Ulrich Rümenapp

Inhibition of Receptor Signaling to Phospholipase D by Clostridium difficile Toxin B ROLE OF Rho PROTEINS

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5′-O-(thio)triphosphate (GTPS)) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF and GTPS, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPS-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.

Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors

Recently, a family of G-protein-coupled receptors named endothelial differentiation gene (Edg) receptor family has been identified, which are specifically activated by the two serum lipids, sphingosine-1-phosphate and lysophosphatidic acid. Sphingosine-1-phosphate can also act intracellularly to release Ca2+ from intracellular stores. Since in several cell types, G-protein-coupled lysophosphatidic acid or sphingosine-1-phosphate receptors mobilize Ca2+ in the absence of a measurable phospholipase C stimulation, it was analysed here whether intracellular sphingosine-1-phosphate production was the signalling mechanism used by extracellular sphingosine-1-phosphate for mobilization of stored Ca2+. Sphingosine-1-phosphate and the low affinity sphingosine-1-phosphate receptor agonist, sphingosylphosphorylcholine, induced a rapid, transient and nearly complete pertussis toxin-sensitive Ca2+ mobilization in human embryonic kidney (HEK-293) cells. The G-protein-coupled sphingosine-1-phosphate receptors, Edg-1, Edg-3 and Edg-5, were found to be endogenously expressed in these cells. Most interestingly, sphingosine-1-phosphate and sphingosylphosphorylcholine did not induce a measurable production of inositol-1,4,5-trisphosphate or accumulation of inositol phosphates. Instead, sphingosine-1-phosphate and sphingosylphosphorylcholine induced a rapid and transient increase in production of intracellular sphingosine-1-phosphate with a maximum of about 1.4-fold at 30 s. Stimulation of sphingosine-1-phosphate formation by sphingosine-1-phosphate and sphingosylphosphorylcholine was fully blocked by pertussis toxin, indicating that extracellular sphingosine-1-phosphate via endogenously expressed G(i)-coupled receptors induces a stimulation of intracellular sphingosine-1-phosphate production. As sphingosine-1-phosphate- and sphingosylphosphorylcholine-induced increases in intracellular Ca2+ were blunted by sphingosine kinase inhibitors, this sphingosine-1-phosphate production appears to mediate Ca2+ signalling by extracellular sphingosine-1-phosphate and sphingosylphosphorylcholine in HEK-293 cells.

p63RhoGEF and GEFT are Rho-specific guanine nucleotide exchange factors encoded by the same gene

Activation of Rho GTPases, which play pivotal roles in diverse cellular functions, is catalysed by specific guanine nucleotide exchange factors (GEFs). We and others (Souchet et al. (2002)) independently cloned a human cDNA encoding a 580 aa protein (p63RhoGEF), which contains a tandem of Dbl homology and pleckstrin homology domains typical for RhoGEFs. In accordance with Souchet et al., recombinant p63RhoGEF interacted with and catalysed GDP/GTP exchange at RhoA, but not Rac1 or Cdc42. Recently, an N-terminally truncated form of p63RhoGEF, termed GEFT, was described as a Rac/Cdc42-specific GEF (Guo et al. 2003). As judged by RT-PCR with specific primers, we were able to detect mRNA variants encoding p63RhoGEF and GEFT within several tissues and cell lines. Apparently, they co-exist within one cell and are derived from the same gene. When expressed in human embryonic kidney cells, both p63RhoGEF and GEFT caused activation of RhoA, but not Rac1 or Cdc42, and induced serum response factor-mediated gene transcription, which was fully blunted by the Rho-inactivating C3 transferase. In line with these data, expression of either p63RhoGEF or GEFT in J82 human bladder carcinoma cells induced the formation of actin stress fibres. We therefore conclude that p63RhoGEF and GEFT are apparently isoforms derived from the same gene and that GEFT, similar to p63RhoGEF, activates RhoA in several cell types.

Rho-specific binding and guanine nucleotide exchange catalysis by KIAA0380, a Dbl family member

Several guanine nucleotide exchange factors (GEFs) for Rho‐GTPases have been identified, all of them containing a Dbl homology (DH) and pleckstrin homology (PH) domain, but exhibiting different specificities to the Rho family members, Rho, Rac and Cdc42. We report here that KIAA0380, a protein with a tandem DH/PH domain, an amino‐terminal PDZ domain and a regulator of G protein signalling (RGS) homology domain, is a specific GEF for RhoA, but not for Rac1 and Cdc42, as determined by GDP release, guanosine 5′‐O‐(3‐thio)triphosphate (GTPγS) binding and protein binding assays. When expressed in J82 cells, DH/PH domain‐containing forms of KIAA0380 induced actin stress fibers, whereas expression of the RGS homology domain prevented lysophosphatidic acid (LPA)‐induced stress fiber formation.

IDENTIFICATION OF G PROTEIN-COUPLED RECEPTORS POTENTLY STIMULATING MIGRATION OF HUMAN TRANSITIONAL-CELL CARCINOMA CELLS

The expression of G protein-coupled receptors inducing calcium mobilization and stimulating cell migration was examined in human transitional-cell carcinoma (J82) cells. Measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) and phospholipase C activity indicated that these cells express several calcium-mobilizing receptors, including those for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, of which only the LPA response was sensitive (∼ 50%) to pertussis toxin (PTX). Migration of J82 cells was strongly stimulated by LPA and thrombin, by 5- to 20-fold, whereas bradykinin, bombesin and histamine were ineffective. Migration induced by either LPA or thrombin was inhibited by the actin cytoskeleton-disrupting agent, cytochalasin B, by the Rho protein-inactivating Clostridium difficile toxin B, by preventing [Ca2+]i transients with an intracellular calcium-chelating agent, and by the phorbol ester, phorbol 12-myristate 13-acetate, which also blocked the LPA- and thrombin-induced [Ca2+]i increases. On the other hand, ADP-ribosylation of Gi type G proteins by PTX abrogated the migratory response to LPA, without affecting the thrombin effect. Similarly, raising cAMP levels inhibited, by about 50%, the LPA- but not the thrombin-induced J82 cell migration. In conclusion, human transitional-cell carcinoma (J82) cells express various G protein-coupled, calcium-mobilizing receptors, out of which only those for LPA and thrombin stimulate cell migration, indicating that phospholipase C-derived second messengers per se are not sufficient for initiating this response. The complex signal transduction processes leading to LPA- and thrombin-stimulated motility of these human carcinoma cells apparently involve several common, essential factors, such as [Ca2+]i changes and Rho protein-regulated reorganization of the cytoskeleton, as well as some distinct components, most notably distinct subtypes of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets.

Muscarinic receptor-stimulated cytosol-membrane translocation of RhoA

© 1997 Federation of European Biochemical Societies.

A role for Rho in receptor- and G protein-stimulated phospholipase C Reduction in phosphatidylinositol 4,5-bisphosphate by Clostridium difficile toxin B

Receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-hydrolyzing phospholipase C (PLC) enzymes by activated α or free βγ subunits of the relevant G proteins. To study whether low molecular weight G proteins of the Rho family are involved in receptor signalling to PLC, we examined the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates Rho proteins, on the regulation of PLC activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) subtype. Toxin B treatment of HEK cells did not affect basal PLC activity, but potently and efficiently inhibited mAChR-stimulated inositol phosphate formation. PLC activation by the endogenously expressed thrombin receptor and by the direct G protein activators, AlFinf4sup−and guanosine 5′-[γ-thio]triphosphate (GTPγS), studied in intact and permeabilized cells, respectively, were also inhibited by toxin B treatment. C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPγS-stimulated PLC activity. Finally, both toxin B and C3 exoenzyme significantly reduced, by 40 to 50%, the total level of PtdIns(4,5)P2 in HEK cells, without affecting the levels of phosphatidylinositol and phosphatidylinositol 4-phosphate. Accordingly, when PLC activity was measured with exogenous PtdIns(4,5)P2 as enzyme substrate, Ca2+- as well as GTPγS- or A1Finf4sup−-stimulated PLC activities were not altered by prior toxin B treatment. In conclusion, evidence is provided that toxin B and C3 exoenzyme, apparently by inactivating Rho proteins, inhibit G protein-coupled receptor signalling to PLC, most likely by reducing the cellular substrate supply.

Sphingolipid receptor signaling and function in human bladder carcinoma cells: inhibition of LPA- but enhancement of thrombin-stimulated cell motility

Abstract. Sphingosine-1-phosphate (SPP) induces a variety of cellular responses, including Ca2+ signaling, proliferation, and inhibition of motility, apparently by acting at specific G protein coupled receptors. Here, the expression, signaling, and motile responses of sphingolipid receptors were examined in human bladder carcinoma (J82) cells, for which lysophosphatidic acid (LPA) and thrombin act as potent agonists. SPP potently and rapidly mobilized Ca2+, stimulated phospholipases C and D, and inhibited cAMP accumulation, without affecting growth of J82 cells, which express the recently identified SPP receptors, Edg-1 and Edg-3. The effects of SPP were mimicked by sphingosylphosphorylcholine (SPPC) and strongly attenuated by pertussis toxin (PTX). SPP and SPPC by themselves induced a small, PTX-sensitive motile response. However, stimulation of cell motility by LPA, which by itself was also PTX-sensitive, was blocked by SPP and SPPC. In contrast, motility stimulation by thrombin, which by itself was PTX-insensitive, was strongly augmented by the sphingolipids in a PTX-sensitive manner. The bidirectional regulation of LPA- and thrombin-stimulated motility was not due to selective alterations in the activation of Rho GTPases which control cell motility. In fact, RhoA activation and Rho-dependent actin stress fiber formation induced by LPA and thrombin were mimicked, but not altered by SPP and SPPC. We conclude that J82 cells express sphingolipid receptors, coupled via G proteins to several signaling pathways. Most importantly, these sphingolipid receptors potently regulate thrombin- and LPA-stimulated motility, but in opposite directions, suggesting that migration of these human bladder carcinoma cells is controlled by a complex network of interacting extracellular ligands.

Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors.

The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C. botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], a substrate and cofactor for PLC and PLD, respectively, were not altered. These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme. In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.

A mammalian Rho-specific guanine-nucleotide exchange factor (p164-RhoGEF) without a pleckstrin homology domain

Rho GTPases, which are activated by specific guanine-nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a recently cloned human cDNA, namely KIAA0337, encoding a protein containing 1510 amino acids (p164). It contains a RhoGEF-specific Dbl homology (DH) domain but lacks their typical pleckstrin homology domain. The expression of the mRNA encoding p164 was found to be at least 4-fold higher in the heart than in other tissues. Recombinant p164 interacted with and induced GDP/GTP exchange at RhoA but not at Rac1 or Cdc42. p164-DeltaC and p164-DeltaN are p164 mutants that are truncated at the C- and N-termini respectively but contain the DH domain. In contrast with the full-length p164, expression of p164-DeltaC and p164-DeltaN strongly induced actin stress fibre formation and activated serum response factor-mediated and Rho-dependent gene transcription. Interestingly, p164-DeltaN2, a mutant containing the C-terminus but having a defective DH domain, bound to p164-DeltaC and suppressed the p164-DeltaC-induced gene transcription. Overexpression of the full-length p164 inhibited M(3) muscarinic receptor-induced gene transcription, whereas co-expression with Gbeta(1)gamma(2) dimers induced transcriptional activity. It is concluded that p164-RhoGEF is a Rho-specific GEF with novel structural and regulatory properties and predominant expression in the heart. Apparently, its N- and C-termini interact with each other, thereby inhibiting its GEF activity.