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 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.

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 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 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 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

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 Export from Pericentriolar Endocytic Recycling Compartment to Cell Surface Depends on Stable, Detyrosinated (Glu) Microtubules and Kinesin

2002 ◽  
Vol 13 (1) ◽  
pp. 96-109 ◽  
Author(s):  
Sharron X. Lin ◽  
Gregg G. Gundersen ◽  
Frederick R. Maxfield
Keyword(s):  
Elevated Temperature ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Endocytic Recycling ◽  
In Cells

A significant fraction of internalized transferrin (Tf) concentrates in the endocytic recycling compartment (ERC), which is near the microtubule-organizing center in many cell types. Tf then recycles back to the cell surface. The mechanisms controlling the localization, morphology, and function of the ERC are not fully understood. We examined the relationship of Tf trafficking with microtubules (MTs), specifically the subset of stable, detyrosinated Glu MTs. We found some correlation between the level of stable Glu MTs and the distribution of the ERC; in cells with low levels of Glu MTs concentrated near to the centriole, the ERC was often tightly clustered, whereas in cells with higher levels of Glu MTs throughout the cell, the ERC was more dispersed. The clustered ERC in Chinese hamster ovary cells became dispersed when the level of Glu MTs was increased with taxol treatment. Furthermore, in a temperature-sensitive Chinese hamster ovary cell line (B104-5), the cells had more Glu MTs when the ERC became dispersed at elevated temperature. Microinjecting purified anti-Glu tubulin antibody into B104-5 cells at elevated temperature induced the redistribution of the ERC to a tight cluster. Microinjection of anti-Glu tubulin antibody slowed recycling of Tf to the cell surface without affecting Tf internalization or delivery to the ERC. Similar inhibition of Tf recycling was caused by microinjecting anti-kinesin antibody. These results suggest that stable Glu MTs and kinesin play a role in the organization of the ERC and in facilitating movement of vesicles from the ERC to the cell surface.

The NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform

2001 ◽  
Vol 114 (6) ◽  
pp. 1101-1113
Author(s):  
M. Gawaz ◽  
F. Besta ◽  
J. Ylanne ◽  
T. Knorr ◽  
H. Dierks ◽  
...  
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Chinese Hamster Ovary Cells ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Cell Surface Expression ◽  
Beta Chain ◽  
Single Chain ◽  
Beta3 Integrin

Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756–759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

2001 ◽  
Vol 358 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Philippe ULSEMER ◽  
Catherine STRASSEL ◽  
Marie-Jeanne BAAS ◽  
Jean SALAMERO ◽  
Sylvette CHASSEROT-GOLAZ ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Ovary Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Vessel Injury ◽  
Von Willebrand

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

scholarly journals Possible domains responsible for intracellular targeting and insulin-dependent translocation of glucose transporter type 4

1995 ◽  
Vol 309 (3) ◽  
pp. 813-823 ◽  
Author(s):  
K Ishii ◽  
H Hayashi ◽  
M Todaka ◽  
S Kamohara ◽  
F Kanai ◽  
...  
Keyword(s):  
Cell Surface ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Terminal Region ◽  
Target Cells ◽  
Glut4 Translocation ◽  
Intracellular Loop ◽  
Intact Cells ◽  
Intracellular Targeting

Translocation of the type 4 glucose transporter (GLUT4) to the cell surface from an intracellular pool is the major mechanism of insulin-stimulated glucose uptake in insulin-target cells. We developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We constructed c-myc epitope-tagged glucose transporter type 1 (GLUT1myc) and found that the GLUT1myc was also translocated to the cell surface of Chinese hamster ovary cells, 3T3-L1 fibroblasts and NIH 3T3 cells, in response to insulin, but the degree of translocation was less than that of GLUT4myc. Since GLUT1 and GLUT4 have different intracellular distributions and different degrees of insulin-stimulated translocation, we examined the domains of GLUT4, using c-myc epitope-tagged chimeric glucose transporters between these two isoforms. The results indicated that, (1) all the cytoplasmic N-terminal region, middle intracellular loop and cytoplasmic C-terminal region of GLUT4 have independent intracellular targeting signals, (2) these sequences for intracellular targeting of GLUT4 were not sufficient to determine GLUT4 translocation in response to insulin, and (3) the N-terminal half of GLUT4 devoid both of cytoplasmic N-terminus and of middle intracellular loop seems to be necessary for insulin-stimulated GLUT4 translocation.

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