the activation of membrane receptors by hormones and growth factors results in the localized generation of intracellular second messengers . the hydrolysis of membrane phospholipids and the generation of biologically active products play important roles in the regulation of cell function and cell fate . well known is the activation of phosphoinositide - specific phospholipase c ( plc ) isoforms , which hydrolyze phosphatidylinositol 4,5-bisphosphate ( pip2 ) , a membrane phospholipid found in all eukaryotic cells ( schmidt et al . stimulation of plc isoforms plays a major role in many early and late cellular responses to receptor activation , including smooth muscle contraction , secretion and neuronal signaling as well as fertilization , cell growth and differentiation ( berridge 2005 ; nishizuka 2003 ) . phospholipase d ( pld ) was first described 60  years ago as a distinct , phospholipid - specific phosphodiesterase activity in cabbage leaves ( hanahan and chaikoff 1948 ) . this pioneering research indicated that pld hydrolyzes phosphatidylcholine to yield phosphatidic acid ( pa ) and choline . the recognition that pld is rapidly and dramatically activated in response to extracellular stimuli in cultured animal cells , now 20  years ago ( bocckino et al . 1987 ; cockcroft 1984 ) , has brought pld signaling to the very forefront of current biological and biomedical research . meanwhile , phosphatidylcholine - hydrolyzing pld has been identified in bacteria , protozoa , fungi , plants and animals , and , due to this widespread distribution , is assumed to be involved in the regulation of fundamental cellular functions . indeed , it has now been established that activation of pld and the generation of pa by a vast number of membrane receptors modulate such a wide array of cellular responses as calcium mobilization , secretion , superoxide production , endocytosis , exocytosis , vesicle trafficking , glucose transport , rearrangements of the actin cytoskeleton , mitogenesis and survival ( cockcroft 2001 ; exton 2002b ; jenkins and frohman 2005 ; liscovitch et al . 2000 ) . pip2 is a critical cofactor for pld , and profoundly affects the activity , membrane localization and receptor activation of both pld isoforms , pld1 and pld2 ( brown et al . thus , reduction of cellular pip2 levels , for instance via scavenging of pip2 by the actin - binding protein fodrin ( lukowski et al . 1998 ) or via forced pip2 hydrolysis by the phosphatase synaptojanin ( chung et al . vice versa , the synthesis of pip2 by phosphoinositide 5-kinase ( pip5k ) isoforms can be directly stimulated by the pld product pa ( jenkins et al . 1992 ) , and this regulation has also been confirmed to occur at the whole cell level ( divecha et al . it is now hypothesized that the reciprocal stimulation of pld and pip5k enzymes enables rapid feed - forward stimulation loops for a localized and explosive generation of pa and pip2 , which may then govern the recruitment and activation of proteins to execute specific cellular tasks , especially membrane trafficking , and changes in the organization of the actin cytoskeleton . the activity and localization of both pld and pip5k are under control of gtpases of the arf and rho families , which are well - defined regulators of membrane transport and actin - reorganization processes . the reciprocal stimulation of pip5k and pld , and the regulation of these enzymes by arf and rho gtpases , point to concerted mechanisms in cellular actions , involving acute , localized pip2 and pa synthesis ( fig .  1 ) . this review will focus on the regulation of pld enzymes by membrane receptors and monomeric gtpases , and on how pld signaling is organized and connected by pip2 metabolism . regulation of pld and pip5k by arf and rho family gtpases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization . both pld and pip5k are stimulated by cell surface receptors and by conventional pkc isoforms , and the latter can become activated after receptor - induced hydrolysis of pip2 by plc . positive feed - forward regulation is achieved by stimulation of pld by pip5k - derived pip2 , and of pip5k by pld - derived pa . activation of arf - gaps by pip2 accelerates the inactivation of arf proteins , and may terminate a round of pa and pip2 synthesis regulation and cellular roles of pld and pip5k . regulation of pld and pip5k by arf and rho family gtpases is essentially involved in the regulation of intracellular vesicle trafficking and actin cytoskeleton reorganization . both pld and pip5k are stimulated by cell surface receptors and by conventional pkc isoforms , and the latter can become activated after receptor - induced hydrolysis of pip2 by plc . positive feed - forward regulation is achieved by stimulation of pld by pip5k - derived pip2 , and of pip5k by pld - derived pa . activation of arf - gaps by pip2 accelerates the inactivation of arf proteins , and may terminate a round of pa and pip2 synthesis most cellular responses following pld activation are probably mediated by the immediate reaction product pa . pa is a multifunctional lipid that can be further metabolized to the bioactive lipids , lysophosphatidic acid ( lpa ) and diacylglycerol ( dag ) , can by itself alter membrane curvature , and can serve as a protein attachment site and affect both cellular localization and activity of various proteins , including raf-1 kinase , protein phosphatase 1 , sphingosine kinase 1 , and mtor ( mammalian target of rapamycin ) , a key regulator of cell growth and proliferation ( jenkins and frohman 2005 ) . pld enzymes can catalyze a transphosphatidylation reaction in which the phosphatidyl moiety of phosphatidylcholine is accepted by primary alcohols , thereby producing stable phosphatidylalcohol instead of pa . this transphosphatidylation reaction is widely applied to measure pld activity in biological samples , and quenching of pa synthesis by primary alcohols has proven extremely helpful to identify the involvement of pld enzymes in cell physiology . in this way , a role for pld has been demonstrated in a variety of signaling processes , such as activation of phosphoinositide ( pi3k , pip5k ) and protein ( akt , erk1/2 ) kinases , calcium mobilization , cytoskeleton remodeling , endocytosis , exocytosis , membrane trafficking , superoxide production , glucose transport , cell migration , cell proliferation , and survival signaling ( exton 2002a ; foster and xu 2003 ) . pld1 has a low basal activity and is extensively regulated by conventional protein kinase c ( pkc , - , - ) isozymes and small gtpases of the arf ( arf1 - arf6 ) and rho ( rhoa , rac1 , cdc42 ) families ( henage et al . pld2 has a higher basal activity than pld1 , but has been shown to respond to arf and pkc as well ( chen and exton 2004 ) . pip2 is recognized to be the most important cofactor for pld , and both pld isoforms are absolutely dependent on pip2 for activity . experiments utilizing inactive pld mutants and rna interference have discriminated isoform - specific pld functions , and showed that pld1 is involved in agonist - induced secretion , actin organization , and cell adhesion and migration ( exton 2002a ; iyer et al . 2006 ; kim et al . 2006 ; vitale et al . 2001 ) , and pld2 in endocytosis and recycling of membrane receptors ( du et al . 2004 ; koch et al . 2006 ; padrn et al . the pld isoforms , both with two splice variants , share an ~50% amino - acid sequence identity ( colley et al . 1997 ; hammond et al . the catalytic core of both pld enzymes are composed of four conserved domains ( domain i - iv ) , and the hkd motifs in the domains ii and iv probably associate together to form a catalytic centre ( xie et al . pld1 is characterized by a 116-amino acid loop region following domain ii , which has been proposed to function as a negative regulatory element ( sung et al . pld1 and pld2 further possess n - terminal ph ( pleckstrin homology ) and px ( phox homology ) domains . 2000 ) , but also to a polybasic pip2 binding motif within the catalytic core ( sciorra et al . 1999 ) , and interaction of pip2 with both domains has been suggested to be involved in membrane targeting of pld as well as stimulation of pld catalytic activity ( du et al . the px domain of pld1 has been reported to preferentially bind to phosphatidylinositol-3,4,5-trisphosphate ( pip3 ) ( lee et al . 2004 ) , but interaction with pi5p has been observed as well ( du et al . recently , it was shown that the px domain of pld has gtpase - activating protein ( gap ) activity towards dynamin , and that pld supports egf receptor endocytosis ( lee et al . the ph and px domains probably contribute to the proper localization of the pld enzymes within cells . in line with a role for pld enzymes in different cellular tasks , pld1 and pld2 pld1 is found throughout the cell , but primarily localizes to perinuclear endosomes and the golgi apparatus ( brown et al . pld2 is almost exclusively present at the plasma membrane in lipid raft fractions ( czarny et al . the localization of pld1 does not seem to be static , and regulated translocation and recycling of the enzyme between cellular compartments may be crucial to its proper functioning . in an elegant study , coordinated subcellular targeting of the lipid binding motifs has been demonstrated to drive this subcellular cycling of pld1 ( du et al . , pld1 was found to translocate from the intracellular compartments to the plasma membrane , and this process was probably dependent on the polybasic pip2 binding site . the ph domain then facilitated entry of pld1 into lipid rafts , a step critical for internalization of the enzyme , whereafter interaction of the px domain with pi5p may control the efficient return of pld1 to the endosomes . hydrolysis of pip2 by plc into the second messengers , inositol-1,4,5-trisphosphate ( ip3 ) and dag , is a general and well - defined answer of cells in response to stimulation of many membrane receptors ( schmidt et al . phosphorylation of pip2 by pi3k results in the rapid accumulation of pip3 , which recruits and activates mediators involved in actin remodeling , mitogenesis and survival ( vanhaesebroeck et al . but it is now recognized that pip2 , as well as other phosphoinositides , are signaling molecules by themselves and can , by binding to unique phosphoinositide - binding sequences , such as the ph and px domains , affect the activity and subcellular localization of many proteins , including many actin regulatory proteins , a wide range of ion channels , and pld ( niggli 2005 ; suh and hille 2005 ; yin and janmey 2003 ) . in this way , pip2 can modulate a remarkable variety of cellular processes , including cortical actin organization , membrane ruffling , vesicle trafficking , gene expression , cell migration and cell survival ( ling et al . subsequent dephosphorylation of pip2 by inositol polyphosphate 5-phosphatases , such as synaptojanin , is believed to terminate local pip2 signaling , for instance in the process of vesicle trafficking ( majerus et al . pip2 may be organized in discrete functional pools within cells , but the existence of pip2 clusters in the plasma membrane is currently under debate . using green fluorescent protein - tagged ph domains or antibodies to visualize pip2 , the lipid was found to concentrate in highly dynamic , actin - rich regions ( tall et al . 2000 ) and lipid rafts ( laux et al . 2000 ; parmryd et al . 2003 ) in the plasma membrane , feeding the idea that spatially organized pip2 synthesis regulates actin polymerization and other cellular processes . the localization of pip2 in rafts is supported by biochemical data ( pike and casey 1996 ) ; however , specific pip2 clustering has been disputed ( van rheenen et al . cdnas encoding three isoforms of pip5k ( designated i , i and i ) with alternative splice variants have been cloned and characterized ( ishihara et al . sequence analysis has shown that pip5k enzymes are related to pip4k enzymes , but that they share no identity with most other lipid ( pi3k and pi4k ) or protein kinases . the sequence similarity between the pip4ks and pip5ks an activation loop spanning the catalytic domain has been shown to determine both substrate specificity and subcellular targeting of pip5ks , which can be swapped by substitution of a single amino acid within this loop ( kunz et al . two dimerization domains were identified , which may contribute to the proper subcellular localization and functioning of the enzyme ( galiano et al . the identification of three pip5k isoforms raised the expectation of a differential regulation of the enzymes by cellular signal transduction components , but up to now the regulatory properties of pip5k - i , i and i appear to be remarkably similar . all pip5k isoforms are stimulated by pa , are extensively regulated by arf and rho gtpases , and inhibited by protein kinase a ( pka ) and pi - stimulated autophosphorylation ( oude weernink et al . nevertheless , evidence has been provided that pip5k isoforms may selectively control functional pip2 pools , which may support particular processes in different cell types . thus , actin reorganization down - stream of rac1 in platelets specifically involves murine pip5k - i ( tolias et al . 2000 ) . human pip5k - i was found to localize in rac1-induced membrane ruffles , and the lim protein ajuba has been identified to interact with and stimulate pip5k - i in leading - edge membrane ruffles in migrating cells ( kisseleva et al . 2005 ) . human pipk - i was detected primarily in cytosolic vesicular structures ( doughman et al . 2003 ) and may synthesize the pip2 pool involved in constitutive endocytosis ( padrn et al . the long - splice variant of pip5k - i , pip5k - i90 , is enriched in neurons and is implicated in the regulation of clathrin coat recruitment , actin dynamics ( wenk et al . 2001 ) and focal adhesion formation ( di paolo et al . 2002 ; ling et al . 2002 ) . in contrast , short pip5k - i87 seems to be the major producer of the pip2 pool that supports receptor - induced ip3 generation ( wang et al . the execution of specific pip2-modulated processes is very probably achieved by an orchestration of appropriate signaling partners within discrete subcellular microdomains , and pld - derived pa as well as the pld enzymes by themselves can contribute to this organization . indeed , both pld1 and pld2 interact with pip5k - i , and pld2 recruits pip5k - i to a submembraneous vesicular compartment ( divecha et al . pld2-derived pa was shown to stimulate pip5k - i splice variants , and the subsequent formation of pip2 to drive the initial stages of integrin - mediated cellular adhesion ( powner et al . 2005 ) . in many processes , the temporal activation and correct localization of pld and pip5k isoforms by monomeric gtpases appears crucial to achieve the spatially organized production of pip2 and pa ( santarius et al . although the direct interaction site on pld for arf has not yet been unequivocally defined , it is well established that arf proteins , particularly arf1 and arf6 , activate both pld enzymes , but especially pld1 ( hammond et al . arf1 is localized to the golgi complex , and is required for proper golgi structure and function . the use of primary alcohols has also pointed to a role for pld in vesicle transport to golgi ( bi et al . 1997 ; ktistakis et al . pld activity has been shown to stimulate the release of nascent secretory vesicles from the trans - golgi network ( chen et al . 1997 ) , and to be required for maintaining the structural integrity and function of the golgi apparatus , but the precise role for pld in vesicle formation is still controversial . pip5k is also a direct effector of arf1 , and an arf1 mutant that selectively activates pip5k , but not pld activity , demonstrated that both pld - derived pa and direct activation of pip5k by arf1 contribute to increased pip2 synthesis ( skippen et al . in permeabilized cells , arf1 has been shown to restore secretion by promoting pip2 synthesis ( fensome et al . arf6 activates pld , and pa has been implicated in the mediation of the effects of arf6 in vesicular trafficking events . a critical role for pld1 in exocytosis has been established in different cell types , including neurons ( humeau et al . 2001 ) , neuroendocrine cells ( vitale et al . 2001 ) and pancreatic  cells ( hughes et al . 2004 ) . pld2 has recently emerged as a mediator of arf - dependent internalization of the -opioid receptor ( koch et al . 2003 ) , and both pld isoforms have been implicated in macrophage phagocytosis ( corrotte et al . 2006 ; iyer et al . in addition , pip5k colocalizes and interacts with , and is directly activated by arf6 at the plasma membrane ( honda et al . 1999 ) , and arf6 and pip2 colocalize on the plasma membrane and on endosomal structures ( brown et al . arf6-organized pip2 turnover at the plasma membrane is apparently involved in regulated secretion ( aikawa and martin 2003 ; brown et al . focal and transient accumulation of pip2 by pip5k is required for phagocytosis as well ( botelho et al . 2000 ; coppolino et al . 2002 ; wong and isberg 2003 ) , and pip2 hydrolysis probably dictates the remodeling of actin necessary for completion of phagocytosis ( scott et al . the synthesis of pip2 is essential for priming the exocytotic apparatus , and the recruitment and activation of pld1 by pip2 seems the primary mechanism for the functional integration of pld1 into the exocytotic pathway ( vitale et al . thus , cd16-induced cytolytic granule secretion mediated by arf6 was shown to involve pip5k - i membrane targeting and activation of both pip5k and pld ( galandrini et al . pip2 also recruits additional proteins  for instance the endocytic proteins ap-2 , epsin and ap180to initiate clathrin - coat formation preceding endocytosis ( ford et al . 2001 ; itoh et al . 2001 ; padrn et al . 2003 ) , and caps ( grishanin et al . 2004 ) to initiate dense - core vesicle exocytosis . direct activation of pip5k - i by arf6 has been shown to stimulate clathrin - coat recruitment to synaptic membranes to allow synaptic vesicle recycling ( krauss et al . pld - derived pa may directly contribute to vesicle fusion in a biophysical manner , as pld cleaves the non - fusogenic lipid , pc , to form the fusogenic lipid , pa . but pa also takes a function as an essential cofactor for pip5k , and disruption of golgi membranes ( sweeney et al . 2002 ) , blockade of clathrin - coat assembly ( arneson et al . 1999 ) and inhibition of arf1-reconstituted secretion ( way et al . 2000 ) after quenching of pa production could be attributed to inhibited pip2 synthesis . thus , both pld and pip2 synthesis seem necessary for membrane trafficking aspects in the endo- and exocytotic machinery . but pld and pip5k also mediate other processes down - stream of arf6 . epidermal growth factor ( egf)-induced membrane ruffling requires arf6-induced pip5k - i translocation to the ruffles and local pip2 production . this leads to the recruitment of pld2 , and pld - derived pa and arf6 may then synergistically activate pip5k ( honda et al . the relationship between arf and pip2 is also bidirectional , as phosphoinositides can regulate arf activity by binding and activating both arf - specific guanine nucleotide exchange factors ( arf - gefs ) ( klarlund et al . 1998 ; paris et al . 1997 ) and arf - gtpase - activating proteins ( arf - gaps ) ( kam et al . 2000 ; nie et al . 2002 ) via their ph domains . the fact that arf - gaps bind pip2 with high affinity and specificity offers an attractive feed - back mechanism for terminating arf activation after a cycle of arf - induced pip2 synthesis . pa formation , especially by pld1 , has been reported to induce stress fibre formation in specific cell types ( cross et al . 1996 ; ha and exton 1993 ; kam and exton 2001 ; porcelli et al . rho proteins , in particular rhoa , rac1 and cdc42 , which control actin cytoskeleton reorganization , exclusively activate pld1 by direct interaction with its c - terminus ( exton 2002b ; powner and wakelam 2002 ) . thus , pld stimulation by rhoa may happen by direct interaction , but may involve indirect , rho - dependent mechanisms as well . inactivation of rho gtpases , with clostridium difficile toxin b or clostridium botulinum c3 exoenzyme , reduced cellular pip2 levels , resulting in inhibiton of receptor - mediated pip2 hydrolysis by plc ( schmidt et al . 1996a ) as well as diminished pld stimulation ( schmidt et al . 1996d ) . as the inhibition of pld signaling after rho inactivation could be largely rescued by the addition of pip2 , rho proteins do seem to affect pld via pip5k regulation ( schmidt et al . pip2 is well - known to associate with and regulate the activity of a plethora of actin - binding proteins that organize actin dynamics ( hilpela et al . 2004 ; yin and janmey 2003 ) , and pa and pip2 may act in concert to mediate rho - dependent actin cytoskeleton remodeling . pip5k isoforms are markedly stimulated by rhoa , rac1 , and cdc42 ( chong et al . 2004a ) , and physically associate with both rhoa ( ren et al . 1996 ) and rac1 ( tolias et al . pip5k isoforms are now seen as critical mediators of rhoa- and rac1-induced actin organization and remodeling ( doughman et al . 2003 ; shibasaki et al . the established rho effector rho - kinase , a serine / threonine kinase , is apparently involved in rho - dependent regulation of both pld ( schmidt et al . 2000 ) , and pip5k was found to play an essential role as down - stream effector of rho and rho - kinase in neurite remodeling ( van horck et al . 2002 ; yamazaki et al . 2002 ) and platelet cytoskeleton assembly ( gratacap et al . 2001 ; yang et al . 2004 ) . but rho may also directly signal to pip5k independently of rho - kinase , as rhoa - induced activation of erm ( ezrin , radixin , moesin ) proteins , that cross - link actin filaments to plasma membranes , was found to be mediated by pip5k , but not by rho - kinase ( matsui et al . pld and pip5k were also demonstrated to collectively mediate rho - induced changes in the actin cytoskeleton . thus , myogenic differentiation induced by arginine - vasopressin , which involves actin fiber formation , is mediated by rho proteins and pld1 , and involves pld - induced pip2 synthesis along the actin fibers ( komati et al . these findings suggest that pld and pip5k enzymes may co - operate down - stream of rho in processes that depend on actin organization . another rho effector , pkc - related protein kinase n ( pkn ) , also directly interacts with pld ( oishi et al . interestingly , components of the actin regulatory machinery , -actin and -actinin , have been found to directly associate with and inhibit the activity of pld isoforms ( lee et al . pld also binds to and is stimulated by filamentous f - actin , and pld1 in particular may act as a signal transduction component responsive to dynamic changes of the actin cytoskeleton ( kusner et al . pkn interacts with -actinin , and pkn may modulate pld signaling by reversing the inhibitory effect of -actinin on pld1 , and by direct interaction with pld1 . in line with the critical role of pa in cellular processes , the enzymatic activity of pld is tightly regulated by a variety of hormones , neurotransmitters , and growth factors . regulation of pld enzymes by membrane receptors , including g protein - coupled receptors ( gpcrs ) and receptor tyrosine kinases ( rtks ) , is complex and mediated by several cytosolic factors , including pkc as well as arf , rho and ras gtpases ( exton 2002b ; liscovitch et al . 2000 ; lpez de jess et al . most receptors that stimulate pld also increase plc activity , leading to activation of the pld regulator pkc , and it was assumed that pld activation might be secondary to plc activation . a physical association between pld with pkc isoforms has been reported , resulting in strong activation of in vitro pld1 activity , and the major interaction site was identified within the n - terminus of pld1 ( park et al . inhibition of pkc was shown to reduce receptor - induced pld responses , and pld1 mutants unresponsive to pkc did respond poorly to activation of gpcrs ( zhang et al . 1999 ) or to active gq proteins ( xie et al . however , stimulation of pld in several receptor systems , including m3 muscarinic and 1-adrenergic receptors , was actually pkc - independent ( balboa and insel 1998 ; muthalif et al . 1997 ; schmidt et al . 1994 ) , suggesting that pld stimulation must not necessarily be secondary to plc stimulation . brefeldin a , an inhibitor of certain arf - gefs , reduced receptor signaling to pld in several cell types , indicating that arf proteins participate in receptor - mediated pld stimulation ( fensome et al . 1998 ; mitchell et al . 1998 ; rmenapp et al . 1995 ; shome et al . likewise , sequestration of arf - gefs by the arf - related protein arp inhibited m3 muscarinic receptor signaling to pld ( schrmann et al . 1999 ) . clostridial toxins and enzymes that specifically inactivate rho proteins and expression of inactive rho mutants have been used to identify the role of rho in signaling to pld . thus , rho proteins were found to be involved in pld stimulation by gpcrs ( m3 muscarinic , bradykinin , sphingosine-1-phosphate and lpa ) , rtks ( pdgf , egf ) , and immunoglobulin ( fcri ) receptors ( hess et al . stimulation of pld by gpcrs was shown to be mediated by both pertussis toxin ( ptx)-insensitive ( gosau et al . 2002 ; schmidt et al . 1994 ) and ptx - sensitive ( cummings et al . g12 family proteins can stimulate pld ( plonk et al . 1998 ) , and rgs ( regulators of g protein signaling ) proteins , that act as  subunit - specific gaps , have been used to position g12 in pld activation by the m3 muscarinic ( rmenapp et al . 2001 ) , the par1 ( fahimi - vahid et al . 2002 ) , and the ca - sensing receptor ( huang et al . 2004 ) , as well as mechanical force ( ziembicki et al . 2005 ) . as forskolin and camp were shown to cause activation of pld via pka and erk1/2 ( ginsberg et al . 1997 ; yoon et al . 2005 ) or , alternatively , via the camp - activated gef for ras - like gtpases , epac and r - ras ( lpez de jess et al . . pld activation is also controlled by -subunits , possibly via src and/or arf6 ( le stunff et al . 1999 ) , but g can also directly interact with and inhibit pld ( preininger et al . 2006 ) . as the precise mechanism of pld stimulation in intact cells was only poorly understood , during the last 10  years our laboratory in essen has focused on the regulation of pld activity by membrane receptors . in hek-293 cells , signaling to pld by a typical gpcr , the m3 muscarinic receptor , and an rtk , the egf receptor , was studied and shown to be executed by several distinct pathways ( fig .  2 ) . in addition , by expressing inactive pld mutants , the m3 muscarinic and the egf receptors were found to signal to individual pld isozymes and to selectively stimulate pld1 and pld2 respectively ( han et al . the m3 muscarinic receptor stimulates both plc and pld via ptx - insensitive mechanisms ( offermanns et al . interestingly , stimulation of pld by the agonist carbachol was not affected by pkc inhibitors , suggesting that activation of pld by the m3 muscarinic receptor was rather independent of plc ( rmenapp et al . expression of -subunits of g proteins and of specific rgs proteins was used to identify the g proteins involved in these pathways , and demonstrated that whereas the m3 receptor signals to plc via gq proteins , activation of pld is mediated by g12 family proteins ( rmenapp et al . pld activation by the m3 receptor , but not by the egf receptor , was further found to be under control of arf ( rmenapp et al . 1995 , 1997 ) as well as rho proteins , particularly rhoa ( schmidt et al . likewise , regulation of mtor by lpa , but not pdgf , involved pld1 activation by rho gtpases ( kam and exton 2004 ) . both arf1 and rhoa were found to become activated after m3 receptor activation ( keller et al . 1995 ) , and a role for rho - kinase in rhoa - controlled pld stimulation could be demonstrated ( schmidt et al . 1999 ) . in further studies , it was shown that activation of pld by rhoa and rho - kinase is mediated by g12 and the tyrosine kinase pyk2 , whereas activation by arf1 is mediated by g13 , pi3k and the arf - gef arno ( han et al . rho proteins were shown to affect signaling to pld by both endothelin-1 and thrombin , apparently by controlling pip2 synthesis , whereas arf selectively affects signaling by the par1 receptor ( fahimi - vahid et al . 2regulation of pld by the m3 muscarinic receptor and receptor tyrosine kinases in hek-293 cells . in human embryonic kidney ( hek-293 ) cells , signaling to pld by the m3 muscarinic receptor and by typical rtks ( egf , pdgf , insulin ) is organized into rather discrete pathways and channeled by particular heterotrimeric g proteins and small gtpases ( orange ) , specific gef proteins ( pink ) and further signaling components ( green ) . ac , adenylyl cyclase ; rock , rho - kinase regulation of pld by the m3 muscarinic receptor and receptor tyrosine kinases in hek-293 cells . in human embryonic kidney ( hek-293 ) cells , signaling to pld by the m3 muscarinic receptor and by typical rtks ( egf , pdgf , insulin ) is organized into rather discrete pathways and channeled by particular heterotrimeric g proteins and small gtpases ( orange ) , specific gef proteins ( pink ) and further signaling components ( green ) . ac , adenylyl cyclase ; rock , rho - kinase pld can directly interact with rala , and a ras / ral signaling cascade was shown to regulate pld responses . in hek-293 cells , ras and rala  but not rho proteins  were located in rtk signaling to pld , and this ras / ral - dependent signaling cascade was found to be dependent on pkc- and a ral - specific gef ( fig . 2 ) ( schmidt et al . 1998 ; voss et al . 1999 ) . rala apparently co - operates with arf ( kim et al . 1998 ; xu et al . 2003 ) and rho proteins ( frankel et al . 1999 ; wilde et al . 2002 ) to achieve full pld activation . likewise , ras proteins were found to modulate pld responses by pdgf ( lucas et al . 2000 ) , and rala to affect egf receptor signaling to pld ( lu et al . 2000 ) . it was recently shown that direct activation of ras - related r - ras by epac is involved in pld stimulation by the m3 muscarinic receptor , apparently by coupling to gs proteins ( lpez de jess et al . 2006 ) , but a contribution of ral proteins to gpcr - induced pld activation has not been found ( meacci et al . collectively , these data demonstrate that heterotrimeric g proteins as well as small gtpases co - ordinate pld activation by specific membrane receptors in particular cell types , and these mechanisms probably contribute to the organization of agonist - induced pa production for the execution of diverse cellular signaling tasks . in addition , the synthesis of pip2 can be directly stimulated by gpcrs ( thrombin , lpa , m3 muscarinic ) as well as rtks ( cochet et al . receptor activation leads to increased association of pip5k with the actin cytoskeleton ( grondin et al . 1991 ; payrastre et al . 1991 ) , and receptor - induced stimulation and cytoskeletal association of pip5k may be directly involved in actin cytoskeletal regulation and initialize the assembly of enzymes into signaling complexes . gpcr - induced stimulation of pip2 synthesis was found to be mediated by pertussis toxin - sensitive gi proteins ( schmidt et al . 1993 ) , but also by g12 and gq proteins ( oude weernink et al . enhanced pip2 synthesis is also caused by conventional pkc isoforms , which may increase pip5k activity by stimulating pip5k dephosphorylation by the okadaic acid - sensitive protein phosphatase 1 ( park et al . in the last decade , pld has taken a firm position as all - round player in cellular signaling events . it is now appreciated that pld and pip5k act together to execute several important cellular functions , including vesicle transport , cytoskeleton dynamics and cell adhesion . because of the reciprocal stimulation of their activities it seems inappropriate to generally assign a conventional  upstairs - downstairs  relationship to pld and pip5k isozymes . the localized generation of the lipid messengers by pld and pip5k , pa and pip2 , is clearly co - ordinated by small gtpases of the arf , rho and ras families . the following picture emerges of how pld and pip5k may co - operate to execute their cellular tasks . particular small gtpases , activated by membrane receptors or cellular factors , bind to pip5k and recruit the enzyme to specific cellular compartments . subsequent activation of pip5k catalytic activity triggers the localized generation of pip2 , which now serves as an anchor for specific proteins , including pld enzymes . the sequestered pld is activated by pip2 and the gtpases , and pld - derived pa now , among other tasks , contributes to the activation of pip5k . this feed - forward regulation loop depends on both pip5k and pld , and quenching of pa formation ( by primary alcohols ) or reduction of pip2 levels ( by plc - mediated hydrolysis or dephosphorylation by phosphatases ) can interrupt the snowball from rolling . pip2 dephosphorylation may be important in the cell as a decisive mechanism to terminate the localized reactions before a cellular avalanche develops . attractive candidates are further specific gefs and gaps for the gtpases , some of which have been shown to be directly regulated by pip2 . pip2-dependent inactivation of the organizing gtpase may then provide the final turn - off signal .