbβ3 activation, we recently developed initial velocity analysis for PAC1 binding. In brief, FITC-PAC1 was added to the activated platelets at indicated time points after stimulation and incubated for only 30 seconds to get the PAC1 binding velocity at the time points in question. The velocity of PAC1 binding reflects the relative numbers of activated αIIbβ3 at the time points. This initial velocity analysis more clearly revealed that PAR1-AP stimulation induced only transient αIIbβ3 activation, whereas PAR4-AP induced long-lasting αIIbβ3 activation. Moreover, the dissociation of α-actinin from αIIbβ3 appears to correlate with the time-dependent changes in the number of activated αIIbβ3. The kinetics of α-actinin-αIIbβ3 interaction was synchronized with tyrosine phosphorylation of α-actinin. When stimulated with PAR1-AP, α-actinin was de-phosphorylated rapidly and re-phosphorylated in late phase. PAR4-AP induced more prolonged de-phosphorylation of α-actinin than PAR1-AP. Thus, these results suggest that the interaction between α-actinin and αIIbβ3 may correlate with inside-out signaling induced by PAR1-AP and PAR4-AP. In platelets from a patient with Glanzmann thrombasthenia the phosphotyrosine profile of α-actinin was almost the same as that of control platelets in both PAR1-AP and PAR4-AP stimulation, confirming that these changes are not mediated αIIbβ3 outside-in signaling. In sharp contrast PAR4-AP stimulation failed to induce the sustained de-phosphorylation of α-actinin in P2Y12-ADP receptor deficient platelets. The blockade of P2Y12 with AR-C69931MX impaired the levels of activated αIIbβ3 induced by PAR4-AP, which correlated with the re-association of α-actinin. To further examine the role for α-actinin in integrin activation, α-actinin was overexpressed in human megakaryoblastic CMK cells and PAR1- AP induced PAC-1 binding to αIIbβ3 was assessed. Initial velocity analysis on CMK cells showed that overexpressed α-actinin inhibited PAR1-AP induced αIIbβ3 activation. These data imply that the binding of α-actinin to αIIbβ3 may regulate the levels of αIIbβ3 activation. Our observations may provide a new molecular framework for understanding the functions of β3 integrins in platelets.

The HPA-1b (Pro33) Isoform of Platelet Integrin αIIbβ3 Is a Prothrombotic Variant: Characterization by Fluorescence Resonance Energy Transfer

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2028-2028
Author(s):  
Abdelouahid El Khattouti ◽  
Volker R. Stoldt ◽  
Rüdiger E. Scharf
Keyword(s):  
Energy Transfer ◽  
Fusion Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatial Separation ◽  
Hek293 Cells ◽  
Β Subunit ◽  
Integrin Αiibβ3 ◽  
Biomechanical Stress ◽  
Cytoplasmic Tails

Abstract Abstract 2028 Background and Objectives: The HPA-1 polymorphism of αIIbβ3 arises from a Leu→Pro exchange at residue 33 of the β3 subunit resulting in HPA-1a (Leu33) or HPA-1b (Pro33). We have documented that patients with coronary artery disease who are carriers of HPA-1b (Pro33) experience their myocardial infarction 5.2 years earlier than HPA-1a/1a (Leu33) patients (J Thromb Haemost 2005; 3: 1522). Based on these observations, it has been postulated that HPA-1b (Pro33) is a prothrombotic variant of αIIbβ3. We have now generated a model overexpressing fluorescent proteins fused with αIIbβ3 in transfected HEK293 cells. Methods: :A yellow protein (YFP) and a cyan fluorescent protein (CFP) were cloned to the C-termini of the β3 and αIIb subunits prior to transfection of HEK293 cells, subsequently expressing the fusion proteins of both HPA-1 isoforms. Using flow cytometry, Western blotting and specific antibodies directed against αIIb or β3, we identified 12 HPA-1a and 11 HPA-1b positive clones. For further experiments only those cell lines expressing equal amounts of fluorescent fusion proteins, i.e. a 140 kD αIIb-CFP and a 113 kD β3-YFP, were used. Results: Functional integrity of both integrin variants and proper membrane insertion were documented by intact activation of transfected HEK293 cells through G protein-coupled receptors with organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol) or direct phorbol 12-myristate 13-acetate-induced stimulation of protein kinase C and by specific binding of Alexa488 fibrinogen to αIIbβ3 in response to inside-out signaling. In the presencence of pertussis toxin or abciximab, activation or ligand binding of αIIbβ3 were completely (>98%) inhibited in both isoforms. Activation of αIIbβ3 stimulates the tyrosine kinase Src, constitutively associated with the the β-subunit of the integrin. To determine whether αIIbβ3-dependent outside-in signaling is responsible for a polymorphism-related modulation, we performed adhesion experiments under static conditions with fibrinogen (50 μg/ml) in the absence or presence of Mn2+ (0.5 mM). Specific activation of the phosphotyrosine motif (Src-pY418), as determined by Western blotting and quantified by densitometry (ratio of Src-pY418/total Src), was 15 + 1.5% higher in HPA-1b than HPA-1a cells in the presence of Mn2+ (n=6 independent experiments, p<0.01). To explore the molecular nature of this difference in terms of putative changes in the allostery of integrin αIIbβ3 with regard to the HPA-1 polymorphism, dynamic measurements were performed using fluorescence resonance energy transfer (FRET). The relative decrease in FRET signal, indicating spatial separation of the cytoplasmic tails of the α- and β-subunit as a consequence of integrin activation, was recorded every minute over 0.5 hrs in transfected HEK293 cells adherent onto fibrinogen. At every time point, the kinetic measurements revealed a significantly faster and more distict (> 5%) decrease in HPA-1b than in HPA-1a cells under static adhesion (p<0.009). Upon exposure of adherent HEK293 cells to increasing shear rates (stepwise elevation from 50 to 1600 sec-1 by doubling the initial shear rate every minute), the spatial separation of the integrin subunits occurred significantly faster and more distinct (> 10%) in HPA-1b (Pro33) than HPA-1a (Leu33) cells in response to shear (p<0.0014). Under the same conditions, the rate of HPA-1b cells still adherent onto immobilized fibrinogen was 80%, while the relative number of residual HPA-1a cells decreased to 20% upon exposure to 1600 sec-1 (p<0.0001). These displacement experiments suggest that the HPA-1b (Pro33) variant is more resistant to biomechanical stress than the HPA-1a (Leu33) isoform. Conclusions: Our findings suggest that the HPA-1 polymorphism can have a significant impact on the activation of αIIbβ3. This is evident from a higher outside-in signaling and a higher resistance to biomechanical stress upon exposure to increasing shear of HPA-1b (Pro33) in comparison with HPA-1a (Leu33) transfectants. The difference in spatial separation of the cytoplasmic tails of the integrin in response to activation, as demonstrated by FRET analyses under static and flow dynamic conditions, reflects allosteric changes that may contribute to the prothrombotic phenotype of the HPA-1b (Pro33) variant. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phosphorylation of Kindlins and the Control of Integrin Function

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 825
Author(s):  
Katarzyna Bialkowska ◽  
Jun Qin ◽  
Edward F. Plow
Keyword(s):  
Beta Subunits ◽  
Post Translational Modification ◽  
Integrin Activation ◽  
High Affinity ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cytoplasmic Tails ◽  
Cell Matrix Interactions ◽  
Integrin Regulation

Integrins serve as conduits for the transmission of information between cells and their extracellular environment. Signaling across integrins is bidirectional, transducing both inside-out and outside-signaling. Integrin activation, a transition from a low affinity/avidity state to a high affinity/avidity state for cognate ligands, is an outcome of inside-signaling. Such activation is particularly important for the recognition of soluble ligands by blood cells but also influences cell-cell and cell-matrix interactions. Integrin activation depends on a complex series of interactions, which both accelerate and inhibit their interconversion from the low to the high affinity/avidity state. There are three components regarded as being most proximately involved in integrin activation: the integrin cytoplasmic tails, talins and kindlins. The participation of each of these molecules in integrin activation is highly regulated by post-translation modifications. The importance of targeted phosphorylation of integrin cytoplasmic tails and talins in integrin activation is well-established, but much less is known about the role of post-translational modification of kindlins. The kindlins, a three-member family of 4.1-ezrin-radixin-moesin (FERM)-domain proteins in mammals, bind directly to the cytoplasmic tails of integrin beta subunits. This commentary provides a synopsis of the emerging evidence for the role of kindlin phosphorylation in integrin regulation.

scholarly journals Structure and Sequence Diversity of the Membrane Distal Region of α-Cytoplasmic Tail in Integrin Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3453-3453
Author(s):  
Aye Myat Myat Thinn ◽  
Jieqing Zhu
Keyword(s):  
Cytoplasmic Tail ◽  
Proximal Region ◽  
Sequence Diversity ◽  
Wild Type ◽  
Integrin Activation ◽  
Cytoplasmic Tails ◽  
Membrane Proximal Region ◽  
Inside Out ◽  
Α5β1 Integrin

Abstract Integrins are α/β heterodimeric cell adhesion receptors with each subunit comprising of a large extracellular domain, a single-spanning transmembrane domain, and usually a short cytoplasmic tail. Different combinations of 18 α and 8 β subunits make up 24 integrin members that recognize diverse extracellular ligands important in numerous biological functions such as immune responses, maintenance of hemostasis, and development. Abnormal activation of integrin is associated with many pathological conditions including thrombosis, inflammatory diseases, as well as tumor-driven cell growth, metastasis, and angiogenesis. Therefore, tight regulation is crucial in integrin activation. Recent structural and functional studies have shown that integrin activation is regulated by the cytoplasmic tails. Studies on the mechanism of integrin activation from inside the cell (namely inside-out activation) have been focused on the β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators such as talin and kindlin. However, the role of α cytoplasmic tail in integrin activation remains elusive. The integrin α cytoplasmic tails share a conserved GFFKR motif at the membrane-proximal region that forms a specific interface with the membrane-proximal region of the β cytoplasmic tail. In contrast, the membrane-distal (MD) regions following the GFFKR motif are diverse significantly both in length, sequence and structure when reported, and their roles in integrin activation have not been well characterized. Our recent studies demonstrated that the α-MD region is required for talin and kindlin-induced activation of αIIb, αV, and αL integrins and suggest that the sequence diversity of the α-MD region might play a role in the regulation of integrin activation. In this study, we further examined the role of α-MD regions in integrin inside-out activation using αIIb, αL, and α5 integrins as platforms. Each MD region of αIIb, αL, and α5 was replaced with those of other α subunits that heterodimerize with β3, β2, and β1 integrins, respectively. β3 subunit forms heterodimers with αIIb and αV integrins. β2 subunit forms heterodimers with αL, αM, αD, and αX integrins. β1 subunit forms heterodimers with α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, and αV integrins. Thus, using these integrin α-chimeras, we were able to systemically study the role of 17 α-MD regions in integrin inside-out activation while retaining the native association of α and β subunits at the cytoplasmic domains. Ligand-mimetic mAb PAC-1, intercellular adhesion molecule-1 (ICAM-1), and human fibronectin were used to measure the talin-head-induced activation of αIIb, αL, and α5 chimeras co-expressed in HEK293FT cells with β3, β2, and β1 integrins, respectively. Conformation-specific monoclonal antibodies were used to report integrin conformational activation. The endogenous α5β1 integrin of HEK293FT cells were knocked out by the CRISPR/Cas9 technology. Our data showed that the chimeric α integrins had different levels of inside-out activation when compared with their corresponding wild-type integrins. Some chimeras such as αIIb-αV, αL-αX, αL-αD, αL-αM, α5-α2, α5-α4, and α5-α9 showed lower integrin activation than the wild types, while other chimeras such as α5-α7 and α5-α10 rendered α5β1 integrin more active than wild type. As a control, the αIIb-α1 and αIIb-αL chimeras all showed higher inside-out activation than wild-type αIIb. Our results suggest that specific amino acids of the α-MD region that immediately follow the GFFKR motif might contribute to integrin inside-out activation, probably through regulating the conformational change of the integrin α transmembrane and cytoplasmic domains. Our study demonstrates an important role of the α-MD region in integrin activation and indicates that structure and sequence diversity of the α-MD region might contribute to the diverse functions of integrins, which are determined by different integrin α subunits. Disclosures No relevant conflicts of interest to declare.

Abstract 24: Integrin αIIb Tail Participates in Inside-Out αIIbß3 Activation by Providing a Steric Lever Arm for Talin

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Feng Ye ◽  
Ang Li ◽  
Qiang Guo ◽  
Weiming Hu
Keyword(s):  
Cell Spreading ◽  
Leukemia Cells ◽  
Amino Acid Residues ◽  
Integrin Activation ◽  
Cell Systems ◽  
Leukocyte Function ◽  
Cytoplasmic Tails ◽  
Inside Out ◽  
Specific Sequences

Increases in ligand binding to integrins (“activation”) play critical roles in platelet and leukocyte function. Integrin activation requires talin and kindlin binding to integrin β cytoplasmic tails. Much research has focused on the conserved GFFKR motif in integrin αIIb tails for its importance in keeping integrins inactive and integrin β cytoplasmic tails and their interacting partners. However, the roles of αIIb tail distal of GFFKR motif are unexplored. Here, we examine the role of αIIb tail distal of GFFKR in talin-mediated inside-out integrin activation, αIIbβ3 outside-in signaling and αIIbβ3-talin interactions. Deletion of amino acid residues after the GFFKR motif in αIIb tail abolished talin-induced inside-out αIIbβ3 activation without affecting αIIbβ3-talin interaction or outside-in αIIbβ3 signaling in modeled cell systems, measured by cell spreading and Src phosphorylation. Addition of non-homologous or non-specific amino acids to the GFFKR motif restored the capacity of talin to activate αIIbβ3 in modeled cells. Moreover, thrombin-stimulated αIIbβ3 activation in megakaryocytic leukemia cells (CMK cells) are similarly abolished by truncation after GFFKR and restored by adding non-specific sequences. Furthermore, Molecular modeling indicates that β3-bound talin sterically clashes with the αIIb tail in the αIIbβ3 complexes, potentially disfavoring the α-β interactions that keep αIIbβ3 inactive. Thus, the αIIb tail sequences distal of GFFKR participate in talin-mediated inside-out αIIbβ3 activation through its steric clashes with β3-bound talin.

scholarly journals Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail

2004 ◽  
Vol 379 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Jun YAMANOUCHI ◽  
Takaaki HATO ◽  
Tatsushiro TAMURA ◽  
Shigeru FUJITA
Keyword(s):  
Distal Region ◽  
Metabolic Energy ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Integrin Activation ◽  
Α Subunit ◽  
Ample Evidence ◽  
Cytoplasmic Tails ◽  
Energy Dependent

Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the α and β tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the α tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membranedistal region of the αIIb tail. Alanine substitution of the RPP residues in the αIIb tail rendered αIIbβ3 constitutively active in a metabolic energy-dependent manner. Although an αIIb/α6Aβ3 chimaeric integrin, in which the αIIb tail was replaced by the α6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the α6A tail inhibited binding of an activation-dependent antibody PAC1. In αIIb/α6Aβ3, deleting the TSDA sequence from the α6A tail or single amino acid substitutions of the TSDA residues inhibited αIIb/α6Aβ3 activation and replacing the membrane-distal region of the αIIb tail with TSDA rendered αIIbβ3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the αIIb tail containing the RPP sequence of the membrane-distal region failed to activate αIIbβ3. These results suggest that the RPP sequence after the GFFKR motif of the αIIb tail suppresses energy-dependent αIIbβ3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by α subunit tails.

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