The monomeric GTPase RhoA, which is a key regulator of numerous cellular processes, is activated by a variety of G protein-coupled receptors, through either G 12 or G q family proteins. Here we report that p63RhoGEF, a recently identified RhoA-specific guanine nucleotide exchange factor, enhances the Rho-dependent gene transcription induced by agonist-stimulated G q/11 -coupled receptors (M 3 -cholinoceptor, histamine H 1 receptor) or GTPase-deficient mutants of G␣ q and G␣ 11 . We further demonstrate that active G␣ q or G␣ 11 , but not G␣ 12 or G␣ 13 , strongly enhances p63RhoGEF-induced RhoA activation by direct protein-protein interaction with p63RhoGEF at its C-terminal half. Moreover, the activation of p63RhoGEF by G␣ q/11 occurs independently of and in competition to the activation of the canonical G␣ q/11 effector phospholipase C ␤. Therefore, our results elucidate a new signaling pathway by which G q/11 -coupled receptors specifically induce Rho signaling through a direct interaction of activated G␣ q/11 subunits with p63RhoGEF.

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 (GTP␥S)) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF 4؊ and GTP␥S, 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 GTP␥S-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.

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTPbinding proteins by AlFy and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F;-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phospholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins. Abbreviations. mAChR, muscarinic acetylcholine receptor; BAPTA/AM, bis-(0-aminophenoxy)-ethane N, N, N' , "-tetraacetic acid tetra(acetoxymethy1ester); Fura-2/AM, Enzymes. Phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); protein kinase C (EC 2.7.1.37).gers, inositol 1,4,5-trisphosphate, which releases sequestered calcium from a subpopulation of the endoplasmic reticulum, and diacylglycerol, which activates protein kinases C [l, 21. These two signals apparently also participate in the transduction of mitogenic signals across the plasma membrane into the nucleus leading to the stimulation of cell growth, DNA synthesis and cell division [2]. Phosphatidylcholine...

Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small-molecular-mass guanine-nucleotide-binding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5'-O-[y-thio]triphosphate (GTPIS]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nucleotide-exchangefactor activity in Golgi membranes, inhibited mAChR-stimulated PLD, whereas basal PLD activity and stimulation by GTPIS] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.

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.