scholarly journals Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

1994 ◽  
Vol 5 (9) ◽  
pp. 977-988 ◽  
Author(s):  
S Kawaguchi ◽  
J M Bergelson ◽  
R W Finberg ◽  
M E Hemler
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Inverse Correlation ◽  
Cytoplasmic Domain ◽  
Negative Regulator ◽  
Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

scholarly journals Specialized functional properties of the integrin alpha 4 cytoplasmic domain.

1995 ◽  
Vol 6 (6) ◽  
pp. 661-674 ◽  
Author(s):  
P D Kassner ◽  
R Alon ◽  
T A Springer ◽  
M E Hemler
Keyword(s):  
Cell Migration ◽  
Shear Flow ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Adhesion Formation ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Positive Effects

For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.

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 Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

1999 ◽  
Vol 10 (8) ◽  
pp. 2507-2518 ◽  
Author(s):  
Yu Shen ◽  
Michael D. Schaller
Keyword(s):  
Cell Adhesion ◽  
Subcellular Localization ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Wild Type ◽  
Critical Determinant ◽  
Single Region ◽  
Dependent Cell ◽  
Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

scholarly journals β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

1998 ◽  
Vol 9 (4) ◽  
pp. 715-731 ◽  
Author(s):  
S. Francesco Retta ◽  
Fiorella Balzac ◽  
Piercarlo Ferraris ◽  
Alexey M. Belkin ◽  
Reinhard Fässler ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Chinese Hamster ◽  
Variable Region ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Β1 Integrin ◽  
Common Region

The β1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms (“common” region) and a distal subdomain specific for each isoform (“variable” region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used β1A and β1B isoforms as well as four mutants lacking the entire cytoplasmic domain (β1TR), the variable region (β1COM), or the common region (β1ΔCOM-B and β1ΔCOM-A). By expressing these constructs in Chinese hamster ovary and β1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol132, 227–238, 1996), we show that β1B, β1COM, β1ΔCOM-B, and β1ΔCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, β1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that β1B interferes in a dominant negative manner with β1A and β3/β5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the β1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the β1B isoform.

scholarly journals Insulin and insulin-like growth factor-1 stimulate dephosphorylation of paxillin in parallel with focal adhesion kinase

1996 ◽  
Vol 314 (2) ◽  
pp. 387-390 ◽  
Author(s):  
Nicky KONSTANTOPOULOS ◽  
Stella CLARK
Keyword(s):  
Amino Acids ◽  
Growth Factor ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Insulin Receptors ◽  
Ovary Cell ◽  
C Terminus

Paxillin and focal adhesion kinase (pp125FAK) co-localize in focal adhesions; recently insulin has been shown to stimulate the dephosphorylation of pp125FAK; however, its effect on paxillin is unknown. We show that insulin and IGF-1 can stimulate the dephosphorylation of paxillin in CHOT (overexpress human insulin receptors) and CHO∆CT69 (overexpress insulin receptors lacking C-terminal 69 amino acids) cells. Furthermore, the insulin-receptor C-terminus is not needed for either insulin or IGF-1 to stimulate paxillin or pp125FAK dephosphorylation in the CHO (Chinese-hamster ovary) cell lines used.

scholarly journals Tetramerization domain of human butyrylcholinesterase is at the C-terminus

1997 ◽  
Vol 327 (3) ◽  
pp. 747-757 ◽  
Author(s):  
M. Renee BLONG ◽  
Elliott BEDOWS ◽  
Oksana LOCKRIDGE
Keyword(s):  
Amino Acids ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Disulphide Bond ◽  
Wild Type ◽  
Flag Epitope ◽  
Bche Activity ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Inactive Protein

Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 μM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571→Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.

scholarly journals Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association

1992 ◽  
Vol 117 (6) ◽  
pp. 1321-1330 ◽  
Author(s):  
AA Reszka ◽  
Y Hayashi ◽  
AF Horwitz
Keyword(s):  
Amino Acids ◽  
Focal Adhesion ◽  
Point Mutations ◽  
Focal Adhesions ◽  
Alpha Helix ◽  
Cytoplasmic Domain ◽  
Amino Acid Sequences ◽  
Immunofluorescent Staining ◽  
Detectable Effect ◽  
Nih 3T3

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.

Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3629-3636 ◽  
Author(s):  
Meghna U. Naik ◽  
Ulhas P. Naik
Keyword(s):  
Cell Migration ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Binding Protein ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Focal Complex ◽  
Platelet Spreading

AbstractPlatelet spreading on the subendothelium in response to vascular injury is fundamental to the regulation of physiologic hemostasis. Previously, we have shown that, when bound to glycoprotein IIb (GPIIb), calcium- and integrin-binding protein (CIB) regulates platelet spreading on immobilized fibrinogen (Fg). In this study, we investigated the signaling events that occur downstream of CIB in the absence of signaling that occurs as a result of granular secretion. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate that CIB induces cell migration. Immunofluorescence analysis of CIB localization indicates that endogenous CIB accumulates in areas of focal adhesions, and its overexpression up-regulates the formation of focal adhesion complexes compared with control cells. Immunoprecipitation analysis indicates that CIB associates with focal adhesion kinase (FAK), a key regulator in focal complex formation, and up-regulates its activity. Overexpression of dominant-negative FAK, FRNK, along with CIB in CHO cells completely inhibits CIB-induced cell migration. Further, confirmation of these data in the platelet system indicates that CIB and FAK associate throughout all stages of platelet spreading but only on Fg binding to GPIIb/IIIa. Taken together, our results suggest that CIB regulates platelet spreading through the regulation of FAK activation. (Blood. 2003;102: 3629-3636)

Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

1993 ◽  
Vol 264 (6) ◽  
pp. L598-L605
Author(s):  
B. Warner ◽  
R. Papes ◽  
M. Heile ◽  
D. Spitz ◽  
J. Wispe
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Lethal Dose ◽  
Chinese Hamster ◽  
Eukaryotic Expression ◽  
Wild Type ◽  
Oxidant Injury ◽  
Sod Activity ◽  
Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

scholarly journals Molecular basis for Ras suppressor-1 recruitment to focal adhesions and stabilization of consensus adhesome complex

2021 ◽  
Author(s):  
Koichi Fukuda ◽  
Fan Lu ◽  
Jun Qin
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Specific Interaction ◽  
Tumor Development ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Biological Data ◽  
Structural Basis ◽  
Lim Domain ◽  
Insight Into

AbstractRas suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating fundamental cell adhesion processes and tumor development. Rsu-1 interacts with a zinc-finger type multi LIM domain-containing adaptor protein PINCH-1 involved in the integrin-mediated consensus adhesome but not with highly homologous isoform PINCH-2. However, the structural basis for such specific interaction and regulatory mechanism remains unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture with eight LRRs shielded by the N- and C-terminal capping modules. We show that a large conserved concave surface of the Rsu-1 LRR domain recognizes the PINCH-1 LIM5 domain, and that the C-terminal non-LIM region of PINCH-2 but not PINCH-1 sterically disfavors the Rsu-1 binding. We further show that Rsu-1 can be assembled, via PINCH-1-binding, into a tight hetero-pentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2 that constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Consistently, our mutagenesis and cell biological data consolidate the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading. Our results provide a crucial molecular insight into Rsu-1-mediated cell adhesion with implication on how it may regulate tumorigenic growth.

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