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Phospholipid signalling: Rho is only ARF the story Michael A. Frohman and Andrew J. Morris

The GTP-binding proteins ARF and Rho control a short to involve alterations in gene expression, indicating number of important cellular processes, such as protein that these events must be controlled through other regula- traffic and cell morphology; there is increasing evidence tory pathways. Thus, Rho proteins, like ARFs, regulate that phospholipase D is a key mediator of these complex processes in cells and in both cases the precise ARF/Rho-regulated events. steps and mechanisms involved have been unclear.

Address: Department of Pharmacological Science and Institute for Cell and Molecular Biology, State University of New York, Stony Brook, That PLD is activated following agonist stimulation has New York 11794-8651, USA. been observed in numerous cell types and with a wide variety of ligands acting via G-protein-coupled receptors or Current Biology 1996, Vol 6 No 8:945–947 receptor tyrosine kinases. While investigating the mecha- © Current Biology Ltd ISSN 0960-9822 nism of PLD activation, several groups observed PLD activities in tissues and cell lines that showed guanine- Small GTP-binding proteins act as switches that control nucleotide dependence, which was subsequently shown to diverse cellular processes. Those in the ADP-ribosylation be mediated by members of the ARF and Rho families. factor (ARF) class direct key steps in intracellular protein Two groups used different approaches to identify ARF as traffic, whereas those in the Rho family regulate gene tran- a GTP-dependent regulator of PLD activity [8,9]. Their scription as well as changes in cell motility and morphology observations were initially made using HL-60 cells as the [1,2]. Several groups have now produced evidence that source of PLD activity, but PLD enzymes with similar these regulatory activities of Rho and ARF proteins may be sensitivity to ARF stimulation (presumably identical or mediated by phosphatidylcholine (PC)-specific phospholi- closely related enzymes) are expressed in other tissues. pase D (PLD). PLD generates phosphatidic acid (PA), The GTP-dependent activation of PLD in neutrophils is which may be an intracellular mediator in its own right and apparently mediated by Rho [10]; furthermore, two Rho- which can be metabolized to form lipids with proven roles related G proteins, Rac1 and Cdc42, can also stimulate as both intercellular and intracellular messengers [3]. several mammalian PLD activities independently of (and synergistically with) other regulators [11,12]. ARF and Rho regulation of PLD activity ARFs were initially identified as activators of cholera toxin We have recently reported [13] the cloning and expression ADP-ribosyl transferase activity towards the ␣ subunit of of the human gene encoding PLD1, a G-protein-regulated the heterotrimeric G protein Gs. There are six mammalian PLD. ARF1, and the Rho proteins RhoA, Rac1 and Cdc42, ARFs, which are largely soluble but can bind to mem- all activate purified PLD1; the purified enzyme can also be branes in a regulated and GTP-dependent manner. ARFs activated by protein kinase C␣ in a novel, kinase-indepen- play an essential role in intracellular membrane traffic, and dent manner. The G-protein activators are approximately have been implicated in both ER-to-Golgi and intra-Golgi equipotent, and the three classes of regulators — ARF, Rho vesicle transport. Reconstitution studies using mammalian and PKC — activate PLD1 synergistically, suggesting that systems have demonstrated that ARF1 regulates the they interact with different sites on the enzyme (our unpub- assembly of the coat protein complex — the coatomer or lished data). Several lines of evidence have suggested the COP — which controls the budding of transport vesicles existence of additional mammalian PLDs (reviewed in from Golgi cisternae [4,5] and presumably also from ER [14]), and our group has recently identified a second mam- membranes. Why there are multiple ARFs is poorly malian PLD, PLD2, which, like PLD1, requires phos- understood, but their existence is presumably important; phatidylinositol 4,5-bisphosphate (PIP2) for activity in vitro ARF6, for example, localizes to the plasma membrane and (unpublished data). Mammalian tissues also contain PIP2- regulates receptor-mediated endocytosis [6]. independent PLD activities which are stimulated by fatty acids such as oleate [15]. It is not known how many mam- Rho family proteins play dynamic roles in the regulation malian PLD enzymes there are, though our molecular of the actin cytoskeleton, thereby controlling various studies [13], which have revealed the existence of a PLD aspects of cell morphology and motility, depending on the multigene family, should help clarify this important issue. particular Rho protein involved. Thus, Rac is required for membrane ruffling and Rho for formation of stress fibers, What is the function of PLD-generated PA? and Cdc42 regulates the formation of actin-containing Understanding the role of PLD in cellular regulation by microspikes called filopodia [7]. The effects of Rho pro- the ARF and Rho proteins will require identification of the teins in microinjected cells occur on a time scale that is too cellular targets and functions of its reaction product, PA. 946 Current Biology 1996, Vol 6 No 8

PA can clearly be metabolized to generate both intracellu- Rothman and colleagues [4] reported that coatomer lar and extracellular messengers — diacylglycerol, arachi- assembly can be reconstituted in washed Golgi membrane donic acid and lysophosphatidic acid (reviewed in [14]) — preparations with the addition of just ARF and coatomer. but it is beginning to appear equally likely that it is also an It is likely, however, that PLD was present in these exper- intracellular mediator in its own right. For example, iments, as it is tightly associated with membranes and several identified (Raf and certain PKC isoforms) and present in significant amounts in the Golgi apparatus [21]. unidentified protein kinases have been reported to be acti- Sternweis, Roth and colleagues [21,22] have begun to vated by PA in vitro [16], although the physiological impor- establish that ARF-activated PLD does indeed have an tance of this type of regulation requires further study [3]. important role in intracellular protein traffic. They have found that ARF-activated PLD is enriched in Golgi mem- As no selective PLD inhibitors have been discovered, branes, and provided several lines of evidence that PA attempts to generate direct evidence supporting in vivo formed through ARF stimulation of PLD plays an impor- roles for PLD-generated PA have entailed the direct tant role in coatomer binding. This suggests the model administration of PA itself to cells, or the use of primary shown in Figure 1, in which activation of Golgi-mem- alcohols which are used as substrates by PLD for a brane-associated PLD1 by ARF leads to recruitment of transphosphatidylation reaction which generates phos- the cytosolic coatomer complex, and consequent alter- phatidylalcohols instead of PA. Exogenously added PA ations in membrane structure leading to vesicle budding. has been shown to increase, and primary alcohols to inhibit, actin polymerization leading to stress-fiber forma- The synergistic effect of Rho on ARF stimulation of PLD1 tion in endothelial cells, suggesting a role for PLD as a activity in vitro suggests that this enzyme may act as an mediator of cytoskeletal reorganization [17]. Primary alco- integrative regulator of both constitutive secretion, pre- hols have also been used to demonstrate that secretion by sumably under the control of ARF, and receptor-regulated HL-60 cells and neutrophils is blocked when PA produc- tion is inhibited, supporting the hypothesis that PLD Figure 1 plays a role in secretion [18,19].

Caveats should be applied, however, to the interpretation of Agonists both of these experimental approaches. PA is normally pro- duced intracellularly by PLD, and it is not clear that exoge- Receptors nously applied PA exerts its actions through normal physiological pathways (as opposed, for example, to stimu- Coated vesicle lating receptors on the cell surface, as lysophosphatidic acid PLC is known to do). And although phosphatidylalchohols Budding ? ? appear to be metabolically inert, they may nonetheless acti- COP vate some PA-dependent processes to an unknown extent. Moreover, the relatively high concentrations of alcohols PKC used in these types of experiment may influence these or other cellular responses through different mechanisms. COP

The role of PLD1 in ARF and Rho-regulated processes GTP Rho PLD1 Given the evidence presented above, it has been tempting ARF GTP to speculate that PLD activation by ARF and Rho reflects PiP2 roles for distinct PLDs in separate pathways — secretion PC PA and signal transduction, respectively. The finding that Golgi PLD1 is regulated by both ARF and Rho, however, sup- membrane Priming Vesiculation ports a blurring of this distinction and suggests that the © Current Biology 1996 two processes may be more integrated than previously rec- ognized. Several lines of evidence support this idea. For PLD1- and ARF-dependent formation of coated vesicles. PLD1 example, most ARF is found in the cytosol in HL-60 cells participates in the ‘priming’ step of coated vesicle formation by [20]; immediately after stimulation with a ligand agonist catalyzing hydrolysis of PC to produce PA, which, perhaps acting in (fMet-Leu-Phe), however, much of the cytosolic ARF concert with ARF, results in coatomer binding to the membrane surface (possibly mediated by a putative ‘coatomer receptor’). becomes membrane-bound in the Golgi apparatus. This Coatomer assembly then results in deformation of the membrane suggests that ARF should be viewed as a participant in the surface (vesiculation), leading to bud formation and release of a coated signal transduction process, and that secretion in this vesicle (budding). We suggest that activation of PLD1 by ARF and Rho (and PKC) links this process of coated-vesicle formation to instance is a regulated consequence of specific agonist- activation of cell-surface receptors. (Adapted from [22].) stimulated events. Dispatch 947

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