Signal transduction by glycophorin A: role of extracellular and cytoplasmic domains in a modulatable process.

Binding of ligands to the extracellular region of the erythrocyte transmembrane protein glycophorin A induces a decrease in membrane deformability. Since the property of membrane deformability is regulated by the skeletal proteins on the cytoplasmic side of the membrane, this suggests that ligand binding may initiate a transmembrane signal. To further study this process, we examined which domains of the extracellular region of glycophorin are involved in signal transduction and whether the cytoplasmic domain of the molecule is necessary for transmitting the signal. Using the ektacytometer, we compared the effect on deformability of four monoclonal antibodies that detect different epitopes on glycophorin A. We found that 9A3 (which recognized the amino terminus of glycophorin) caused a 5.8-fold increase in rigidity, R-10 and 10F7 (which recognized epitopes in the mid-region of the extracellular domain) caused a 10.8-fold increase in rigidity and B14 (which binds to glycophorin close to the membrane) caused a 18-fold increase in rigidity. Further, a direct relationship was observed between the degree of antibody-induced rigidity and the amount of glycophorin A that became associated with the skeletal proteins in a Triton shell assay. In Miltenberger V erythrocytes, which contain a hybrid sialoglycoprotein with no cytoplasmic domain, antibody binding did not induce an increase in rigidity. These results imply that glycophorin A is capable of a modulatable form of transmembrane signaling that is determined by the extracellular domain to which the ligand binds, and the cytoplasmic domain of glycophorin A is crucial for this process.

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Lateral diffusion of wild-type and mutant Ld antigens in L cells.

The Journal of Cell Biology ◽

10.1083/jcb.99.6.2333 ◽

1984 ◽

Vol 99 (6) ◽

pp. 2333-2335 ◽

Cited By ~ 31

Author(s):

M Edidin ◽

M Zuniga

Keyword(s):

Plasma Membrane ◽

Diffusion Coefficients ◽

Transmembrane Protein ◽

Transmembrane Proteins ◽

Lateral Diffusion ◽

Cytoplasmic Domain ◽

Histocompatibility Antigen ◽

Wild Type ◽

Major Histocompatibility Antigen ◽

Cytoplasmic Side

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

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Regulation of chemotaxis by the cytoplasmic domain of tissue factor

Thrombosis and Haemostasis ◽

10.1160/th04-07-0405 ◽

2005 ◽

Vol 93 (01) ◽

pp. 27-34 ◽

Cited By ~ 23

Author(s):

Matilda Johnell ◽

Brit Sorensen ◽

Lars Petersen ◽

Carl-Henrik Heldin ◽

Agneta Siegbahn

Keyword(s):

Signal Transduction ◽

Tissue Factor ◽

Intracellular Signaling ◽

Signal Transduction Pathway ◽

Cytoplasmic Domain ◽

Cellular Migration ◽

Boyden Chamber ◽

Transduction Pathway ◽

Low Levels

SummaryWe previously demonstrated that FVIIa bound to tissue factor (TF) induces a hyperchemotactic response towards PDGF-BB. The aim of the present study was to investigate the role of the cytoplasmic domain of TF in cell migration. Porcine aortic endothelial (PAE) cells expressing human PDGF β -receptors (PAE/ PDGFR β ) were transfected for expression of human wildtype TF (PAE/PDGFRβ ,TF), a construct lacking the cytoplasmic domain (PAE/PDGFRβ ,TF ∆ cyto), a construct with alanine replacement of serine 258 (PAE/PDGFRβ ,TFS258A), or a construct with alanine replacement of serine 253, 258 and 263 in the cytoplasmic domain (PAE/PDGFRβ ,TF3SA). All stably transfected cell lines expressed functional TF. Chemotaxis was analyzed in a modified Boyden chamber assay. PAE/PDGFRβ ,TF cells stinulated with FVIIa migrated towards a 100-fold lower concentration of PDGF-BB than in the absence of FVIIa,however,hyperchemotaxis was not seen in PAE/PDGFRβ ,TF ∆ cyto cells. PAE/ PDGFR β /TFS258A and PAE/PDGFRβ ,TF3SA cells responded to low levels of PDGF-BB, but migrated a significantly shorter distance than PAE/PDGFRβ ,TF cells. Thus, hyperchemotaxis towards PDGF-BB is likely to depend in part on phosphorylation of the cytoplasmic domain of TF.We conclude that the cytoplasmic domain of TF plays a pivotal role in modulating cellular migration response.Our results suggest that the FVIIa/TF complex mediates intracellular signaling by alternative signal transduction pathway(s).

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Role of the carboxyl-terminal half of the extracellular domain of the human thyrotropin receptor in signal transduction

Endocrinology ◽

10.1210/en.131.2.548 ◽

1992 ◽

Vol 131 (2) ◽

pp. 548-552 ◽

Cited By ~ 9

Author(s):

Y. Nagayama

Keyword(s):

Signal Transduction ◽

Extracellular Domain ◽

Thyrotropin Receptor ◽

Carboxyl Terminal

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Role of Cytoplasmic Domain Serines in Intracellular Trafficking of Furin

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0653 ◽

2004 ◽

Vol 15 (6) ◽

pp. 2884-2894 ◽

Cited By ~ 27

Author(s):

Florencia B. Schapiro ◽

Thwe Thwe Soe ◽

William G. Mallet ◽

Frederick R. Maxfield

Keyword(s):

Plasma Membrane ◽

Steady State ◽

Transmembrane Protein ◽

Interleukin 2 ◽

Chimeric Protein ◽

Cytoplasmic Domain ◽

Serine Phosphorylation ◽

Endocytic Recycling ◽

Late Endosomes

Furin is a transmembrane protein that cycles between the plasma membrane, endosomes, and the trans-Golgi network, maintaining a predominant distribution in the latter. It has been shown previously that Tac-furin, a chimeric protein expressing the extracellular and transmembrane domains of the interleukin-2 receptor α chain (Tac) and the cytoplasmic domain of furin, is delivered from the plasma membrane to the TGN through late endosomes, bypassing the endocytic recycling compartment. Tac-furin also recycles in a loop between the TGN and late endosomes. Localization of furin to the TGN is modulated by a six-amino acid acidic cluster that contains two phosphorylatable serines (SDSEED). We investigated the role of these serines in the trafficking of Tac-furin by using a mutant chimera in which the SDS sequence was replaced by the nonphosphorylatable sequence ADA (Tac-furin/ADA). Although the mutant construct is internalized and delivered to the TGN, both the postendocytic trafficking and the steady-state distribution were found to differ from the wild-type. In contrast with Tac-furin, Tac-furin/ADA does not enter late endosomes after being internalized. Instead, it traffics with transferrin to the endocytic recycling compartment, and from there it is delivered to the TGN. As with Tac-furin, Tac-furin/ADA is sorted from the TGN into late endosomes at steady state, but its retrieval from the late endosomes to the TGN is inhibited. These results suggest that serine phosphorylation plays an important role in at least two steps of Tac-furin trafficking, acting as an active sorting signal that mediates the selective sorting of Tac-furin into late endosomes after internalization, as well as its retrieval from late endosomes back to the TGN.

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CRB3 Binds Directly to Par6 and Regulates the Morphogenesis of the Tight Junctions in Mammalian Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e03-04-0235 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1324-1333 ◽

Cited By ~ 190

Author(s):

Céline Lemmers ◽

Didier Michel ◽

Lydie Lane-Guermonprez ◽

Marie-Hélène Delgrossi ◽

Emmanuelle Médina ◽

...

Keyword(s):

Epithelial Cells ◽

Tight Junction ◽

Tight Junctions ◽

Transmembrane Protein ◽

Direct Interaction ◽

Extracellular Domain ◽

Cytoplasmic Domain ◽

Epithelial Morphogenesis ◽

Neurotrophin Receptor ◽

Tight Junction Formation

Crumbs is an apical transmembrane protein crucial for epithelial morphogenesis in Drosophila melanogaster embryos. A protein with all the characteristics for a Crumbs homologue has been identified from patients suffering from retinitis pigmentosa group 12, but this protein (CRB1) is only expressed in retina and some parts of the brain, both in human and mouse. Here, we describe CRB3, another Crumbs homologue that is preferentially expressed in epithelial tissues and skeletal muscles in human. CRB3 shares the conserved cytoplasmic domain with other Crumbs but exhibits a very short extracellular domain without the EGF- and laminin A-like G repeats present in the other Crumbs. CRB3 is localized to the apical and subapical area of epithelial cells from the mouse and human intestine, suggesting that it could play a role in epithelial morphogenesis. Indeed, expression of CRB3 or of a chimera containing the extracellular domain of the neurotrophin receptor p75NTR and the transmembrane and cytoplasmic domains of CRB3 led to a slower development of functional tight junctions in Madin-Darby canine kidney cells. This phenotype relied on the presence of CRB3 four last amino acids (ERLI) that are involved in a direct interaction with Par6, a regulator of epithelial polarity and tight junction formation. Thus, CRB3, through its cytoplasmic domain and its interactors, plays a role in apical membrane morphogenesis and tight junction regulation.

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Promotion of Neurite and Filopodium Formation by CD47: Roles of Integrins, Rac, and Cdc42

Molecular Biology of the Cell ◽

10.1091/mbc.e04-01-0019 ◽

2004 ◽

Vol 15 (8) ◽

pp. 3950-3963 ◽

Cited By ~ 63

Author(s):

Motoaki Miyashita ◽

Hiroshi Ohnishi ◽

Hideki Okazawa ◽

Hiroyasu Tomonaga ◽

Akiko Hayashi ◽

...

Keyword(s):

Transmembrane Protein ◽

Neuroblastoma Cells ◽

Axon Extension ◽

Entire Structure ◽

Signaling Mechanisms ◽

Extracellular Region ◽

Fc Fusion Protein ◽

Neurite Formation ◽

Filopodium Formation

Axon extension during development is guided by many factors, but the signaling mechanisms responsible for its regulation remain largely unknown. We have now investigated the role of the transmembrane protein CD47 in this process in N1E-115 neuroblastoma cells. Forced expression of CD47 induced the formation of neurites and filopodia. Furthermore, an Fc fusion protein containing the extracellular region of the CD47 ligand SHPS-1 induced filopodium formation, and this effect was enhanced by CD47 overexpression. SHPS-1–Fc also promoted neurite and filopodium formation triggered by serum deprivation. Inhibition of Rac or Cdc42 preferentially blocked CD47-induced formation of neurites and filopodia, respectively. Overexpression of CD47 resulted in the activation of both Rac and Cdc42. The extracellular region of CD47 was sufficient for the induction of neurite formation by forced expression, but the entire structure of CD47 was required for enhancement of filopodium formation by SHPS-1–Fc. Neurite formation induced by CD47 was also inhibited by a mAb to the integrin β3 subunit. These results indicate that the interaction of SHPS-1 with CD47 promotes neurite and filopodium formation through the activation of Rac and Cdc42, and that integrins containing the β3 subunit participate in the effect of CD47 on neurite formation.

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Modulation of Env Content in Virions of Simian Immunodeficiency Virus: Correlation with Cell Surface Expression and Virion Infectivity

Journal of Virology ◽

10.1128/jvi.78.13.6775-6785.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 6775-6785 ◽

Cited By ~ 73

Author(s):

Eloísa Yuste ◽

Jacqueline D. Reeves ◽

Robert W. Doms ◽

Ronald C. Desrosiers

Keyword(s):

Cell Surface ◽

Simian Immunodeficiency Virus ◽

Stop Codon ◽

Transmembrane Protein ◽

Fold Increase ◽

Surface Expression ◽

Cytoplasmic Domain ◽

Cell Surface Expression ◽

Molar Ratio ◽

Immunodeficiency Virus

ABSTRACT Specific mutations were created in the cytoplasmic domain of the gp41 transmembrane protein of simian immunodeficiency virus strain 239 (SIV239). The resultant strains included a mutant in which Env residue 767 was changed to a stop codon, a double mutant in which positions 738 and 739 were changed to stop codons, another mutant in which a prominent endocytosis motif was changed from YRPV to GRPV by the substitution of tyrosine 721, and a final combination mutant bearing Q738stop, Q739stop, and Y721G mutations. The effects of these mutations on cell surface expression, on Env incorporation into virions, and on viral infectivity were examined. The molar ratio of Gag to gp120 of 54:1 that we report here for SIV239 virions agrees very well with the ratio of 60:1 reported previously by Chertova et al. (E. Chertova, J. W. Bess, Jr., B. J. Crise, R. C. Sowder II, T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur, J. Virol. 76:5315-5325, 2002), although they were determined by very different methodologies. Assuming 1,200 to 2,500 Gag molecules per virion, this corresponds to 7 to 16 Env trimers per SIV239 virion particle. Although all of the mutations increased Env levels in virions, E767stop had the most dramatic effect, increasing the Env content per virion 25- to 50-fold. Increased levels of Env content in virions correlated strictly with higher levels of Env expression on the cell surface. The increased Env content with the E767stop mutation also correlated with an increased infectivity, but the degree of change was not proportional: the 25- to 50-fold increase in Env content only increased infectivity 2- to 3-fold. All of the mutants replicated efficiently in the CEMx174 and Rh221-89 cell lines. Although some of these findings have been reported previously, our findings show that the effects of the cytoplasmic domain of gp41 on the Env content in virions can be dramatic, that the Env content in virions correlates strictly with the levels of cell surface expression, and that the Env content in virions can determine infectivity; furthermore, our results define a particular change with the most dramatic effects.

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Role of the adapter protein SLP-76 in GPVI-dependent platelet procoagulant responses to collagen

Blood ◽

10.1182/blood-2002-04-1234 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2839-2844 ◽

Cited By ~ 17

Author(s):

Lorie Leo ◽

Jorge Di Paola ◽

Barbi A. Judd ◽

Gary A. Koretzky ◽

Steven R. Lentz

Keyword(s):

Signal Transduction ◽

Signal Transduction Pathway ◽

Annexin V ◽

Fold Increase ◽

Surface Expression ◽

Procoagulant Activity ◽

Adapter Protein ◽

Glycoprotein Vi ◽

Granule Release

The adapter protein SLP-76 is a critical mediator of signal transduction via the platelet collagen receptor glycoprotein VI (GPVI) and its coreceptor FcRγ. We tested the hypothesis that SLP-76 is required for collagen-induced procoagulant responses in murine platelets. Platelets from SLP-76 null (SLP-76−/−) or heterozygous (SLP-76+/−) mice were activated with the GPVI agonist convulxin, and surface expression of P-selectin (a marker of granule release) and annexin V binding (a marker of procoagulant phospholipid) were determined by flow cytometry. Convulxin induced surface expression of P-selectin in SLP-76+/− platelets, but not SLP-76−/− platelets (P < .01), and failed to stimulate annexin V binding to either SLP-76+/−or SLP-76−/− platelets. Platelet procoagulant activity was measured in a prothrombinase assay. Convulxin did not stimulate procoagulant activity in either SLP-76+/− or SLP-76−/− platelets, but fibrillar collagen produced a 1.9-fold increase in procoagulant activity in both SLP-76+/− and SLP-76−/− platelets (P < .001 versus unstimulated platelets). Similar results were obtained with platelets from FcRγ null mice, for which collagen, but not convulxin, induced procoagulant activity (P < .01). Costimulation with thrombin and collagen produced a further (2.3-fold) increase in procoagulant activity in SLP-76+/− platelets (P < .05), but not in SLP-76−/− platelets. SLP-76−/− platelets also exhibited less annexin V binding than SLP-76+/−platelets after costimulation with thrombin and convulxin (P < .05). These findings demonstrate that an intact GPVI/FcRγ/SLP-76 signal transduction pathway is not essential for platelet procoagulant activity induced by collagen but is necessary for maximal procoagulant response to costimulation with thrombin plus collagen. Thus, both GPVI-dependent and GPVI-independent pathways contribute to collagen-induced platelet procoagulant activity.

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The Characterization of Microparticle Phenotypes in Stored Red Blood Cell Units.

Blood ◽

10.1182/blood.v120.21.2282.2282 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2282-2282

Author(s):

Edward D Tran ◽

Xiaoping Wu ◽

Grady R. Blacken ◽

Jose A. Lopez ◽

Jing-fei Dong ◽

...

Keyword(s):

Tissue Factor ◽

Blood Cells ◽

Annexin V ◽

Extracellular Domain ◽

Cytoplasmic Domain ◽

Band 3 ◽

Glycophorin A ◽

Exact Nature ◽

Domain Specific ◽

Inside Out

Abstract Abstract 2282 The transfusion of red blood cells (RBC) is associated with adverse outcomes in critically ill patients. One hypothesis explaining this observation involves the accumulation of potentially inflammatory/prothrombotic microparticles (MPs), released from cellular membranes during unit processing and storage. Despite their implication, the exact nature of MPs within RBC units remains incompletely defined. Here, we characterized the MP phenotypes present within stored RBC units and quantified the various populations. We directly sampled 11 O-positive non-leukoreduced RBC unit bags and isolated MPs by sequential centrifugation. We used a two-step process to detect MPs by flow cytometry. First, we used commercially available Megamix beads to define MPs according to their size during flow cytometric analysis and Trucount beads to quantify MP counts/mL (reported as median [IQR]). MPs ≤1 μm in size were subsequently classified into the following populations: total annexin V-positive, tissue factor-positive (CD142), and those staining for RBC- (intra/extracellular domain-specific glycophorin A, and band-3), leukocyte- (CD45), platelet- (CD42b), and endothelial-specific (CD144) markers. The total concentration of MPs was 1.52×109 MPs/mL [1.39×109]; however, only 4.3% were Annexin V-positive (5.16×107 MPs/mL [7.27×107]), and 11.7% were tissue factor-positive (4.24×107 MPs/mL [1.11×108]). Likewise, MPs with leukocyte-, platelet-, and endothelial-specific markers accounted for only 0.05% (4.44×105 MPs/mL [7.83×105]), 0.17% (1.99×106 MPs/mL [3.11×106]), and 0.06% (7.58×105 MPs/mL [8.04×105]), respectively (Figure 1). The amount of RBC-derived MPs varied depending on the antibody used. While only 4.2% of MPs (3.40×107 MPs/mL [2.91×107]) stained positive for the extracellular domain of glycophorin A, 41.3% (8.20×108 MPs/mL [4.06×108]) and 20.6% MPs (3.03×108 MPs/mL [3.53×108]) stained positive for the cytoplasmic domain of glycophorin A and band-3 respectively (Figure 1). The concentration of MPs positive for the cytoplasmic domain of glycophorin A was significantly greater their extracellular domain-specific counterparts (p<0.01). The results indicate that MPs in stored RBC units are primarily derived from RBCs, but also from other blood cells and vascular endothelial cells. A strong stain for the cytoplasmic domains of two RBC markers (cytoplasmic domain-specific glycophorin A and band 3) suggest the presence of a unique inside-out MP phenotype, potentially derived by RBC membrane “flipping” during microvesiculation. Ongoing studies are required to confirm these results and focus on 1) the mechanism of MP formation and (patho)physiological significance of inside-out RBC MPs and 2), the association between these MPs and the adverse effects seen in patients receiving RBC transfusions. Disclosures: No relevant conflicts of interest to declare.

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