Cellular Signalling Vol. 4 No. 1, pp. 11-23, 1992. Printed in Great Britain.

0898-6568/92 $5.00+ .00 © 1992 Pergamon Press plc

MINI REVIEW MODULATION OF HORMONE-SENSITIVE PHOSPHOLIPASE C G. GUILLON,* B. MOUILLACand A. L. SAVAGE Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Rue de la Cardonille, 34094 Montpellier, Cedex 5, France (Received 27 September 1991; and accepted 12 October 1991) Key words: Phospholipase C, G protein, GTP, calcium, inositol phospholipids, phosphorylation, tyrosyl protein kinase, EGF, cyclic AMP, ion fluxes, pertussis toxin, platelets, polyphosphoinositol lipids, depolarization, secretion, vasopressin.

Ins(1,4,5)P 3 generation and calcium release and the correlation between physiological events and inositol phosphate or diacylglycerol production have received a lot of attention and have been extensively reviewed [4, 5]. During the course of all these studies, the complexity of this signal transduction system has become apparent and, as for many biological systems, there exist positive and negative feedback loops which modulate and control the activity of phospholipase C and the physiological response. The aim of this review is to highlight those components of the signal transduction network which are likely to be involved in these feedback regulations and to discuss how their modulation may regulate the activity of agonist-stimulated phospholipase C. Since the activation of phospholipase C requires agonist : receptor interaction, G protein activation and an adequate supply of the PIP 2 substrate, the obvious targets for these feedback mechanisms are therefore the receptor, the G protein, PIP s and of course the phospholipase C enzyme itself.

1. INTRODUCTION IN RECENT years a link has been established between the interaction of Ca2+-mobilizing agonists with their receptors and changes in the activity of the phosphodiesterase enzyme, phospholipase C. The signal encoded in the agonist : receptor binding is relayed across the plasma membrane to phospholipase C via a guanine nucleotide binding protein (G protein) and upon activation, phospholipase C hydrolyses a minor component of membrane inositol phospholipids, phosphatidylinositol 4,5 bisphosphate (PIPE). Two molecules are produced from this hydrolysis: Ins(l,4,5)P 3 and diacylglycerol. DAG activates the Ca 2÷ and phospholipid dependent protein kinase C (PKC) and Ins(1,4,5)P3 diffuses into the cytosol and releases Ca 2+ from the endoplasmic reticulum. Thus, activation of phospholipase C generates two intracellular messengers which transmit the biological signal associated with the agonist to the intracellular environment (see Ref. [I] for review). Many studies have been devoted to the characterization of the components of this signal transduction system and to date at least three G protein subtypes [2] and five phospholipase C isoenzymes [3] have been described, although the significance of this diversity is unclear. Similarly the relationship between

2. REGULATION AT THE H O R M O N A L RECEPTOR LEVEL In the case of calcium mobilizing hormones, it has been demonstrated in many biological systems that there is a linear relationship between receptor occupancy and phospholipase C activation [6]. This implies

* A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

11

12

G. GUILLONet

that all modifications of receptor density and/or alteration of receptor properties such as phosphorylation may alter the hormone-stimulated inositol phosphates accumulation. Such phenomena may explain cross regulations among transmembrane signalling pathways and are illustrated below. WRK1 cells, a rat mammary tumour cell line, have vasopressin receptors which are coupled to PLC [7]. Prolonged exposure of the cells to dexamethasone induces a time- and dose-dependent increase in vasopressin-induced inositol phosphate accumulation. The potentiating effect of this steroid is mainly due to its action upon receptor synthesis, since (1) binding experiments showed that dexamethasone increases the density of vasopressin receptors and (2) treatment with cycloheximide, an inhibitor of protein synthesis, completely suppressed the steroid effect (G. Guillon, unpublished observations). Similarly, in rat basophilic leukemia cells, it has been demonstrated that addition of dexamethasone to the culture medium potentiated PLC activation in response to an adenosine analogue [8]. As for WRK1 cells, this enhanced responsiveness could be partly attributed to an increase in receptor number. Similarly cq-stimulated inositol phosphate accumulation was markedly increased in cortical slices from ovariectomized rats which had received repeated injections of oestradiol but both basal and carbamylstimulated phospholipase C activity were unchanged. As for dexamethasone, this effect of oestradiol probably reflects an increase in the number of ~ binding sites [9]. Many hormone receptors can be phosphorylated by protein kinase C and such phosphorylations have been shown, in some systems, to correlate with alteration in ligand binding properties. This probably explains why tumour promoting phorbol esters (activator of protein kinase C) generally decrease agonist-induced PIP 2 hydrolysis and calcium mobilization. In DDT-MF2 cells, for example, PMA inhibits norepinephrine stimulation of inositol lipid metabolism and also decreases the affinity of agonist binding to ~t~ receptors [10]. This altera-

al.

tion is closely related to phosphorylation of the ~ adrenergic receptor. Such alterations of calcium mobilizing receptors by agents which stimulate PKC may also explain the homologous desensitization phenomena observed in other systems [11-13]. The current hypothesis for desensitization involves diacylglycerol which is generated during hormone-induced PIP 2 hydrolysis and is an endogenous activator of PKC. Activated PKC may phosphorylate the hormone receptor and uncouple it from the G protein involved in PLC activation. This hypothesis has been confirmed in some systems; for example, in WRKI cells, staurosporine, a PKC inhibitor, is able to reverse the early phase of homologous desensitization (A. L. Savage, unpublished observations) and in rat glomerulosa cells, staurosporine potentiates the hormone-induced accumulation of inositol phosphates [13]. However, we cannot exclude the possibility that PKC also acts via the phosphorylation of the coupling G protein. This hypothesis will be discussed in the following section. In many systems, increasing the intracellular concentration of cAMP exerts a negative effect on hormone-sensitive phospholipase. In WRK1 cells, inositol phosphate accumulation induced by vasopressin can be greatly reduced by prior exposure of the cells to cholera toxin, forskolin (activators of adenylate cyclase) or dibutyril cAMP [14]. Similarly, in rat glomerulosa cells, corticotropin, which interacts with a receptor which is positively coupled to adenylate cyclase, also reduces the vasopressin-stimulated inositol phosphate accumulation [15]. In both cases, these inhibitory effects are due mainly to a reduction of the vasopressin receptor density thus reducing the pool of hormone receptors available for PLC activation and so reducing inositol phosphate accumulation. The effect of cAMP on receptor binding may be a result of phosphorylation via protein kinaseA and although this hypothesis was confirmed by Bouvier e t al. [16] for ~ adrenergic receptor, we cannot exclude a direct effect of cAMP at the level of receptor synthesis as discussed for the follitropin receptor [17].

Modulation of hormone-sensitive PLC 3. REGULATIONS AT THE G PROTEIN LEVEL In common with the adenylate cyclase system, the phosphoinositol signal pathway possesses distinct G proteins which differ in their sensitivity to bacterial toxins and couple the hormone receptors to the effector, phospholipase C (reviewed in Ref. [18]). In human platelets [19], alteration of Gi (the inhibitory G protein coupled to adenylate cyclase) by phosphorylation alters its ability to inhibit adenylate cyclase and so, by analogy, it is conceivable that a modification of the G proteins involved in PLC activation affects phospholipid turnover and may be responsible for some of the regulatory effects observed. The following examples illustrate this possibility in more detail. In intact mast cells and in HL60 cells, mastoparan, a toxin from wasp venom, induces a dose-dependent stimulation of PI breakdown [20]. This stimulation is pertussis-toxin-sensitive and so it was postulated that mastoparan, in some specific tissues, stimulates PI turnover by interacting with an IAP-sensitive G protein. Recent data favour this hypothesis since mastoparan interacts with a G protein and stimulates its GTPase activity [21]. Mastoparan therefore by-passes the receptor and stimulates PLC activity via activation of the Gi protein. Neomycin, an aminoglycoside antibiotic, probably stimulates inositol phosphate accumulation in permeabilized human platelets [22] by a similar mechanism, since this molecule is also able to stimulate GTPase activity in rat mast cells [23] in a pertussis toxin sensitive manner and to increase the affinity of the chemoattractant receptor in HL60 cells [24]. Retinoic acid was shown to inhibit rapidly and specifically the GTPyS-sensitive phospholipase C from permeabilized HL60 cells [25]. Since there was no effect on the basal level of inositol phosphate production, it was suggested that retinoic acid interfered at the level of the coupling G protein. The inhibitory effect of this substance on PI metabolism is probably of physiological importance since it seems to

13

correlate with its ability to induce cell differentiation. At least two distinct types of G proteins couple hormonal receptors to PLC; an lAP-sensitive G protein, such as Go and Gi and an IAP-insensitive one, such as Gq [26, 27]. All these proteins are heterotrimeric (0tflj, with a GTP binding site on the 0t subunit [18]. The fly subunits, which do not stimulate PLC, may be involved in the interaction of the G protein with the hormone receptor. Thus hormones activate PLC by interacting with their specific receptors; GTP then binds to the 0t subunit of the coupling G protein which subsequently dissociates into ~tGTP and fly. ~GTP may then activate PLC. Such a mechanism implies an equilibrium between the ct and fly subunits and so increasing the amount of fly should reduce the free concentration of ~ and so reduce PLC activity. Experiments using purified bovine brain fly protein confirm this hypothesis [28]. These results resemble those described for the adenylate cyclase systems. The inhibitory effect of fly upon adenylate cyclase activity represents a mechanism by which some hormones inhibit adenylate cyclase [29]. It is tempting to consider that some hormone inhibitions of PLC described earlier [30] could be explained by such a mechanism. Changes in the intracellular concentration of cGMP may also affect PI metabolism. However, it is impossible to generalize about these effects since the response to cGMP varies according to the system studied. In membrane preparations derived from bovine aortic smooth muscle cells, cyclic GMP was shown to inhibit both vasopressin-sensitive PLC and GTPase activities [31]. ATP was found to be absolutely essential for this effect. This suggests that cGMP stimulates a cGMP-dependent protein kinase which presumably phosphorylates the coupling G protein since neither the vasopressin receptor nor the basal PLC activities were modified. Such results imply that agents which increase the intracellular concentration of cGMP (nitroprusiate or atrial natriuretic factor) may be involved in a feedback inhibition of PI hydrolysis [32]. However, in cultured

14

G. GUILLON et al.

pituitary cells [33] increasing the cGMP concentration led to an increase in basal inositol phosphate accumulation and in rat hepatocytes [34], 8-bromo-cGMP treatment affected neither the basal nor vasopressin-stimulated levels of inositol phosphate formation. Among the other cyclic nucleotides, cAMP inhibits sodium fluoride-sensitive phospholipase C activity in human platelets [35] and mouse fibroblasts [36]. It has been implied from studies in mouse fibroblasts and neuroblastoma cells that cAMP-dependent protein kinase phosphorylates a low molecular mass G protein probably involved in PLC activation [36, 37]. As for cGMP however, different results have been obtained on rat hepatocyte. In these cells, 8-bromo-cAMP potentiates the vasopressinstimulated inositol phosphate accumulation [34]. The inhibitory effect of protein kinase C activators on hormone-sensitive PLC is now well established [11-13] and phosphorylation of the coupling G protein involved in these transduction mechanisms remains an attractive hypothesis for this inhibition. In human platelets, the thrombin receptor is coupled to PLC via G proteins. G i is involved in these mechanisms but as the thrombin-stimulated inositol phosphate accumulation is only partially inhibited by IAP treatment, other IAP-insensitive G proteins are probably involved. In this system [38], PKC activation by phorbol esters or homologous desensitization induced by a preincubation with high concentration of thrombin were shown to reduce thrombin-activated inositol phosphate accumulation and to phosphorylate ct G protein. It is thus tempting to consider that, in this system, the phosphorylation of G proteins, induced by PKC activation, represents the mechanism for homologous desensitization [38]. However, in these systems, activation of PKC, either by phorbol ester or homologous desensitization, only marginally affects PLC activities stimulated by GTP),S or NaF despite the fact that such treatments greatly inhibit hormone-activation of PI turnover [13]. There are two possible explanations for this; (1) the phosphorylation of the receptor uncouples it

from the G protein or (2) the phosphorylation of the G protein prevents its interaction with the receptor without affecting its coupling to the PLC and so does not affect the catalytic activity of the enzyme. Results from studies on DDT l MF 2 cells [39] favour the first hypothesis since phosphorylation of the cq receptor by PKC prevents its coupling with the G protein. However, we cannot exclude the second possibility since the 'ctq family' of IAP and CT insensitive G proteins involved in these mechanisms has not been studied extensively and, as shown in Table 1, unlike cti-like G proteins, little is known about the phosphorylation of the '~q' G protein. 4. REGULATION AT THE PHOSPHOLIPASE C LEVEL Recently different PLC isoenzymes have been purified and cloned, which has greatly increased our knowledge of the enzymatic, immunological and physiochemical properties of these enzymes [3]. This information should improve our understanding of how these enzymes are activated in response to receptor occupation and how they can be regulated. In this section we will try to analyse how a modification of the PLC structure (a phosphorylation for example) or a modification of the PLC cofactor (calcium) may affect and regulate the activity of hormone-sensitive PLC.

Regulation of phospholipase C by phosphorylation Activators of protein kinase C such as phorbol esters, inhibit hormone-stimulated but not basal PLC activity [11, 13] and although PKC phosphorylates ct, fl, ), and 6 isoforms of PLC (see Ref. [3] and Table 2) it is unclear whether or not these phosphorylations are physiological regulations since few in vitro studies have shown that phosphorylation affects the catalytic activity of PLC [40]. Perhaps in vivo, such a phosphorylation prevents the interaction of the catalytic subunit of the PLC with the coupling G protein which

Modulation of hormone-sensitive PLC

15

TABLE 1. PHOSPHORYLATIONOF G~ PROTEINSINVOLVEDIN SECONDMESSENGERGENERATION Tissue

Effector

Rat hepatocyte

AVP, ATII, ai2 glucagon, 8bromo-cAMP, PMA PMA az

Human platelet Human Thrombin platelet Dictostelium cAMP Human PKC platelet

G protein phosphorylated

ai G~2 ~i

G,, protein Effect of phosphorylation unphosphorylated

Reference

ai3, as

79

ai -

-

-

-

-

-

flT~i

may explain why activation of PKC only inhibits hormone-induced accumulation of inositol phosphate and not basal levels. Agents which increase intracellular cyclic AMP concentration like cholera toxin, forskolin or hormones positively coupled to adenylate cyclase, also seem to reduce the activity of agonist-sensitive phospholipase C. As seen in Table 2, when cAMP is increased, PLC is phosphorylated on serine residues only but no modification of basal PLC V activity was observed in vitro after phosphorylation. However, in NCB-20 cells, a neuro tumour cell line [37], the situation was reversed; no phosphorylation of PLCII isoenzyme was detected when intracellular cAMP concentration was raised despite a significant reduction in inositol phosphate accumulation, suggesting that (1) cAMP may affect other levels of the PI metabolism cascade, or (2) that PKA only phosphorylates specific isoforms of PLC. Recently some very interesting data concerning the regulation of PLC activity by phosphorylation have come from studies with growth factors. Growth factors are known to stimulate tyrosine kinase activity, phosphorylation of certain PLC isoenzymes and inositol phosphate metabolism (see Table 2). All these effects appear to be linked. In NIH 3T3 cells, Kim etal., demonstrated that PDGF, on binding to its receptor, associates with and CELLS 4:I-B

Loss of ability of GTPv to inhibit forskolinstimulated adenylate cyclase activity Possible attenuation of PLC activation -

-

Loss of ability of somatostatin to inhibit adenylate cyclase activity

80 81 82 19

phosphorylates tyrosine and serine residues of PLCyt which then increases inositol phosphate production [41]. Substituting one tyrosine residue (Tyr 783) on PLCTI with phenylalanine completely suppressed the PDGF-induced PI metabolism but the mutation did not affect the interaction between PLC and the P D G F receptor. From these data, it was concluded that PDGF-induced inositol phosphate accumulation requires the association of P D G F receptor with PLCy~ which is then phosphorylated on a tyrosine residue [41]. Similarly Margolis et al. demonstrated that when EGF binds to its specific receptor on A431 cells, PLCII interacts with the EGF receptor and is rapidly phosphorylated on a tyrosine residue [42]. As a result of this phosphorylation, Ins(1,4,5)P3 is produced and calcium is released from intracellular stores. Since tyrosine phosphorylation of PLCy~ increases its catalytic activity both in vivo and in vitro [40], it is tempting to conclude that tyrosine kinase receptors promote PI metabolism indirectly via the phosphorylation of PLC subunits. This effect may not be limited to growth factor receptors because in human T cells, an antibody to CD 3 complex also increases the phosphorylation of PLCT~ and, as for EGF and P D G F receptors, this phosphorylation is probably responsible for the inositol phosphate accumulation observed. However T antigen receptors, unlike

16

G. GUILLON et al.

TABLE 2. PHOSPHORYLATION OF PHOSPHOLIPASE C

Cell line

Effector

Rat gliomai cell (C6Bul)

cAMP

PLC isoform phosphorylated

Influence of PLC phosphorylation PLC isoform not or effector on PI phosphorylated metabolism

PLC PLCfl and 6 phosphorylated on serine residue

Murine Fibroblast Choleratoxin PLC z (BAL B/c3T3) forskolin phosphorylated on serine residue

Murine Fibroblast Phorbol (NIH 3T3) ester

PLC~ and 3 PLC fl phosphorylated on serine and tyrosine residues

Human T cell

PLCTI PLCfl, phosphorylat~i on serine and tyrosine residues

T cell antigen

Murine Fibroblast PDGF (NIH 3T3) Murine Fibroblast EGF (NIH 3T3)

PLCTI phosphorylated on serine and tyrosine residues PLC?t pbosphorylated on tyrosine residues

Rat liver epithelial EGF cell (WB)

PLC~

Rat NGF pheochromocytom (PC 12)

PLC),

E G F or P D G F receptors, does not exhibit a tyrosine activity [43]. It is perhaps worth mentioning that the ability of E G F to stimulate inositol phosphate production seems to depend on the cell type. Rat hepatocytes, for example,

In vitro, no effect on PLC catalytic activity In intact cells, inhibition of hormonalinduced inositol phosphate accumulation. In vitro, inhibition of sodium fluoride sensitive PLC, with no alteration of PDGF-stimulated inositol phosphate accumulation. In vitro, no effect on PLC catalytic activity In intact cells, inhibition of hormonal induced inositol phosphate accumulation. In intact cells, activation of antigenstimulated inositol phosphate accumulation Tyrosine phosphorylation was essential for PDGF activation of PLCy~. In vitro, activation of PLC ~,~ activity.

In intact cells activation of inositol phosphate accumulation. In intact cells, activation of inositol, phosphate accumulation

Reference 83

36

84

43 and 85

41

40

86

87

possess E G F receptors and their stimulation leads to an increase in intracellular Ca 2+ concentration without accumulation of IP3 (see Ref. [44] and also A . L . Savage and B . R . Martin, unpublished observations). One

Modulation of hormone-sensitivePLC possible explanation for this is that the phospholipase C isoenzyme present in hepatocytes is not a substrate for tyrosine kinase.

Regulation of phospholipase C by Ca 2+ The phospholipase C enzyme requires calcium to hydrolyse polyphosphoinositides and increasing free calcium concentration in the incubation medium leads to a dose-dependent activation of inositol phosphate accumulation [45]. The concentration of calcium which allows half activation of PIP2-PLC (ECs0) is about 0.1-1 #M depending on the system tested. This value is in the range of intracellular free calcium, thus all molecules which modify intracellular calcium concentration may affect the PIP2-PLC activities and be responsible for positive or negative regulations. The following examples illustrate such possibilities. In many cell lines, adding ionomycin to the incubation medium increases both intracellular calcium concentration and inositol phosphate accumulation [46]. This last effect is probably due to calcium influx generated by ionomycin since it disappears if extracellular calcium is omitted from the incubation medium. These results may explain why in some biological systems a calcium influx is often associated with an increase in inositol phosphate accumulation [47]. In human skin fibroblast for example, removing extracellular sodium, which promotes calcium influx via the Na+/Ca 2+ exchange, evokes large increases in cytosolic free calcium and inositol phosphates accumulation [48]. Similarly in HIT-T15 cells, an insulin secreting cell line, glucose is able to stimulate inositol phosphate accumulation but since this effect is blocked by the calcium channel antagonist, verapamil, and can be mimicked by depolarization, the authors concluded that the increase in phospholipase C activity is secondary to an increase in cytosolic calcium concentration [49]. Recently, a lot of interesting data on the activation of phospholipase C by calcium has been obtained from studies using rat glomerulosa cells. In these cells, vasopressin-stimulated phospholipase C activity is reduced in the

17

absence of external calcium. Furthermore, both 45Ca2+ influx and inositol phosphate accumulation can be totally and partially inhibited, respectively, by treating the cells with IAP. In membranes, however, IAP has no effect on vasopressin-stimulated inositol phosphate accumulation although phospholipase C remains sensitive to Ca 2÷. In intact cells, therefore, there appear to be two mechanisms for stimulating phospholipase C; a direct stimulation of phospholipase C as a result of receptor binding and secondly, an activation of the enzyme which occurs as a result of IAP-sensitive, vasopressin-stimulated Ca 2÷ influx. This CaZ+-influx pathway is not functional in acellular systems, which explains why phospholipase C activity is not sensitive to lAP treatment in membrane preparations [47]. Calcium activation of phospholipase C may also be induced in the rat glomerulosa cells through the activation of the ACTH receptor which is positively coupled to adenylate cyclase but, when activated, also stimulates calcium influx via an L-type channel. Inositol phosphate accumulation is stimulated only at low doses of ACTH and it has been proposed that phospholipase C activated by Ca 2+ influx because there is no ACTH-stimulated inositol phosphate accumulation in the presence of the non-specific calcium channel blocker CoCI 3 [50]. In these last examples, Ca 2÷ entry has been induced by a variety of means and in each case phospholipase C activity is increased. In support of this role for Ca 2÷ as an activator of phospholipase C, it has been shown that reducing calcium influx or intracellular calcium concentration inhibits PI turnover. Recent studies on rat pituitary cells illustrate this point. These cells possess TRH, ATI and dopaminergic D2 receptors. Treatment of these cells with dopamine inhibits both TRH or ATl-stimulated calcium influx and inositol phosphate accumulation [51, 52]. This inhibition of inositol phosphate production is linked to the suppression of calcium influx since agonist-inositol phosphate production is reduced in the absence of extracellular calcium or in the presence of nimodipine, a calcium

18

G. GUILLONet al.

channel antagonist. Similar dopaminergic regulations of phospholipase C activity have been described in human trophoblast [53] and on rat glomerulosa cells [54]. This effect of dopamine is perhaps of physiological importance since it may explain the inhibitory mechanism of dopamine on secretion processes. Similarly, galanin, a neuropeptide, was shown to reduce carbachol stimulation of inositol phosphate accumulation in rat ventral hippocampus by lowering calcium influx [55]. These results suggest also that regulation of calcium channel activity may indirectly modulate PI turnover by affecting the intracellular calcium concentration. The role that Ca 2+ plays in the regulation of phospholipase C in the rat hepatocyte seems to be slightly different from other cells. Renard e t al. [56] used quin 2 to monitor and fix the cytosolic Ca 2+ concentration to known values in intact hepatocytes and showed that PIP2-sensitive phospholipase C was partially dependent on Ca 2÷ at concentrations below basal values but was not affected when Ca 2+ was raised from 0.2 to 1/~M with ionomycin. Similarly Wallace and Fain [57], working with liver plasma membranes, showed that Ca 2÷ concentrations as high as 6 #M had no effect on PIP 2 breakdown whereas during cell activation the concentration of intracellular Ca 2÷ rises from 0.2 #M to 1.5/~M [58]. In rat hepatocytes therefore, it appears that phospholipase C requires a basal, internal Ca :+ concentration (0. lq2.2 #M) for activity but raising Ca 2+ alone within physiological limits does not stimulate PIP: breakdown. 5. REGULATION OF PHOSPHOLIPASE C ACTIVITY AT THE SUBSTRATE LEVEL

In many cell systems the inositol phospholipid pool is of limited size and rapid resynthesis of the phospholipase C substrate, Ptdlns(4,5) bisphosphate, is essential for maintaining hormone-stimulated phospholipase C activity. Clearly, therefore, if the rate of supply of the substrate is altered, phospholipase C activity will change. There are several exogenous substances which can regulate phospholipase C

activity at the substrate level by interfering either with the inositol phospholipid pool itself or the enzymes responsible for its generation. For example, neomycin is known to interact with inositol phospholipids [59] thereby hindering the supply of the substrate to phospholipase C and so reducing its turnover [60]. Similarly, an antibody to Ptdlns(4,5)P2 has been developed which specifically interacts with the lipid and suppresses its hydrolysis by phospholipase C [61]. This antibody blocked mitogenesis in PDGF and bombesin-treated NIH 3T3 cells [61] and abolished PDGF-induced Ca2+-induced Ca 2÷ mobilization in vascular smooth muscle cells [62]. Similarly, profilin, an actin binding protein, inhibits cytosolic phospholipase C activity in platelets by competing with PLC for interaction with PIP 2 [63]. Furthermore, a variety of substances such as polyamines, histones and polylysines can increase the activity of Ptdlns 4-kinase in A431 cells and Ptdlns(4)P kinase in neutrophils can be activated by leukotriene B 4 and chemoattractants, although the physiological relevance of these activations is not known (reviewed in Refs [64, 65]). Endogenous compounds such as cyclic AMP may also regulate Ptdlns kinase activities. In yeast mutants of the ras gene, for example, which produce low levels of cAMP, Ptdlns and Ptdlns(4)P kinase activities are low but addition of cAMP increases the incorporation of 32p into polyphosphoinositol lipids [66]. However, addition of exogenous cAMP or agents which increase intracellular cAMP concentrations may, in many eukaryotic cells, inhibit Ptdlns kinases [64]. Whether or not these effects are due to PKA activation or to a direct effect of cAMP or its derivative (namely adenosine) remains controversial [67]. Moreover, data from rat brain imply that phosphatidylinositol 4-phosphate kinase activity may be regulated by a GTPyS and thus by a GTP binding protein [68]. Studies in human platelets have shown that the tumour promoting phorbol ester (TPA), when used at concentrations which stimulate PKC, increases the pool of Ptdlns(4)P and

Modulation of hormone-sensitivePLC Ptdlns(4,5)P 2 but decreases the Ptdlns pool. The authors of this study suggested that TPA activates both the Ptdlns and Ptdlns(4)P kinases and proposed a positive feedback regulation of agonist-sensitive PIP:-PLC such that diacylglycerol, produced by the primary action of the agonist on PLC, activates PKC which then stimulates Ptdlns kinase to produce more PIP 2. This replenishment of the Ptdlns(4,5)P2 pools would sustain PLC activity and the hormone response [69]. Recently it has been shown that hormone receptors exhibiting tyrosine kinase activity are often closely associated with Ptdlns kinases. In mouse B82L cells for example, Cochet et al. [70] found that the EGF receptors interact with phosphoinositol lipid kinases and addition of EGF resulted in tyrosine phosphorylation of Ptdlns(4)P-5-kinase and an increase in its activity. In Chinese hamster ovary cells [71] which overexpress insulin receptors, addition of insulin produced a very large stimulation of Ptdlns kinase activity which was related to the insulin receptor tyrosine kinase activity, suggesting that Ptdlns kinases may be substrates of the insulin receptor tyrosine kinase. PDGF receptors which similarly exhibit a tyrosine kinase activity were also found to be associated with the phospholipid kinase but in this case it was linked to Ptdlns(3)P kinase which produces a novel phospholipid,

•• I

v

Ins (l,4,s)Ps OAG

Ptdlns(3)P (reviewed in Ref. [63]). Since increasing the amount of PLC substrates may increase the intracellular concentration of second messengers generated by PLC, these data may explain how activation of tyrosine kinase receptors is able to stimulate indirectly inositol lipid metabolism in many cell systems. However it is not yet clear if this Ptdlns kinase phosphorylation alone is sufficient to activate Ptdlns kinase activities. 6. INTEGRATION OF MULTIPLE REGULATIONS: AN OVERVIEW At least four distinct mechanisms activate phosphatidylinositol lipid turnover. The most widespread mechanism involves the interaction of the hormone receptor with phospholipase C via a coupling G protein (Fig. 1, I). Some other hormones or regulating molecules, for example molecules which activate kinases, stimulate inositol phosphate accumulation via the phosphorylation of the catalytic unit of PLC (Table 2). Two types of mechanism have been described: the hormone receptor complex has an intrinsic tyrosine kinase activity and PLC is directly activated by this complex (Fig. 1, II); alternatively the hormone receptor complex activates a tyrosine kinase distinct from the hormone receptor and, once activated, this kinase which is often a protein encoded by an

C1¢"

,.,.

ATP

PIP2

1 C

m (1,4,S)Ps DAG

®

19

®

Ins {1/i,5~8

DAG

@

®

F[~. 1. Mechanisms of phospholipase C activation. Abbreviations: G--G protein; PLC--phospholipase C; PLC, P--phosphorylated PLC; DAG---diacylglycerol; TKR--tyrosine kinase receptor; NTRK--non tyrosinekinase receptor; TK--tyrosine kinase.

20

G. GUILt~Net al.

oncogene, phosphorylates the PLC and activates it (Fig. 1, III). Growth factors and cell antigens generally stimulate PI metabolism by such mechanisms [41-43]. At the present time, only the molecular mechanisms involved in G protein activated PLC have been studied in depth and, as a consequence, the majority of studies are devoted to the modulation of hormonal sensitive PLC concerned with these systems. As summarized in this article, the modification of any part of this multienzyme complex (hormone receptor, coupling G protein and PLC) may affect the activation processes, e.g. modulation of hormone receptor density, modification of the catalytic properties of the coupling G protein or phospholipase C and regulation of the pool of PLC substrate. Regulation at the level of the myo-inositol transport across the plasma membrane may also represent another level of regulation since (1) many cells need extracellular myo-inositol to survive and divide, (2) some hormones and Didemnine B, an extract from cyclodepsipeptide isolated from Carribean tunicate, affect intracellular inositol influx (M.N. Dufour and G. Guillon, unpublished results), and (3) protein kinase C activators stimulate inositol transport in HL60 cells [72]. The regulation at the level of the inositol phosphate phosphatases also represent another interesting possibility for future study. These possibilities for regulation may in part explain the cross talk between the different cellular signalling pathways. The best studied is the influence on PI metabolism of agents which increase intracellular cyclic AMP. The various ways in which cAMP affects hormone-sensitive PLC activities are summarized in Fig. 2. Whether or not all these regulatory steps are present in the same cell remains to be shown although this is unlikely since, according to the cell type studied, an increase in cAMP can either potentiate inositol phosphate production [34] and IP3-induced calcium release or inhibit this response [15, 35]. Similarly, as suggested in a recent study on human platelets, 8-bromocGMP inhibits thrombin-stimulated IP

'i

FIG. 2. Cross-talk between the cAMP and inositol phosphates pathways. Abbreviations: R~--receptor coupled to phospholipase C; G--G protein involved in activation of PLC; PLC--phospho!ipase C; R2--receptor coupled to adenylate cyclase; AC--adenylate cyclase; Gs--G protein involved in activation of AC; PKA--protein kinase A; DAG--diacylglycerol; PI--phosphatidyl inositol; PIP--phosphatidyl inositol 4 phosphate; PIP2--phosphatidyl inositol 4,5 bisphosphate.

accumulation suggesting that agents which increase intracellular cGMP may also be considered as modulators of PI metabolism [31, 33]. If the second messengers generated by hormone transduction systems other than the phospholipase C/Ca 2+ signalling pathway can affect PIP 2 hydrolysis, then the products of PIP 2 hydrolysis can also modulate the production of other signal molecules. For example, diacylglycerol and phorbol esters (PKC activators) enhance hormone and GTP-stimulated adenylate cyclase activity in some mammalian cell lines [73]. Phosphorylation by PKC of the inhibitory GTP binding protein 'Gi' [18] or the catalytic unit of the adenylate cyclase probably explains these sensitization effects [74]. On the other hand phorbol esters phosphorylate the f12 adrenergic receptor thereby uncoupling it from the stimulatory GTP binding protein 'G s' and reducing isoproterenol-sensitive adenylate cyclase activity [73]. The regulation of the PI metabolism induced by other mechanisms, Sections 3 and 4, has not been extensively studied since it has only recently been reported that receptors exhibiting

Modulation of hormone-sensitivePLC tyrosine kinase activity can phosphorylate and activate PLC. Moreover, other kinases of the src family are associated with the tyrosine kinase receptor, which raises the possibility that these additional kinases may play a direct or indirect role in the regulation of PLCy tyrosine phosphorylation and its activation (reviewed in Ref. [75]). Whether PLC is activated by Ca 2+ or whether increases in Ca :+ concentration simply modulates its activity remains an open question. In some systems the evidence favours a regulatory role for Ca 2+. In pituitary cells, for example, dopamine by acting on D 2 receptors reduces calcium influx and thereby decreases hormone-stimulated inositol phosphate accumulation [51, 52]. In many other systems a calcium ionophore such as ionomycin, which does not interact with hormone sensitive PLC, stimulates PLC activity [45, 46]. Similarly, in cerebral cortex slices, the excitatory amino acid quisqualate stimulates inositol phosphate accumulation following ionotropic receptorinduced depolarization and Ca 2+ entry. This PLC response is highly dependent upon external calcium unlike the muscarinic response in the same cells which involves a coupling G protein [76]. These examples show how calcium activation of PLC regulates PI metabolism, but in many systems it is difficult to separate clearly a direct activation of PLC via its interaction with a receptor activated G protein, from a calcium stimulation of the catalytic subunit since (1) Insl,4,5P 3 generated by the first mechanism releases Ca 2+ from the endoplasmic pool which may in turn reactivate PLC [45], and (2) many hormone receptors like vasopressin, angiotensin I, 0q adrenergic, excitatory amino acids both stimulate calcium influx and PLC activation [47, 77]. In conclusion, PLC can be activated directly or indirectly via different mechanisms and many drugs, hormones or ions may modulate PI-turnover by affecting different steps of the enzymatic cascade which leads to PLC activation. However, cAMP, for example, may either activate or inhibit PLC activity depending on the cell type considered, which indicates that

21

there is a large degree of variability in the control of PI-metabolism. This may be accounted for by (1) the existence of distinct isoforms of PLC which exhibit similar catalytic activity but have rather distinct immunological and physicochemical parameters and exhibit poor homology in their sequences [3], (2) the variation in the nature of the G proteins which couple hormone receptors to PLC but differ in their molecular weight and in their ability to be ADP-ribosylated by pertussis toxin [2, 18, 26], and (3) a distinct cellular distribution of the different PLC isoforms and G proteins. For example Go is more abundant in neural tissues [78] and PLC isoforms are expressed differently between and within tissues and in individual cells (reviewed in Ref. [3]). Thus the variation in the modulation of phospholipase C between different cells can probably be explained by the presence of different combinations of at least five distinct PLC isoenzymes and three G proteins. The recent data concerning the influence of tyrosine kinase receptors and non-receptor tyrosine kinases of the src family in the phosphorylation of PLC also contribute to the complexity of such regulatory mechanisms. Thus by characterizing the combination of PLC isoform and G protein present in a given cell, we may reach a better understanding of how the hormone-response in that cell can be modulated. Acknowledgements--We are grateful to Mrs PAOLUCCI and J. HUET for typing the manuscript, Mrs M. PASSAMAfor drawing the figures and Mr S. JARD for many stimulating discussions. A.L.S. is a recipient of a Wellcome Trust Fellowship.

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