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Biomedical subjects

E Mekada

Publications and source records attributed to E Mekada.

At least 37 records · Page 2Linked to original sources
PubMed2000

Integrin alpha 2 beta 1-dependent EGF receptor activation at cell-cell contact sites.

Certain integrins including alpha 2 beta 1 and alpha 3 beta 1 localize to intercellular binding sites, and thus may participate in cell-cell interactions. We demonstrated here the physical and functional associations of integrin alpha 2 beta 1 with epidermal growth factor receptor (EGFR) at intercellular adhesion sites. Immunoprecipitation with anti-integrin alpha 2 antibodies or anti-integrin beta 1 antibody resulted in preferential coprecipitation of EGFR from A431 cell lysates, while anti-EGFR antibody coprecipitated integrin alpha 2 beta 1. Chemical crosslinking confirmed the association of integrin alpha 2 beta 1 and EGFR. Colocalization of integrin alpha 2 beta 1 and EGFR at cell-cell contact sites was observed by double immunofluorescence staining of A431 cells. EGF-induced EGFR stimulation did not affect the association of integrin alpha 2 beta 1 and EGFR. However, immunostaining with the antibody specific to activated-EGFR revealed that EGFR localized at cell-cell contact sites are phosphorylated even in serum-depleted conditions, while EGFR localized to other sites is totally dephosphorylated in the same conditions. The EGFR phosphorylation in cell-cell contact sites observed in a serum-depleted culture was abrogated with a function-blocking antibody of integrin alpha 2, but not with a non-function-blocking alpha 2 antibody or function-blocking alpha 3 antibody. Moreover, the EGFR phosphorylation in serum-depleted conditions was not observed in suspended cells, or largely abrogated in sparse cells, indicating that cell-cell adhesion is required for EGFR phosphorylation. These results indicate that integrin alpha 2 beta 1 not only physically associates with EGFR but also functions in serum-independent EGFR activation at cell-cell contact sites. The present results shed a new light on the role of intercellular integrins in cell-cell interactions.

Cell Communication↗
PubMed2000

Domain analysis of the tetraspanins: studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding.

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.

Animals↗
PubMed1999

Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) transduces mitogenic signals through the EGF receptor (EGFR). There are two forms of HB-EGF, the membrane-anchored form (pro-HB-EGF) and the soluble form (sHB-EGF). We studied the biological activity of pro-HB-EGF by using a model in which pro-HB-EGF-expressing effector cells was co-cultured with EGFR-expressing target cells. The DER cell, an EGFR-expressing derivative of the interleukin-3-dependent hematopoietic 32D cell line, grows well in the presence of EGF or sHB-EGF without IL-3. When DER cells were co-cultured on a monolayer of Vero-H cells overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis were induced in the DER cells even in the presence of excess amounts of EGF or sHB-EGF. Such growth inhibition of DER cells was abrogated when specific antagonists for pro-HB-EGF were added in the culture medium or when direct contact of DER cells with Vero-H cells was prevented, indicating that pro-HB-EGF is involved in this inhibitory effect. Pro-HB-EGF-induced apoptosis of DER cells was also observed even in the presence of IL-3. This rules out the possibility of simple competition between soluble EGFR ligands and pro-HB-EGF. Moreover, 32D cells expressing EGFR mutant composed of the extracellular and the transmembrane domain of EGFR and the cytoplasmic domain of erythropoietin receptor did not undergo apoptosis by co-culture with Vero-H cells, indicating that the inhibitory signal induced by pro-HB-EGF-expressing Vero-H cells is mediated to DER cells via EGFR and that the cytoplasmic domain of EGFR is essential for pro-HB-EGF-induced apoptosis. From these results, we concluded that pro-HB-EGF has unique biological activity through cell-cell contact that is distinct from the activity of sHB-EGF.

Animals↗
PubMed1998

A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor.

The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is the heparin-binding EGF-like growth factor (HB-EGF) that exists in a membrane-anchored form which is converted to a soluble form upon cell stimulation with TPA, an activator of protein kinase C (PKC). We show that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta, suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the proHB-EGF ectodomain.

ADAM Proteins↗
PubMed1998

Diphtheria toxin translocation across endosome membranes. A novel cell permeabilization assay reveals new diphtheria toxin fragments in endocytic vesicles.

By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADP-ribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.

Animals↗
PubMed1998

N-myc transactivates RCC1 gene expression in rat fibroblast cells transformed by N-myc and v-ras.

Oncogenic cooperation was found between the N-myc and v-ras oncogenes in a rat fibroblast cell line, 3Y1. To investigate the specific role of N-myc in the transformation, we established transformed cell lines that expressed N-myc under a controllable promoter. Using these cells, we found that constitutive expression of N-myc is necessary to maintain the transformation, and that the expression level of N-myc is closely correlated with the transformation. Since another myc family gene, c-myc, directly activates expression of RCC1, which has important functions for eukaryotic cell proliferation, we focused on the relationship between N-myc and RCC1. Cells transformed by N-myc and v-ras expressed several times more RCC1 mRNA than the parent 3Y1 cells, and the expression of RCC1 changed in a parallel with the expression of N-myc. Gel retardation analysis and experiments with reporter plasmids constructed from a DNA fragment of the RCC1 gene indicated that the N-Myc protein controls expression of RCC1 by binding directly to CACGTG elements in the RCC1 gene. These results suggest that N-myc can directly transactivate expression of RCC1, a c-myc target gene.

Animals↗
PubMed1997

Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis.

Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.

Amino Acid Sequence↗
PubMed1997

Immune system-related CD9 is expressed in mouse central nervous system myelin at a very late stage of myelination.

CD9 is a tetra-membrane-spanning glycoprotein involved in cell adhesion, migration, and proliferation in both the immune and the immature nervous system. In this study, CD9 expression was detected in myelin of mouse brain, starting at postnatal day 16. The amount of CD9 protein continuously increased with age and persisted in the adult brain. It appeared later than myelin proteolipid protein (PLP), a typical late myelin marker. Mature oligodendrocytes were abundant in CD9, although it was also detected in astrocytes and microglial cells in vitro. CD9 appeared at the end of the myelination process and was localized along the outermost membrane of compact myelin. CD9 is known to associate with integrins, which are candidate receptors for extracellular matrix and transmit extracellular signals into the cells. Taken together, CD9 at the surface of central nervous system (CNS) mature myelin may have a unique function to facilitate signal transduction and enhance myelin membrane adhesion to extracellular matrices at very late stages of development.

Animals↗
PubMed1997

c-myc activates RCC1 gene expression through E-box elements.

Proto-oncogene c-myc is implicated in proliferation of mammalian cells. Although c-Myc protein has been demonstrated to function as a transcription factor recognizing an E-box (CACGTG) element, few c-myc-regulated genes have been identified and the specific role of c-myc is still unclear. RCC1 is necessary for mammalian cells to proliferate. Four CACGTG elements exist within 1.3 kb downstream of the major transcription start site for the human RCC1 gene in HeLa cells. Stimulation of HeLa cells with serum increased c-myc expression and RCC1 expression. Therefore the relationship between the expression of RCC1 and c-myc was investigated. Rat 3Y1 cells overexpressing c-myc contained about twice as much RCC1 mRNA as control cells. When a chimeric protein comprised of c-myc and the estrogen binding domain of estrogen receptor was activated by addition of 4-hydroxytamoxifen (OHT), expression of RCC1 mRNA increased twofold. To examine whether c-Myc functions through the CACGTG elements, a DNA fragment of RCC1 intron 4, exon 5 and part of intron 5 was joined to firefly luciferase cDNA to construct a reporter plasmid. In transient expression experiments using HeLa cells, co-transfection with c-myc stimulated the luciferase activity up to 2.5-fold in a dose-dependent manner. When the CACGTG elements in the reporter plasmid were destroyed, stimulation by c-myc was not observed. The four CACGTG elements did not contribute equally to the stimulation by c-myc. Gel retardation experiments suggest that c-Myc with Max binds to the CACGTG elements in the context of the RCC1 gene sequence in vitro. These results indicate that c-Myc can regulate expression of RCC1 through the E-box elements.

Animals↗
PubMed1996

Amino-terminal processing of cell surface heparin-binding epidermal growth factor-like growth factor up-regulates its juxtacrine but not its paracrine growth factor activity.

Human heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expressed on Chinese hamster ovary (CHO) cells is synthesized as a 19-kDa major, and 22- and 27-kDa minor, membrane-anchored precursors (proHB-EGF). In contrast, the 27-kDa species is major and the 19- and 22-kDa ones are minor in mouse proHB-EGF. The juxtacrine growth factor activities of human and mouse proHB-EGFs on CHO cells toward EP170.7 cells in co-culture are significantly different. To investigate the relationship between the juxtacrine growth factor activities and the molecular species, we prepared human-mouse chimeras. Chimeras that have the human amino-terminal sequence with a mouse EGF-like domain showed approximately 8-fold up-regulation of the juxtacrine growth factor activity and the predominance of a 19-22-kDa major species. In contrast, chimeras that have the mouse amino-terminal sequence with a human EGF-like domain showed approximately 5-fold down-regulation of the juxtacrine activity and the predominance of the 27-kDa major species. A Gly32.HB-EGF (117-amino acid form), which is amino-terminally extended, induced the same mitogenic activity as that of Arg73.HB-EGF (75-amino acid form), which is amino-terminally truncated. These results strongly suggested that amino-terminal processing of human proHB-EGF would be required for up-regulation of its juxtacrine growth factor activity, but not for its paracrine activity.

Amino Acid Sequence↗
PubMed1996

Expression and distribution of CD9 in myelin of the central and peripheral nervous systems.

CD9 is a member of the newly identified tetra-membrane-spanning protein family. We show here that CD9 is a constituent of myelin in the central and peripheral nervous systems. Expression of CD9 was detected in human cerebral white matter and sciatic nerve by Northern and Western blotting. Myelin in the central and peripheral nervous systems was strongly stained with a monoclonal antibody against human CD9 antigen in paraffin-embedded sections. CD9 was detected in adult nervous tissue but not in developing brain at less than 20 weeks of gestation. Immunohistochemical studies indicated that expression of CD9 is correlated with myelination and is somewhat delayed compared with expression of myelin basic protein, a major component protein of myelin. In the central nervous system, CD9 was detected along the outermost membrane of compact myelin but not inside compact myelin or the periaxonal region. Although the membrane-anchored form of heparin-binding epidermal-growth-factor-like growth factor (proHB-EGF), which is identical to the diphtheria toxin receptor, forms a complex with CD9 in some human and monkey cell lines, proHB-EGF was not detected in myelin immunocytochemically. The distribution of CD9 in the outer surface of myelin and its relatively late developmental appearance suggest that CD9 may interact with the extracellular matrix or cell adhesion molecules and participate in the maintenance of the entire myelin sheath.

Aging↗
PubMed1995

Heparin-like molecules on the cell surface potentiate binding of diphtheria toxin to the diphtheria toxin receptor/membrane-anchored heparin-binding epidermal growth factor-like growth factor.

Diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF), has a high affinity for heparin. We studied the effect of heparin-like molecules on the binding of diphtheria toxin (DT) to DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate (HS) proteoglycans were about 15 times less sensitive to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin was added to the culture medium, DT sensitivity of the mutant cells was fully restored. Studies of binding of 125I-labeled DT to HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA indicated that the increased sensitivity to DT after addition of heparin is due to increased binding of DT to cells. Vero cells display a relatively large amount of heparan sulfate residues compared to CHO-K1 cells or L cells. Enhancement of DT binding by the addition of heparin was also observed with CHO-K1 cells and L cells that had been transfected with human DTR/proHB-EGF cDNA, but the degree of enhancement was less than that observed with the HS-deficient CHO cells. Addition of heparin did not affect DT binding or DT sensitivity of Vero cells. Heparin-dependent binding was observed when intact Vero cells were treated with heparitinase or when the cell membrane was solubilized with a neutral detergent. Scatchard plot analysis for the binding of DT to a recombinant HB-EGF in vitro and to L cells expressing human DTR/proHB-EGF revealed that heparin increases the affinity of DTR/proHB-EGF for DT but does not change the number of binding sites. Although DRAP27/CD9 is known to enhance DT binding to DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9 increase DT binding by independent mechanisms. Thus, heparin-like molecules, probably in the form of heparan sulfate proteoglycan on the cell surface, are a third factor required for maximal DT binding activity of cells.

Animals↗
PubMed1995

Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.

Amino Acid Sequence↗
PubMed1995

The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-like growth factor.

The membrane-anchored heparin-binding EGF-like growth factor precursor (proHB-EGF)/diphtheria toxin receptor (DTR) belongs to a class of transmembrane growth factors and physically associates with CD9/DRAP27 which is also a transmembrane protein. To evaluate the biological activities of proHB-EGF/DTR as a juxtacrine growth factor and the biological significance of its association with CD9/DRAP27, the mitogenic activity of proHB-EGF/DTR was analyzed using stable transfectants of mouse L cells expressing both human proHB-EGF/DTR and monkey CD9/DRAP27, or either one alone. Juxtacrine activity was assayed by measuring the ability of cells in co-culture to stimulate DNA synthesis in an EGF receptor ligand dependent cell line, EP170.7. LH-2 cells expressing human proHB-EGF/DTR stimulated EP170.7 cell growth moderately. However, LCH-1 cells, a stable co-transfectant expressing both human proHB-EGF/DTR and monkey CD9/DRAP27 cDNAs, dramatically unregulated the juxtacrine growth factor activity of proHB-EGF/DTR approximately 25 times over that of LH-2 cells even though both cell types expressed similar levels of proHB-EGF/DTR on the cell surface. Anti-CD9/DRAP27 antibodies which were not able to neutralize the mitogenic activity of soluble HB-EGF suppressed LCH-1 cell juxtacrine growth activity to the same extent as did anti-HB-EGF neutralizing antibodies and CRM 197, specific inhibitors of human HG-EGF. These findings suggest that optimal expression of the juxtacrine growth activity of proHB-EGF/DTR requires co-expression of CD9/DRAP27. These studies also indicate that growth factor potentiation effects which have been observed previously for soluble growth factors also occurs at the level of cell surface associated growth factors.

Amino Acid Sequence↗
PubMed1995

Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin alpha 3 beta 1 at cell-cell contact sites.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family of growth factors, which interact with EGF receptor to exert mitogenic activity. The membrane-anchored form of HB-EGF, proHB-EGF, is biologically active, providing mitogenic stimulation to neighboring cells in a juxtacrine mode. ProHB-EGF forms a complex with diphtheria toxin receptor-associated protein (DRAP27)/CD9, a tetra membrane-spanning protein that upregulates the juxtacrine mitogenic activity of proHB-EGF. We explored whether other proteins associate with DRAP27/CD9 and proHB-EGF. Immunoprecipitation with anti-DRAP27/CD9 resulted in preferential coprecipitation of integrin alpha 3 beta 1 from Vero cell, A431 cell and MG63 cell lysates. Anti-integrin alpha 3 or anti-integrin beta 1 coprecipitated DRAP27/CD9 from the same cell lysates. Chemical cross-linking confirmed the physical association of DRAP27/CD9 and integrin alpha 3 beta 1. Using Vero-H cells, which overexpress HB-EGF, we also demonstrated the association of proHB-EGF with DRAP27/CD9 and integrin alpha 3 beta 1. Moreover, colocalization of proHB-EGF, DRAP27/CD9, and integrin alpha 3 beta 1 at cell-cell contact sites was observed by double-immunofluorescence staining. At cell-cell contact sites, DRAP27/CD9 was highly coincident with alpha-catenin and vinculin, suggesting that DRAP27/CD9, proHB-EGF, and integrin alpha 3 beta 1 are colocalized with adherence junction-locating proteins. These results indicate that direct interaction of growth factors and cell adhesion molecules may control cell proliferation during the cell-cell adhesion process.

Animals↗
PubMed1995

Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor: conversion from juxtacrine to paracrine growth factor activity.

Vero cell heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (proHB-EGF). Localization and processing of proHB-EGF, both constitutive and 12-O-tetradecanoylphorbol 13-acetate (TPA)-inducible, was examined in Vero cells overexpressing recombinant HB-EGF (Vero H cells). Flow cytometry and fluorescence immunostaining demonstrated that Vero cell proHB-EGF is cell surface-associated and localized at the interface of cell to cell contact. Cell surface biotinylation and immunoprecipitation detected a 20- to 30-kDa heterogeneous proHB-EGF species. Vero H cell surface proHB-EGF turned over constitutively with a half-life of 1.5 h. Some of the 20- to 30-kDa cell surface-associated proHB-EGF was processed and a 14-kDa species of bioactive HB-EGF was released slowly, but most of the proHB-EGF was internalized, displaying a diffuse immunofluorescent staining pattern and accumulation of proHB-EGF in endosomes. Addition of TPA induced a rapid processing of proHB-EGF at a Pro148-Val149 site with a half-life of 7min. The TPA effect was abrogated by the protein kinase C inhibitors, staurosporine and H7. Kinetic analysis showed that loss of cell surface proHB-EGF is maximal at 30 min after addition of TPA and that proHB-EGF is resynthesized and the initial cell surface levels are regained within 12-24 h. Loss of cell surface proHB-EGF was concomitant with appearance of 14- and 19-kDa soluble HB-EGF species in conditioned medium. Vero H cell-associated proHB-EGF is a juxtacrine growth factor for EP170.7 cells in coculture. Processing of proHB-EGF resulted in loss of juxtacrine activity and a simultaneous increase in soluble HB-EGF paracrine mitogenic activity. It was concluded that processing regulates HB-EGF bioactivity by converting it from a cell-surface juxtacrine growth factor to a processed, released soluble paracrine growth factor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗