VPS33B, a Member of the Sec1/Munc18 Protein Family, Binds to Integrin Beta Subunits and Is Required for αIIbβ3 Outside-in Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 340-340
Author(s):  
Binggang Xiang ◽  
Guoying Zhang ◽  
Shaojing Ye ◽  
Cai Huang ◽  
Jun Liu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cho Cells ◽  
Human Platelet ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Integrin Activation ◽  
Clot Retraction ◽  
Domain Fusion ◽  
Inside Out

Abstract Integrins play fundamental roles in many biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. Integrin activation is essential for cell adhesion, spreading, survival, proliferation, and migration. Integrins are heterodimeric transmembrane glycoproteins composed of a and b subunits. The function of integrins is modulated by bi-directional transmembrane signaling: inside-out and outside-in signaling, which are mediated through the interactions between integrin a or b subunit cytoplasmic tails and intracellular proteins and can be regulated by many different biochemical signaling pathways. αIIbβ3 is a major integrin expressed in megakaryocytes and platelets. Antagonists of αIIbβ3 are potent anti-thrombotic drugs and new inhibitors targeting αIIbβ3 are under preclinical testing or large patient trials to treat acute coronary syndromes. Integrin αIIbβ3 activation involves binding of proteins, including talin, kindlins, Src kinase, and Gα13 to the cytoplasmic domain of its β subunit. To gain insight into αIIbβ3signaling and to identify new proteins that regulate αIIbβ3 activation, agarose beads conjugated with glutathione S-transferase (GST)-β3 integrin cytoplasmic domain fusion protein (GST-β3CD) or GST-αIIb integrin cytoplasmic domain fusion protein (GST-αIIbCD) were incubated with human platelet lysates. Proteins pulled down by the GST-β3CD or GST-αIIbCD beads were subjected to mass spectrometric analysis. We found that the GST-β3CD but not the GST-αIIbCD beads specifically pulled down a previously unreported protein, the vacuolar protein sorting-associated protein 33B (VPS33B), encoded by the VPS33B gene. To verify that VPS33B is in a complex with αIIbβ3, we infected Chinese hamster ovary (CHO) cells stably expressing αIIbβ3 (A5 cells) with an adenovirus containing FLAG epitope-tagged human VPS33B cDNA. Cells were allowed to adhere and spread on fibrinogen. FLAG-VPS33B exhibited a significant intracellular colocalization with aIIbb3. Immunoblotting analysis revealed a specific association of b3 with precipitated FLAG-VPS33B. The specific interaction was also confirmed by reciprocal immunoprecipitation using a specific β3antibody. Furthermore, in an in vitro binding assay, we were able to pull down VPS33B from the lysates of CHO cells overexpressing VPS33B by the GST-β3CD beads. The interaction appears to be direct, since we were also able to pull down purified FLAG-VPS33B with GST-β3CD and GST-β1CD proteins. The pulldown assay showed that the β3 fragment spanning residues 716~730 bound efficiently to FLAG-VPS33B. In contrast, the fragments containing either residues 729~762 or residues 744~762 fragments failed to do the same. More importantly, using the rabbit polyclonal antibody against VPS33B, we were able to coimmunoprecipitate endogenous b3 subunit with VPS33B from human platelet lysate. Thus, we identified a novel binding protein of integrin β3. Next, we investigated the role of VPS33B in integrin activation using a recombinant integrin activation model of CHO cells. Overexpression of VPS33B in CHO cells expressing αIIbβ3 (A5 cells) markedly potentiated cell spreading on fibrinogen and F-actin formation. To establish a role of VPS33B in integrin activation in platelets, we created a mouse model with megakaryocyte- and platelet-specific deletion of VPS33B. Platelets lacking VPS33B were defective in spreading on fibrinogen. VPS33B-/- platelets failed to support clot retraction. On the other hand, thrombin-induced fibrinogen binding to platelets and platelet aggregation were not affected by the loss of VPS33B. Collectively, these results demonstrate an essential role of VPS33B in αIIbβ3 outside-in signaling but is not requried for integrin inside-out signaling. We further demonstrate that VPS33B promotes αIIbβ3 outside-in signaling through RhoA and Rac1 activation, leading to clot retraction and cell spreading, respectively. Therefore, our results for the first time establish vesicle trafficking proteins as an important novel class of modifiers of integrin function in platelets and cells. 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 Functional role of the cytoplasmic domain of the integrin alpha 5 subunit

1993 ◽  
Vol 122 (1) ◽  
pp. 209-221 ◽  
Author(s):  
JS Bauer ◽  
J Varner ◽  
C Schreiner ◽  
L Kornberg ◽  
R Nicholas ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cho Cells ◽  
Functional Role ◽  
Cell Spreading ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Alpha Subunit ◽  
Wild Type ◽  
Cytoskeletal Organization

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

A Dual Role for the α-Subunit KVGFFKR Motif of αIIbβ3: Integrin Activation and Intracellular Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1545-1545
Author(s):  
Kelly Aylward ◽  
Marc Devocelle ◽  
Niamh Moran
Keyword(s):  
Platelet Aggregation ◽  
Ligand Binding ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Intracellular Signaling ◽  
Dual Role ◽  
Cytoskeletal Reorganization ◽  
Integrin Activation ◽  
Α Subunit

Abstract The platelet-specific integrin αIIbβ3 plays a critical role in platelet aggregation and pathological thrombosis. Integrin affinity and ligand binding are regulated by the highly conserved αIIb membrane-proximal motif 989KVGFFKR995. We have recently shown that this motif is dependent on the presence of two phenylalanines (FF) for its activity. In order to investigate the role of KVGFFKR on integrin transmembrane signaling we used two parallel systems: (1) stable Chinese Hamster Ovary (CHO) cells expressing mutant αIIbβ3 integrins and (2) platelets treated with synthetic palmitylated (pal) peptides corresponding to the seven amino acid motif. In CHO cells, we chose cytoskeletal reorganization as a means to explore outside-in signaling. Alanine substitutions were introduced to the α-subunit KVGFFKR domain and co-expressed with wildtype β3. Cells were stably transfected with wildtype αIIb(992FF993)/β3, αIIb(992AA993)/β3 and αIIb (992AF993)/β3 to produce the FF, AA and AF cells respectively. Their ability to reorganize their cytoskeleton upon adhesion to fibrinogen was then determined. Even though double alanine substitution produced a constitutively activated integrin, the AA cells were unable to give rise to cytoskeletal reassembly as seen in the FF and AF cells. Using phalloidin as a marker, the AA cells displayed polymerized F-actin but failed to show the elaborate elongated stress fibers formed in the FF and AF cells. To further investigate the role of the KVGFFKR motif on downstream signaling events, we focused on using pal-peptides in platelets. We have shown that in addition to stimulating platelet aggregation presumably by facilitating the spatial separation of the integrin cytoplasmic tails, pal-KVGFFKR (pal-FF) induced tyrosine phosphorylation even in the absence of ligand (EDTA:5mM) or (ReoPro:10μg/ml). The tyrosine phosphoproteome associated with alanine-substituted peptides pal-KVGAFKR (pal-AF) and pal-KVGFAKR (pal-FA) was similar to that of pal-FF. However there is a remarkable absence of a specific 100kDa band (probably α-actinin) in the phosphoprotein profile in response to pal-KVGAAKR (pal-AA) both with peptide treatment alone and in the presence of TRAP. A closer look at ppFAK125 revealed that its tyrosine phosphorylation is also inhibited by pal-AA. Since α-actinin and ppFAK125 phosphorylation are closely linked events it supports α-actinin as the 100kDa missing phosphoprotein. However, pal-AA did not inhibit ppSyk72or ppSrc60 activation. Moreover pal-AA was identified as a potent antagonist, inhibiting platelet aggregation, PAC-1 binding and tyrosine phosphorylation. In summary, a double alanine substitution of the α-subunit membrane proximal domain disturbs cytoskeletal reorganization downstream, even though this substitution produces a constitutively activated integrin. This suggests a signaling role for the conserved α-integrin motif in addition to regulating integrin affinity. Furthermore in platelets, pal-FF peptide, by mimicking the endogenous αIIb KVGFFKR sequence can both activate the integrin and contribute to an intracellular signaling response even when ligand binding is absent. Taken together, both the stable cell system and pal-peptides in platelets support a role for the KVGFFKR domain in outside-in signaling. Also since pal-AA is an antagonist of integrin function it highlights the complexity of the proximal regulation of αIIbβ3 activation and suggests a dual role for this motif in integrin activation and intracellular signaling.

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.

Reversible Association of 14-3-3 with Platelet Glycoprotein Ibα Regulates the Ability of Cells to Adhere, Roll and Spread On VWF

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1061-1061
Author(s):  
Adam D. Munday ◽  
Jose A. Lopez
Keyword(s):  
Amino Acids ◽  
Cho Cells ◽  
Platelet Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Stress Fibers ◽  
Platelet Glycoprotein ◽  
Shear Rates ◽  
Platelet Spreading

Abstract Abstract 1061 Platelet adhesion to sites of vascular injury is required for the arrest of bleeding. Initial platelet adhesion is mediated by binding of von Willebrand factor (VWF) to the platelet glycoprotein (GP) Ib-IX-V complex, leading to the activation of integrin α IIbβ3 and other molecules that mediate firm adhesion, spreading and thrombus formation. The GPIb-IX-V complex comprises 4 polypeptides: GPIbα, GPIbβ, GPIX and GPV, in a 2:4:2:1 stoichiometry. Only the first three polypeptides are required for full VWF binding function. GPIbα is a 610 amino acid polypeptide that binds every known complex ligand within its N-terminal 300 amino acids. The cytoplasmic domain comprises 96 amino acids and contains binding sites for filamin, PI 3-kinase and the scaffolding protein 14-3-3. The association of 14-3-3 with the GPIbα cytoplasmic domain regulates the affinity for VWF. Typically, 14-3-3 requires phosphoserine- or phosphothreonine-containing motifs to bind target proteins. One such motif is in the GPIbα cytoplasmic domain surrounding Ser609, which is phosphorylated and known to bind 14-3-3. Mutation of Ser609 to Ala abrogates 14-3-3 association, which has been proposed to reduce the ability of GPIbα to bind VWF. Platelet aggregation results in the dissociation of 14-3-3 from a subpopulation of GPIbα. Ser609 also becomes dephosphorylated upon platelet spreading. To dissect further the functional roles of 14-3-3 association with GPIbα, we expressed in Chinese hamster ovary (CHO) cells GPIb-IX complexes (GPIbα, GPIbβ, and GPIX) containing either wild type GPIbα, or GPIbα mutants S609A or S609E. In other proteins, mutation of Ser to Glu at the 14-3-3 binding site mimics phosphoserine, recapitulates 14-3-3 binding and often prevents 14-3-3 dissociation. We first assessed the ability of the WT and mutant GPIbα to associate with 14-3-3. As expected, we detected little 14-3-3 binding to GPIbα S609A. GPIbα S609E bound 14-3-3 to the same extent as did WT GPIbα, indicating that the Glu substitution was able to mimic Ser phosphorylation at residue 609. We then assessed the ability of the CHO cells to attach to and roll on VWF under flow over a wide range of shear rates. At 3.26 and 10 dyne/cm2 the α 609A and α 609E cells rolled twice as fast as the WT cells. Both CHO cells and platelets display a characteristic velocity nadir as shear rates increase. The α 609A and α 609E cell showed defective shear-enhanced adhesion; their slowest velocity was ∼3-fold faster than the WT cells. Because GPIbα is dephosphorylated upon platelet spreading, we also assessed the effect of the mutations on cell spreading on VWF. All three cell lines adhered similarly to VWF but a higher percentage of α 609A cells spread (67% vs 58% for WT and α 609E). Of the spread cells, the α S609E cells spread less well; their spread area was 15% less than the WT and α S609A cells. The morphology of the adherent, spread cells was dramatically different among the different cell lines. WT cells displayed a few filopodial extensions along with punctate phalloidin staining indicative of focal adhesions. In some cases the cells displayed stress fibers. The α S609A cells extended more and longer filopodia than the WT cells but had fewer focal adhesions and more stress fibers. The CHO α S609E cells extended thin filopodia that tended to be polarized at two sides of the cell body, and had fewer focal adhesions and no stress fibers. We also examined the effect of the mutations on localization of the GPIb-IX complex to lipid raft membrane microdomains, which is required for platelet adhesion to VWF. Raft GPIbα was reduced by 40% in the S609A cells but increased 1.6-fold in the S609E cells. In summary, lack of 14-3-3 association decreased raft localization of the complex, reduced shear-induced cell adhesion, but increased cell spreading. Stable 14-3-3 association increased raft localization, but decreased shear-induced cell adhesion and decreased the ability of cells to fully spread. Together, our results demonstrate that regulated 14-3-3 association mediated by the phosphorylation status of S609 is required for coordinated adhesion, and cell spreading. Together, our results demonstrate that the functions of the GPIb-IX complex are regulated by the ability of GPIbα Ser609 to both bind and release 14-3-3 and suggest that it is not 14-3-3 binding per se that regulates GPIbα function. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
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.

The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1531-1531
Author(s):  
Xi Mo ◽  
Jose A. Lopez ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Transmembrane Domain ◽  
Complex Structure ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Expression Level ◽  
Platelet Glycoprotein ◽  
Cellular Expression

Abstract Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.

Electron Microscopic Analysis of Subcellular Distribution and Translocation of Ca ION in Human Platelet

1979 ◽  
Author(s):  
M. Kazama ◽  
T. Daimon ◽  
K. Nakamura ◽  
J. Matsuda ◽  
I. Naito ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Human Platelet ◽  
Microscopic Analysis ◽  
Clot Retraction ◽  
Potassium Oxalate ◽  
Electron Microscopic ◽  
Platelet Suspension ◽  
Dense Bodies ◽  
Transmission Electron

For the analysis of the subcellular distribution of Ca ion in human platelet, the washed platelet suspension was fixed in the potassium antimonate-OsO4 method or in the potassium oxalate-glutaraldehyde method without post-fixation with OSO4. The STEM-images were observed with scanning image system fitted with the transmission electron microscope without electron staining.The dense deposition of antimonate or oxalate was found in plasmalemma, the membrane of open canalicular system, mitochondria, α-granules and dense bodies. It was revealed with the energy dispersive type electron probe x-ray microanalyzer system that the deposition of oxalate was exclusively composed of Ca.In the process of platelet aggregation with various agents, α-granules and dense bodies were expelled out together with Ca, which suggested the least role of these ion depositions in the aggregation and clot retraction. The translocation of antimonate deposits was not observed in the Verapamil-treated platelets even after the addition of aggregation agents. This phenomenon indicated that this drug inhibited the release of Ca from the Ca-storing organelles. The distribution of Ca was identical in the platelets of a case of thrombasthenia and the translocation of Ca was not observed with the addition of various aggregating agents.

scholarly journals Contrasting roles for integrin beta 1 and beta 5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration.

1994 ◽  
Vol 125 (2) ◽  
pp. 447-460 ◽  
Author(s):  
R Pasqualini ◽  
M E Hemler
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Detailed Comparison ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Cytoplasmic Domains ◽  
Migration Assay ◽  
Inside Out

To carry out a detailed comparison of the roles of integrin beta 1 and beta 5 cytoplasmic domains, we expressed both wild type beta 1 and chimeric beta 1/5 constructs in CHO cells. In the latter, the cytoplasmic domain of beta 1 was replaced with that of beta 5. The human beta 1 and beta 1/5 constructs appeared at similar levels at the cell surface (mostly as alpha 5 beta 1 heterodimers) and contributed equally to CHO cell adhesion to fibronectin. However, beta 1 but not beta 1/5 localized to focal adhesion-like structures when CHO cells were spread on fibronectin. Furthermore, only the beta 1-CHO cells showed increased proliferation in response to fibronectin plus an integrin-activating anti-beta 1 antibody, and showed increased appearance of 32P-labeled protein (p90) that correlated with proliferation. In sharp contrast, the beta 1/5-CHO cells were notably more migratory than beta 1-CHO cells in a transwell haptotactic migration assay. These results indicate that the beta 1 and beta 5 integrin subunit cytoplasmic domains can translate similar adhesive information into highly contrasting subsequent events. Thus, we have established that "inside-out" and "outside-in" integrin signaling pathways are regulated by fundamentally distinct mechanisms. In addition, we suggest that the same properties of the beta 1 cytoplasmic domain that promote recruitment to visible focal adhesion-like structures may also be conductive to cell proliferation. Conversely, the properties of the beta 5 tail that make it less likely to localize into focal adhesion-like structures may contribute to enhanced cell migration.

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