scholarly journals The Cytoplasmic Domain of the Integrin α9 Subunit Requires the Adaptor Protein Paxillin to Inhibit Cell Spreading but Promotes Cell Migration in a Paxillin-independent Manner

2001 ◽  
Vol 12 (10) ◽  
pp. 3214-3225 ◽  
Author(s):  
Bradford A. Young ◽  
Yasuyuki Taooka ◽  
Shouchun Liu ◽  
Karen J. Askins ◽  
Yasuyuki Yokosaki ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intracellular Signaling ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cytoplasmic Domains ◽  
Independent Manner ◽  
Ovary Cells

The integrin α9 subunit forms a single heterodimer, α9β1. The α9 subunit is most closely related to the α4 subunit, and like α4 integrins, α9β1 plays an important role in leukocyte migration. The α4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the α9 cytoplasmic domain has similar effects, a series of chimeric and deleted α9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an α9β1-specific ligand. Like α4, the α9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the α9 cytoplasmic domain and to a similar level as α4. In paxillin −/− cells, α9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the α9-paxillin interaction prevented α9 from inhibiting cell spreading but had no effect on α9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin α subunits enhance migration and inhibit cell spreading are distinct and that the α9 and α4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

scholarly journals Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia

1998 ◽  
Vol 111 (2) ◽  
pp. 249-260 ◽  
Author(s):  
J.O. Gonatas ◽  
Y.J. Chen ◽  
A. Stieber ◽  
Z. Mourelatos ◽  
N.K. Gonatas
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Ovary Cells

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.

The cytoplasmic domain of syndecan-1 is not required for association with Triton X-100-insoluble material

1994 ◽  
Vol 107 (6) ◽  
pp. 1571-1581 ◽  
Author(s):  
H.M. Miettinen ◽  
M. Jalkanen
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type Mouse ◽  
Cytoplasmic Domain ◽  
Insoluble Residue ◽  
Wild Type ◽  
Ovary Cells ◽  
Triton X

Cell surface heparan sulfate proteoglycans such as syndecan-1 bind various extracellular matrix proteins and have been suggested to interact with the cytoskeleton. Such interactions are thought to be important for stabilizing cell morphology. Syndecan-1 resists extraction with Triton X-100. This insolubility was reported not to be affected by removal of the glycosaminoglycan chains, suggesting that the insolubility is not due to binding to the extracellular matrix, but rather to an association with the actin cytoskeleton (Rapraeger, A., Jalkanen, M. and Bernfield, M. (1986) J. Cell Biol. 103, 2683–2696). To examine further the interaction of syndecan-1 with the Triton X-100-insoluble residue, we expressed wild-type mouse syndecan-1 and a cytoplasmic deletion mutant (tail-less) in Chinese hamster ovary cells. We observed that both the wild-type and the tail-less syndecan-1 were partly insoluble in Triton X-100. The insolubility was not affected by increasing temperature (37 degrees C or 50 degrees C) or by cytochalasin D. Removal of the glycosaminoglycan chains from the ectodomain, however, resulted in complete Triton X-100 solubility, unlike previous reports. Syndecan-1 could also be released into the Triton X-100-soluble fraction by addition of heparin or heparan sulfate to the extraction medium. We conclude that the cytoplasmic domain of syndecan-1 is not responsible for Triton X-100 insolubility. Instead, our results indicate that Triton X-100 insolubility is caused by an interaction of syndecan-1 molecules with other cellular and/or extracellular molecules mediated by the heparan sulfate chains.

scholarly journals A molecular switch that controls cell spreading and retraction

2007 ◽  
Vol 179 (3) ◽  
pp. 553-565 ◽  
Author(s):  
Panagiotis Flevaris ◽  
Aleksandra Stojanovic ◽  
Haixia Gong ◽  
Athar Chishti ◽  
Emily Welch ◽  
...  
Keyword(s):  
Wound Repair ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Spreading ◽  
Clot Retraction ◽  
Ovary Cells ◽  
Dependent Cell ◽  
Mediate Cell ◽  
Rhoa Signaling ◽  
Cell Retraction

Integrin-dependent cell spreading and retraction are required for cell adhesion, migration, and proliferation, and thus are important in thrombosis, wound repair, immunity, and cancer development. It remains unknown how integrin outside-in signaling induces and controls these two opposite processes. This study reveals that calpain cleavage of integrin β3 at Tyr759 switches the functional outcome of integrin signaling from cell spreading to retraction. Expression of a calpain cleavage–resistant β3 mutant in Chinese hamster ovary cells causes defective clot retraction and RhoA-mediated retraction signaling but enhances cell spreading. Conversely, a calpain-cleaved form of β3 fails to mediate cell spreading, but inhibition of the RhoA signaling pathway corrects this defect. Importantly, the calpain-cleaved β3 fails to bind c-Src, which is required for integrin-induced cell spreading, and this requirement of β3-associated c-Src results from its inhibition of RhoA-dependent contractile signals. Thus, calpain cleavage of β3 at Tyr759 relieves c-Src–mediated RhoA inhibition, activating the RhoA pathway that confines cell spreading and causes cell retraction.

scholarly journals The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1758-1766 ◽  
Author(s):  
T Fujimoto ◽  
RP McEver
Keyword(s):  
Endothelial Cells ◽  
Membrane Trafficking ◽  
Cell Activation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Ovary Cells ◽  
Phosphatase Inhibitors ◽  
Protein Kinase C Inhibitors ◽  
Activated Platelets

Abstract P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.

scholarly journals The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

1994 ◽  
Vol 124 (5) ◽  
pp. 705-715 ◽  
Author(s):  
RJ Garippa ◽  
TW Judge ◽  
DE James ◽  
TE McGraw
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Amino Terminus ◽  
Ovary Cells ◽  
Major Function ◽  
Amino Terminal ◽  
Human Transferrin Receptor

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.

scholarly journals A Second Leukotriene B4 Receptor, Blt2

2000 ◽  
Vol 192 (3) ◽  
pp. 421-432 ◽  
Author(s):  
Takehiko Yokomizo ◽  
Kazuhiko Kato ◽  
Kan Terawaki ◽  
Takashi Izumi ◽  
Takao Shimizu
Keyword(s):  
Intracellular Signaling ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Identity ◽  
Leukotriene B4 ◽  
Cellular Functions ◽  
Ovary Cells ◽  
Reading Frame ◽  
Antiinflammatory Therapy ◽  
Novel Target

Leukotriene B4 (LTB4) is a potent chemoattractant and activator of both granulocytes and macrophages. The actions of LTB4 appear to be mediated by a specific G protein–coupled receptor (GPCR) BLT1, originally termed BLT (Yokomizo, T., T. Izumi, K. Chang, Y. Takuwa, and T. Shimizu. 1997. Nature. 387:620–624). Here, we report the molecular cloning of a novel GPCR for LTB4, designated BLT2, which binds LTB4 with a Kd value of 23 nM compared with 1.1 nM for BLT1, but still efficiently transduces intracellular signaling. BLT2 is highly homologous to BLT1, with an amino acid identity of 45.2%, and its open reading frame is located in the promoter region of the BLT1 gene. BLT2 is expressed ubiquitously, in contrast to BLT1, which is expressed predominantly in leukocytes. Chinese hamster ovary cells expressing BLT2 exhibit LTB4-induced chemotaxis, calcium mobilization, and pertussis toxin–insensitive inhibition of adenylyl cyclase. Several BLT1 antagonists, including U 75302, failed to inhibit LTB4 binding to BLT2. Thus, BLT2 is a pharmacologically distinct receptor for LTB4, and may mediate cellular functions in tissues other than leukocytes. BLT2 provides a novel target for antiinflammatory therapy and promises to expand our knowledge of LTB4 function. The location of the gene suggests shared transcriptional regulation of these two receptors.

scholarly journals Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

1997 ◽  
Vol 8 (7) ◽  
pp. 1219-1232 ◽  
Author(s):  
A A Reszka ◽  
J C Bulinski ◽  
E G Krebs ◽  
E H Fischer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Mitogen Activated Protein Kinase ◽  
Ovary Cells ◽  
Morphological Alterations ◽  
Cytoskeletal Regulation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs.

scholarly journals PECAM-1/CD31 Trans-homophilic Binding at the Intercellular Junctions Is Independent of Its Cytoplasmic Domain; Evidence for Heterophilic Interaction with Integrin αvβ3 in Cis

2000 ◽  
Vol 11 (9) ◽  
pp. 3109-3121 ◽  
Author(s):  
Cindy W.Y. Wong ◽  
Guido Wiedle ◽  
Christoph Ballestrem ◽  
Bernhard Wehrle-Haller ◽  
Susanne Etteldorf ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Splice Variants ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integrin Αvβ3 ◽  
Cytoplasmic Domain ◽  
Cell Junctions ◽  
Binding Property ◽  
Cytoplasmic Domains

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin αvβ3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin αvβ3 associates with PECAM-1 on the same cell surface as in a cis manner.

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.

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