Trudi Hengeveld - Academia.edu (original) (raw)
Papers by Trudi Hengeveld
Journal of Cell Biology, 1998
Gap junctions mediate cell–cell communication in almost all tissues, but little is known about th... more Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase ...
Current Biology, 2001
Gap junctions are specialized cell-cell junctions that Results and discussion Increasing evidence... more Gap junctions are specialized cell-cell junctions that Results and discussion Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. They are composed of connexin proteins, which form part of a multiprotein complex. For example, the c-Src and v-Src tyrosine kinases can bind directly to and phostransmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the phorylate the Cx43 C-terminal tail (CT) via SH2 and SH3 domain interactions [6, 7]. Furthermore, the very most widely expressed connexin member, has been implicated in the regulation of Cx43 channel C-terminal residues of Cx43 interact with the second PDZ domain of the Zona-Occludens-1 (ZO-1) protein, which gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little may recruit regulatory proteins into gap junctions [8, 9]. Thus, the Cx43 complex might fulfill functions that are is known about binding partners. To identify Cx43interacting proteins, we performed pull-down not necessarily related to the control of gap-junctional communication (GJC), a possibility that has received little experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 attention to date. tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding To identify new Cx43 binding proteins, lysates of 35 Sto Cx43 is specific in that it is not observed with Met/Cys-labeled Rat-1 cells were subjected to pull-down three other connexins. We established that a 35assays using the C-terminal tail of Cx43 fused to GST as amino acid juxtamembrane region in the Cx43 tail, an affinity matrix (GST-Cx43CT; Figure 1). Results of which contains a presumptive tubulin binding motif, a typical experiment are shown in Figure 2. The most is necessary and sufficient for microtubule binding. prominently labeled protein that interacts with Cx43CT, Immunofluorescence and immunoelectron but not with GST alone, has an apparent MW of about microscopy studies reveal that microtubules 55 kDa (Figure 2a). When analyzed by two-dimensional extend to Cx43-based gap junctions in contacted SDS-PAGE/iso-electric focusing, at least 12 distinct cells. However, intact microtubules are Cx43CT-interacting protein spots were detected, among dispensable for the regulation of Cx43 gapwhich was an intensely labeled 55-kDa spot (pI value junctional communication. Our findings suggest 4.8; Figure 2b). In large-scale pull-down experiments, we that, in addition to its well-established role as a detected a 55-kDa protein doublet on Coomassie-stained channel-forming protein, Cx43 can anchor gels that corresponds to the 35 S-labeled 55-kDa protein microtubule distal ends to gap junctions and thereby (Figure 2c). The lower 55-kDa band was subjected to might influence the properties of microtubules in tryptic digestion. Sequence analysis of three distinct pepcontacted cells. tides revealed that the protein is identical to rat -tubulin (Figure 2d). A trace amount of peptide was found to Addresses: *Division of Cellular Biochemistry and † Division of Cell represent ␣-tubulin. Since ␣-tubulin migrates slightly
The EMBO journal, Jan 15, 1996
Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytos... more Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through di...
The EMBO journal, Jan 2, 1996
Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membr... more Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membra...
The Journal of biological chemistry, Jan 5, 1993
Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitope... more Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common a...
Progress in Biophysics and Molecular Biology
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasm... more Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasma levels of low- and very-low-density lipoproteins, while high-density lipoproteins were strongly reduced. Changes in cholesteryl ester fatty acid profiles indicated an accumulation of HDL-like particles rather than LDL in the low-density fractions. By intravenous injection of [14C]cholesteryl ester-labeled HDL into tumor-bearing mice, conversion of HDL into lipoproteins of low density was demonstrated.
The Journal of Cell Biology, 1994
Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activat... more Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA-and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.
The Journal of Cell Biology, 1990
The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during bio... more The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfidelinked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and
The Journal of Cell Biology, 1998
Gap junctions mediate cell-cell communication in almost all tissues, but little is known about th... more Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is con-trolled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca 2+ , protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.
Journal of Biological Chemistry, 2001
Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by ce... more Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK ؉ ) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK ؉ and, to a lesser extent, with wild-type c-Src, but not with kinasedead c-Src. Mutation of residue Cx43 Tyr 265 (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK ؉ in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43⌬263) that lacks residue Tyr 265 . Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr 265 , resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.
FEBS Letters, 1992
The monoclonal antit~:~ly, MONO, recognizes an epitolm on the O protein ~o.subunit [van der Voom ... more The monoclonal antit~:~ly, MONO, recognizes an epitolm on the O protein ~o.subunit [van der Voom ¢t al,, submitted] and readily immunopr¢. edpitates heterotrimeric Go proteins from sotubilizad, crude bovine ~rain membrane, as ~.11 as from a purifmd bovine brain G pmt¢in pn:pamtion. Upon incubation of the immunoprocipitat~ with GTP),$, all ,aF.subunits arc released from the ao,.lubunit. Thus. binding of MONO to the Go protein do~ not apixar to interfere with r¢lr.asa or bound GDP, binding of GTPTS or GTP'/$.induced subtmit distociation. However, we have I~en unable to indue~ a similar dissociation of Go using its ph~iolot:ioal activator, GTP. Surprisingly. we did not obsarv¢ any dissociation of Go (bound to MONO) upon dilution in a range from :500 to S aM. $in~ an appar¢nt X.t of cto-GDP for binding/~7 of 340-390 aM has tame r¢lmrled [(1989) J. Biol. Chem. 264, 20688-206961 our results would sugg~t that binding of MONO to the ao-subunit ind~ an increatsed affinity of'~.GDP for.~y. Alternatively. th~,m r~ults could 1.~ oxplain~ if. under the conditions used, the K,~ of =o-G DP for.~F vatr¢ at I¢ast two orders of rna~nitud~ lower thole ~timatcd previously.
Journal of Neurobiology, 1999
... DOI: 10.1002/(SICI)1097-4695(199912)41:4<495::AID-NEU5>3.0.CO;2-K. Copyright © ... more ... DOI: 10.1002/(SICI)1097-4695(199912)41:4<495::AID-NEU5>3.0.CO;2-K. Copyright © 1999John Wiley & Sons, Inc. Issue. Journal of Neurobiology. ... Correspondence: Masayuki Yamashita, Nara Medical University, 840 Shijo-cho, Kashihara 634-8521, Japan. Publication History. ...
Current Biology, 2001
Loss of membrane potential (membrane as much as 50 mV due to activation of a Cl Ϫ conductance tha... more Loss of membrane potential (membrane as much as 50 mV due to activation of a Cl Ϫ conductance that is distinct from the Cl Ϫ channels regulated by cytodepolarization) is one of the earliest and most striking responses of quiescent cells to stimulation solic [Ca 2ϩ ], volume, or voltage [3]. Activation of the Cl Ϫ conductance is resistant to pertussis toxin; it correlates with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and with enhanced phosphoinositide hydrolysis and RhoAdependent f-actin remodeling [3], but causal relationships, thrombin [1-3]. Membrane depolarization is due to the activation of a chloride conductance [3]. While if there are any, are unknown. To dissect the underlying G protein pathway(s) and to gain insight into the physiothis response has received relatively little attention in the past, it is clear that the acute loss of membrane logical consequences of GPCR-induced membrane depolarization, we set out to examine the response in N1Epotential may have important physiological consequences [4-6]. However, the dissection of the 115 and NG105-18 neuronal cells. These cells express multiple G q -coupled receptors, while they show G 12/13 -
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane li... more GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane lipid fluidities of these cells and of normal lymphoid cells were estimated by fluorescence polarization, using the probe diphenylhexatriene and by measuring the (free) cholesterol/phospholipid molar ratio in whole cells. The results indicate that the membrane fluidity (reciprocal of the lipid structural order) of the lymphoma cells increases in the order of their location: peripheral blood less than spleen less than mesenterial lymph node less than ascites fluid. The membrane fluidities of normal lymphocytes from thymus, mesenterial lymph node and spleen were about the same, but higher than of peripheral blood lymphocytes, and between those of the lymphoma cells from lymph node and spleen. These results are confirmed by more extensive analysis on purified plasma membranes from the splenic and ascitic GRSL lymphoma cells and from normal splenocytes and thymocytes. The significantly higher lipid order parameter found in the GRSL plasma membrane isolated from the spleen as compared to those from the ascites cells could be fully explained by the differences measured in the major chemical determinants of the fluidity, i.e., the cholesterol/phospholipid ratio, the sphingomyelin content and the degree of saturation of the fatty acyl groups of the phospholipids. It was also found that the cholesterol/phospholipid ratio in erythrocyte membranes isolated from the peripheral blood of the tumor bearers was higher than in those from normal control mice. The observed differences in membrane fluidity between distinct subsets of tumor cells may be relevant to the sensitivity of these cells to immune attack or to drugs.
Current Biology, 2001
Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. Th... more Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. They are composed of connexin proteins, which form part of a multiprotein complex. For example, the c-Src and v-Src tyrosine kinases can bind directly to and phostransmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the phorylate the Cx43 C-terminal tail (CT) via SH2 and SH3 domain interactions [6, 7]. Furthermore, the very most widely expressed connexin member, has been implicated in the regulation of Cx43 channel C-terminal residues of Cx43 interact with the second PDZ domain of the Zona-Occludens-1 (ZO-1) protein, which gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little may recruit regulatory proteins into gap junctions [8, 9]. Thus, the Cx43 complex might fulfill functions that are is known about binding partners. To identify Cx43interacting proteins, we performed pull-down not necessarily related to the control of gap-junctional communication (GJC), a possibility that has received little experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 attention to date. tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding To identify new Cx43 binding proteins, lysates of 35 S-
Journal of Cell Biology, 1998
Gap junctions mediate cell–cell communication in almost all tissues, but little is known about th... more Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase ...
Current Biology, 2001
Gap junctions are specialized cell-cell junctions that Results and discussion Increasing evidence... more Gap junctions are specialized cell-cell junctions that Results and discussion Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. They are composed of connexin proteins, which form part of a multiprotein complex. For example, the c-Src and v-Src tyrosine kinases can bind directly to and phostransmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the phorylate the Cx43 C-terminal tail (CT) via SH2 and SH3 domain interactions [6, 7]. Furthermore, the very most widely expressed connexin member, has been implicated in the regulation of Cx43 channel C-terminal residues of Cx43 interact with the second PDZ domain of the Zona-Occludens-1 (ZO-1) protein, which gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little may recruit regulatory proteins into gap junctions [8, 9]. Thus, the Cx43 complex might fulfill functions that are is known about binding partners. To identify Cx43interacting proteins, we performed pull-down not necessarily related to the control of gap-junctional communication (GJC), a possibility that has received little experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 attention to date. tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding To identify new Cx43 binding proteins, lysates of 35 Sto Cx43 is specific in that it is not observed with Met/Cys-labeled Rat-1 cells were subjected to pull-down three other connexins. We established that a 35assays using the C-terminal tail of Cx43 fused to GST as amino acid juxtamembrane region in the Cx43 tail, an affinity matrix (GST-Cx43CT; Figure 1). Results of which contains a presumptive tubulin binding motif, a typical experiment are shown in Figure 2. The most is necessary and sufficient for microtubule binding. prominently labeled protein that interacts with Cx43CT, Immunofluorescence and immunoelectron but not with GST alone, has an apparent MW of about microscopy studies reveal that microtubules 55 kDa (Figure 2a). When analyzed by two-dimensional extend to Cx43-based gap junctions in contacted SDS-PAGE/iso-electric focusing, at least 12 distinct cells. However, intact microtubules are Cx43CT-interacting protein spots were detected, among dispensable for the regulation of Cx43 gapwhich was an intensely labeled 55-kDa spot (pI value junctional communication. Our findings suggest 4.8; Figure 2b). In large-scale pull-down experiments, we that, in addition to its well-established role as a detected a 55-kDa protein doublet on Coomassie-stained channel-forming protein, Cx43 can anchor gels that corresponds to the 35 S-labeled 55-kDa protein microtubule distal ends to gap junctions and thereby (Figure 2c). The lower 55-kDa band was subjected to might influence the properties of microtubules in tryptic digestion. Sequence analysis of three distinct pepcontacted cells. tides revealed that the protein is identical to rat -tubulin (Figure 2d). A trace amount of peptide was found to Addresses: *Division of Cellular Biochemistry and † Division of Cell represent ␣-tubulin. Since ␣-tubulin migrates slightly
The EMBO journal, Jan 15, 1996
Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytos... more Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through di...
The EMBO journal, Jan 2, 1996
Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membr... more Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membra...
The Journal of biological chemistry, Jan 5, 1993
Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitope... more Monoclonal antibodies were raised against purified bovine and human brain G-proteins. The epitopes recognized by three monoclonal antibodies (MONO, 3C2, and 3E7) were mapped by expressing defined parts of the bovine G(o) alpha-cDNA in bacteria, followed by immunoblotting. All three antibodies recognize the recombinant bovine alpha o-protein, but at distinct sites. The epitopes of MONO and 3C2 were mapped between alpha o amino acids 80 and 145, and both antibodies recognize alpha o exclusively. Heterotrimeric G(o)-proteins as well as guanosine 5'-3-O-(thio)triphosphate-liganded free alpha o-subunits are readily immunoprecipitated by these monoclonal antibodies. Binding of MONO or 3C2 does not affect ADP-ribosylation of the alpha o-subunit by pertussis toxin. Apparently, the antibodies do not bind to or induce large conformational changes in regions of the alpha o-subunit that are involved in association with beta gamma-subunits or ADP-ribosylation. 3E7 behaves as an anti common a...
Progress in Biophysics and Molecular Biology
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1985
Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasm... more Outgrowth of the transplanted GRSL lymphoma in GR mice yielded several-fold increased blood plasma levels of low- and very-low-density lipoproteins, while high-density lipoproteins were strongly reduced. Changes in cholesteryl ester fatty acid profiles indicated an accumulation of HDL-like particles rather than LDL in the low-density fractions. By intravenous injection of [14C]cholesteryl ester-labeled HDL into tumor-bearing mice, conversion of HDL into lipoproteins of low density was demonstrated.
The Journal of Cell Biology, 1994
Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activat... more Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA-and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.
The Journal of Cell Biology, 1990
The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during bio... more The interactions between transferrin (Tf) and transferrin receptor (Tfr) as they occur during biosynthesis were studied in the human hepatoma cell line HepG2, which synthesizes both. Early during biosynthesis the Tfr monomer is converted to a disulfidelinked Tfr dimer. The Tfr monomer is not able to bind Tf, but Tf binding is observed as soon as the covalent Tfr dimer is formed and can take place in the ER. The Tf-Tfr complex is transported through the Golgi reticulum and trans-Golgi reticulum (TGR) and
The Journal of Cell Biology, 1998
Gap junctions mediate cell-cell communication in almost all tissues, but little is known about th... more Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is con-trolled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca 2+ , protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.
Journal of Biological Chemistry, 2001
Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by ce... more Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK ؉ ) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK ؉ and, to a lesser extent, with wild-type c-Src, but not with kinasedead c-Src. Mutation of residue Cx43 Tyr 265 (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK ؉ in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43⌬263) that lacks residue Tyr 265 . Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr 265 , resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.
FEBS Letters, 1992
The monoclonal antit~:~ly, MONO, recognizes an epitolm on the O protein ~o.subunit [van der Voom ... more The monoclonal antit~:~ly, MONO, recognizes an epitolm on the O protein ~o.subunit [van der Voom ¢t al,, submitted] and readily immunopr¢. edpitates heterotrimeric Go proteins from sotubilizad, crude bovine ~rain membrane, as ~.11 as from a purifmd bovine brain G pmt¢in pn:pamtion. Upon incubation of the immunoprocipitat~ with GTP),$, all ,aF.subunits arc released from the ao,.lubunit. Thus. binding of MONO to the Go protein do~ not apixar to interfere with r¢lr.asa or bound GDP, binding of GTPTS or GTP'/$.induced subtmit distociation. However, we have I~en unable to indue~ a similar dissociation of Go using its ph~iolot:ioal activator, GTP. Surprisingly. we did not obsarv¢ any dissociation of Go (bound to MONO) upon dilution in a range from :500 to S aM. $in~ an appar¢nt X.t of cto-GDP for binding/~7 of 340-390 aM has tame r¢lmrled [(1989) J. Biol. Chem. 264, 20688-206961 our results would sugg~t that binding of MONO to the ao-subunit ind~ an increatsed affinity of'~.GDP for.~y. Alternatively. th~,m r~ults could 1.~ oxplain~ if. under the conditions used, the K,~ of =o-G DP for.~F vatr¢ at I¢ast two orders of rna~nitud~ lower thole ~timatcd previously.
Journal of Neurobiology, 1999
... DOI: 10.1002/(SICI)1097-4695(199912)41:4<495::AID-NEU5>3.0.CO;2-K. Copyright © ... more ... DOI: 10.1002/(SICI)1097-4695(199912)41:4<495::AID-NEU5>3.0.CO;2-K. Copyright © 1999John Wiley & Sons, Inc. Issue. Journal of Neurobiology. ... Correspondence: Masayuki Yamashita, Nara Medical University, 840 Shijo-cho, Kashihara 634-8521, Japan. Publication History. ...
Current Biology, 2001
Loss of membrane potential (membrane as much as 50 mV due to activation of a Cl Ϫ conductance tha... more Loss of membrane potential (membrane as much as 50 mV due to activation of a Cl Ϫ conductance that is distinct from the Cl Ϫ channels regulated by cytodepolarization) is one of the earliest and most striking responses of quiescent cells to stimulation solic [Ca 2ϩ ], volume, or voltage [3]. Activation of the Cl Ϫ conductance is resistant to pertussis toxin; it correlates with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and with enhanced phosphoinositide hydrolysis and RhoAdependent f-actin remodeling [3], but causal relationships, thrombin [1-3]. Membrane depolarization is due to the activation of a chloride conductance [3]. While if there are any, are unknown. To dissect the underlying G protein pathway(s) and to gain insight into the physiothis response has received relatively little attention in the past, it is clear that the acute loss of membrane logical consequences of GPCR-induced membrane depolarization, we set out to examine the response in N1Epotential may have important physiological consequences [4-6]. However, the dissection of the 115 and NG105-18 neuronal cells. These cells express multiple G q -coupled receptors, while they show G 12/13 -
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane li... more GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane lipid fluidities of these cells and of normal lymphoid cells were estimated by fluorescence polarization, using the probe diphenylhexatriene and by measuring the (free) cholesterol/phospholipid molar ratio in whole cells. The results indicate that the membrane fluidity (reciprocal of the lipid structural order) of the lymphoma cells increases in the order of their location: peripheral blood less than spleen less than mesenterial lymph node less than ascites fluid. The membrane fluidities of normal lymphocytes from thymus, mesenterial lymph node and spleen were about the same, but higher than of peripheral blood lymphocytes, and between those of the lymphoma cells from lymph node and spleen. These results are confirmed by more extensive analysis on purified plasma membranes from the splenic and ascitic GRSL lymphoma cells and from normal splenocytes and thymocytes. The significantly higher lipid order parameter found in the GRSL plasma membrane isolated from the spleen as compared to those from the ascites cells could be fully explained by the differences measured in the major chemical determinants of the fluidity, i.e., the cholesterol/phospholipid ratio, the sphingomyelin content and the degree of saturation of the fatty acyl groups of the phospholipids. It was also found that the cholesterol/phospholipid ratio in erythrocyte membranes isolated from the peripheral blood of the tumor bearers was higher than in those from normal control mice. The observed differences in membrane fluidity between distinct subsets of tumor cells may be relevant to the sensitivity of these cells to immune attack or to drugs.
Current Biology, 2001
Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. Th... more Increasing evidence indicates that gap-junctional Cx43 is mediate intercellular communication. They are composed of connexin proteins, which form part of a multiprotein complex. For example, the c-Src and v-Src tyrosine kinases can bind directly to and phostransmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the phorylate the Cx43 C-terminal tail (CT) via SH2 and SH3 domain interactions [6, 7]. Furthermore, the very most widely expressed connexin member, has been implicated in the regulation of Cx43 channel C-terminal residues of Cx43 interact with the second PDZ domain of the Zona-Occludens-1 (ZO-1) protein, which gating by growth factors [3-5]. The Cx43 tail contains various protein interaction sites, but little may recruit regulatory proteins into gap junctions [8, 9]. Thus, the Cx43 complex might fulfill functions that are is known about binding partners. To identify Cx43interacting proteins, we performed pull-down not necessarily related to the control of gap-junctional communication (GJC), a possibility that has received little experiments using the C-terminal tail of Cx43 fused to glutathione-S-transferase. We find that the Cx43 attention to date. tail binds directly to tubulin and, like full-length Cx43, sediments with microtubules. Tubulin binding To identify new Cx43 binding proteins, lysates of 35 S-