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.

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.

Integrin β3 Membrane-Proximal and -Distal Residues Cooperatively Regulate Talin-Mediated αIIbβ3 Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1848-1848
Author(s):  
Jun Yamanouchi ◽  
Takaaki Hato ◽  
Hiroshi Fujiwara ◽  
Yoshihiro Yakushijin ◽  
Masaki Yasukawa
Keyword(s):  
Ligand Binding ◽  
Double Mutant ◽  
Distal Region ◽  
Proximal Region ◽  
Structural Rearrangements ◽  
Wild Type ◽  
Integrin Activation ◽  
Integrin Αiibβ3 ◽  
Membrane Proximal Region ◽  
Constitutively Active

Abstract Integrin αIIbβ3 exists in a low affinity state in resting platelets and requires activation for high affinity binding with soluble ligands. Activation of αIIbβ3 is tightly linked to structural rearrangements of the αIIbβ3 molecule that is initiated from the cytoplasmic tails of the αIIb and β3 subunits. The β3 membrane-distal region has been shown to interact with many signaling and cytoskeletal molecules, and considered as a trigger point of integrin activation. The interaction of the β3 tail with a cytoplasmic protein, talin, largely contributes to integrin activation. In view of the link between integrin activation and allosteric structural rearrangements of integrins, one would expect that structural changes in the β3 membrane-distal region containing binding sites for intracellular proteins would be relayed to the membrane-proximal region, leading to αIIbβ3 activation. However, there has been no evidence that structural rearrangement of the β3 membrane-distal region is directly linked to integrin activation. No activating mutation has so far been reported in the β3 membrane-distal region despite numerous reports of loss-of-function mutants in this region. In this context, a previously reported αIIbβ3 mutant in which the β3 tail was replaced by the β1 tail was noteworthy. This chimeric integrin, αIIbβ3/β1, was constitutively active. Because the β1 and β3 subunits have relatively high sequence homology in their membrane-proximal regions, we reasoned that the residues differing between the β1 and β3 membrane-distal regions may be responsible for αIIbβ3 activation. To identify such residues, we produced 13 αIIbβ3 mutants in which the individual or group residues in the β3 tail were substituted with the corresponding β1 tail residues. The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of fibrinogen and PAC1, a ligand-mimetic anti-αIIbβ3 antibody. Among them, only β3I719M and E749S mutants bound significant PAC1 and fibrinogen binding without any stimulation and the RGDS peptide abolished binding of these ligands, indicating a constitutively active state. The similar effect was observed with I719A and E749A mutants. Moreover, the I719M/E749S double mutant showed more PAC1 binding than the single mutants, reaching the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of M719 or S749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation, indicating talin-mediated activation of mutant integrins. Since I719 is located at the β3 membrane-proximal region, it is likely that the I719 mutation disrupts the well-known membrane-proximal clasp to maintain integrins at a low affinity state. On the other hand, E749 is located at the β3 membrane-distal region. This result provides experimental evidence that structural perturbation of the β3 membrane-distal region is linked to integrin activation. Moreover, our result showed that the mutational effects of the membrane-proximal I719 and the membrane-distal E749 residues were additive and talin-dependent, suggesting that the β3 membrane-proximal and –distal regions cooperatively regulate talin-mediated αIIbβ3 activation. This finding is consistent with a recent model of talin-induced αIIbβ3 activation in which talin cooperatively interacts with the β3 membrane-proximal and distal regions.

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

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Mitali Das ◽  
Sujay Ithychanda ◽  
Kamila Bledzka ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Cell Migration ◽  
K562 Cells ◽  
Cell Types ◽  
Suppressive Effect ◽  
Integrin Activation ◽  
Intracellular Signals ◽  
Integrin Binding Site ◽  
Multiple Cell ◽  
Β3 Integrin ◽  
Inside Out

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.

scholarly journals Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors

1999 ◽  
Vol 345 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Annet HAMMACHER ◽  
John WIJDENES ◽  
Douglas J. HILTON ◽  
Nicos A. NICOLA ◽  
Richard J. SIMPSON ◽  
...  
Keyword(s):  
Proximal Region ◽  
Leukaemia Inhibitory Factor ◽  
Binding Affinities ◽  
Wild Type ◽  
Correct Orientation ◽  
Fibronectin Type Iii ◽  
Membrane Proximal Region ◽  
Stimulating Factor ◽  
Fibronectin Type

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of 125I-labelled IL-6 to (GR-FNIII)gp130, or of 125I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, 125I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.

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.

scholarly journals Cytotoxic T Lymphocyte Antigen 4 (Ctla-4) Engagement Delivers an Inhibitory Signal through the Membrane-Proximal Region in the Absence of the Tyrosine Motif in the Cytoplasmic Tail

1999 ◽  
Vol 190 (6) ◽  
pp. 765-774 ◽  
Author(s):  
Chiaki Nakaseko ◽  
Shoichiro Miyatake ◽  
Tomohiko Iida ◽  
Satoru Hara ◽  
Ryo Abe ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
T Lymphocyte ◽  
Cell Activation ◽  
Cytotoxic T Lymphocyte ◽  
Interleukin 2 ◽  
Cytoplasmic Tail ◽  
Proximal Region ◽  
Lymphocyte Antigen ◽  
Membrane Proximal Region

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a T cell costimulation receptor that delivers inhibitory signals upon activation. Although the tyrosine-based motif (165YVKM) within its cytoplasmic tail has been shown to associate in vitro with Src homology 2 domain–containing tyrosine phosphatase (SHP-2) and phosphatidylinositol 3 kinase upon phosphorylation, the mechanism of negative signaling remains unclear. Here, we report a new mechanism of negative signaling based on the analysis of murine T cell clones transfected with various mutants of CTLA-4. Upon T cell activation by cross-linking with anti-CD3 and anti-CD28 antibodies, CTLA-4 engagement inhibited both proliferation and interleukin 2 production in tyrosine mutants as well as in wild-type CTLA-4 transfectants. Furthermore, the mutant CTLA-4 lacking most of the cytoplasmic region strongly suppressed interleukin 2 production as well. These data suggest that negative signals by CTLA-4 could be mediated through the membrane-proximal region of CTLA-4 but not through the YVKM motif and that the association of CTLA-4 with SHP-2 is not required for CTLA-4–mediated suppression of T cell activation.

scholarly journals A membrane proximal region of the integrin alpha5 subunit is important for its interaction with nischarin

2004 ◽  
Vol 377 (2) ◽  
pp. 449-457 ◽  
Author(s):  
Suresh K. ALAHARI ◽  
Hani NASRALLAH
Keyword(s):  
Cell Migration ◽  
Mutational Analysis ◽  
Cytoplasmic Tail ◽  
Proximal Region ◽  
Integrin Subunit ◽  
Protein Fragments ◽  
Yeast Two Hybrid System ◽  
Membrane Proximal Region ◽  
Two Hybrid ◽  
Minimal Region

In a previous study [Alahari, Lee and Juliano (2000) J. Cell Biol. 151, 1141–1154], we have identified a novel protein, nischarin, that specifically interacts with the cytoplasmic tail of the α5 integrin subunit. Overexpression of this protein profoundly affects cell migration. To examine the nischarin–α5 interaction in detail, and to find the minimal region required for the interaction, several mutants of nischarin and α5 were created. The results obtained for the yeast two-hybrid system indicate that a 99-aminoacid region of nischarin (from residues 464 to 562) is indispensable for the interaction. Also, we demonstrate that the membrane proximal region (from residues 1017 to 1030) of the α5 cytoplasmic tail is essential for the interaction. To characterize more directly the properties of the interaction between nischarin and α5, we performed surface-plasmon resonance studies in which peptides were immobilized on the surface of a sensor chip, and the recombinant nischarin protein fragments were injected. Consistent with the two-hybrid results, recombinant nischarin binds well to immobilized α5 peptides. In addition, mutational analysis revealed that residues Tyr1018 and Lys1022 are crucial for α5–nischarin interactions. These results provide evidence that nischarin is capable of directly and selectively binding to a portion of the α5 cytoplasmic domain. Further studies demonstrated that the minimal α5 binding region of nischarin does not affect cell migration.

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.

scholarly journals TGF-β maintains dormancy of prostatic stem cells in the proximal region of ducts

2005 ◽  
Vol 170 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Sarah N. Salm ◽  
Patricia E. Burger ◽  
Sandra Coetzee ◽  
Ken Goto ◽  
David Moscatelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transforming Growth Factor ◽  
Distal Region ◽  
Proximal Region ◽  
Cell Compartment ◽  
Androgen Ablation ◽  
Androgen Withdrawal ◽  
Prostatic Diseases ◽  
Prostatic Ducts

We have previously shown that prostatic stem cells are located in the proximal region of mouse prostatic ducts. Here, we show that this region responds differently to transforming growth factor (TGF)-β than the distal ductal region and that under physiological conditions androgens and TGF-β are crucial overall regulators of prostatic tissue homeostasis. This conclusion is supported by the observations showing that high levels of TGF-β signaling are present in the quiescent proximal region of ducts in an androgen-replete animal and that cells in this region overexpress Bcl-2, which protects them from apoptosis. Moreover, androgen ablation reverses the proximal-distal TGF-β signaling gradient, leading to an increase in TGF-β signaling in the unprotected distal region (low Bcl-2 expression). This reversal of TGF-β–mediated signaling accompanies apoptosis of cells in the distal region and gland involution after androgen withdrawal. A physiological TGF-β signaling gradient (high proximally and low distally) and its functional correlates are restored after androgen replenishment. In addition to highlighting the regulatory role of androgens and TGF-β, these findings may have important implications for the deregulation of the stem cell compartment in the etiology of proliferative prostatic diseases.

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