Biochimica et Biophysica Acta, 1092 (1991) 49-71 © 1991 Elsevier Science Publishers B.V. 016%4889/91/$03.50 ADONIS 0167488991001198

49

Minirev|ew

BBAMCR 12881

The PtdIns-PLC superfarni! y and signal transduction Eric Meldrum, Peter J. Parker and Amanda Carozzi Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London (U.K.) (Received 2 August 1990) (Revised manuscript received 10 October 1990)

Key words: Phosphoinositide; Phospholipase C; Phosphatidylinositol; Signal transducation

Contents

II.

The phosphoinositides and their importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inositoi phospholi~iOb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Inositol phosphoi'pids in signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 49 50

Characteristics of the Ptdlns-PLC superfamily members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Initial studies on Ptdlns-PLCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Current members of the Ptdlns-PLC superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Biochemical properties of Ptdlns-PLCs in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52 52 53 56

III. Receptor mediated stimulation of PtdIns-PLC activity in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Basal Ptdlns-PLC activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Receptor types which couple to PtdIns-PLC enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. G protein coupled PtdIns-PLC activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ptdlns-PLC activities coupled to tyrosine kinase receptors . . . . . . . . . . . . . . . . . . . . . . . . . . .

59 59 60 61 63

IV. Heterologous regulation of Ptdlns-PLC activity in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64

V. Summaryand perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 67 67 67

I. The phosphoinositides and their importance I.A. Inositol phospholipids The phosphoinositides are a small group of membrane phospholipids which are unique in that their myo-inositol headgroup can be phosphorylated at multiAbbreviations: Ptdlns-PLC, phosphoinositide specific phospholipase C; Ptdlns, phosphatidylinositol; Ptdlns(4)P, phosphatidylinositol 4phosphate; Ptdlns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Cyclns(l:2,4,5)P3, inositol 1,2-cyclic 4,5-trisphosphate; GTP¥S, guanosine 5'-O-(3-thiotriphosphate); DAG, diacylglycerol; PA, phosphatidic acid; PMA, phorbol12-myristate-13-acetate; A kinase, cAMP dependent protein ~nase; PKC, protein kinase C; mACHR, muscarinic acetylchofine receptor. Correspondence: A. Carozzi, Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, U.K.

pie sites [1]. They form a minor component of most, if not all, eukaryotic membranes, with the three most predominant phosphoinositides being phosphatidylinositol (Ptdlns) which forms the majority, phosphatidylinositoi 4-monophosphate (Ptdlns(4)P) and phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2); the numbers referring to the position of the phosphates on the inositol ring (Fig. 1). Like most glycerophospholipids, Ptdlns, Ptdlns(4)P and Ptdlns(4,5)P2 are comprised of an sn-l,2-diacylglycerol backbone with the headgroup linked to the 3 carbon, in this instance, through the D-1 hydroxyl of the inositol ring (reviewed in Ref. 3). Collectively, these three lipids usually constitute less than 8% of total m e m b r a n e phospholipids [1] and were originally isolated together as a mixture referred to as the 'diphos-

50

oo Ip_o_

oI O=P-O-

I

3

o

O ~) OHI~I I'~(~ .j.i "~(~'I~) H2

O

O

O mPII - O -

O-'PII - O -

0

--Idnas¢ phosphalase

~1 H ~ .__CH i 2 H_____

I

O-

0

_kinase

H I H~ - - C "~G H2

pllcx~phal~

0

0

0

Phoq}halidylinosild (Ptdlns) (rnon~~nositide)

Phoq~alidylinosilol & p h o s p h a t e (Pldlns(4)P) (diphosphoinositide)

Phosphalidylinosilol 4,5-bisphosphate (Ptdlns(4,5)P2) (triphosphoinosilide)

Fig. 1. Structure of the phosphoinositides in brain. The phosphoinositides are typical phospholipids in that they comprise an sn-l,2-diacyiglycerol backbone with the 3-carbon of the glycerol covalently attached, via a phosphate, to the headgroup which in this case is inositol. Analysis of the fatty acids within the diacylglycerol of brain [2] (but not necessarily other tissues) has indicated that a large proportion of the phosphoinositides possess stearate (C18:0) on the 1 carbon and arachidonate (C20:4) on the 2 carbon of glycerol. The only structural distinction between these phosphoinositides is the degree of phosphorylation on positions 4 and 5 of the inositol ring, allowing these three lipids to interconvert through a series of phosphorylation/dephosphorylation reactions.

phoinositide fraction' from bovine brain [4]. They have been shown to interconvert via a series of successive phosphorylation and dephosphorylation reactions [5] which are governed by specific kinases and phosphatases [6]. The cytoplasmic or plasma membrane location of these enzymes has indicated that these three lipids readily interconvert in the plasma membrane where the metabolism of one of the phosphoinositides has the potential to influence the levels of all three (Fig. 1.) [7]. Other inositol containing membrane lipids have been isolated from both eukaryotes and prokaryotes including the complex sphingolipids (reviewed in [!]), the mannosides of Ptdlns found in mycobacteria [8,9] dimyristoyl-Ptdlns which has been shown to be linked via a glycan bridge to ectoproteins [10] and the recently reported phosphoinositides phosphorylated on the three position of the inositol ring [11,105,106]. While these lipids, too, are potential substrates for phospholipases, this review is concerned predominantly with phospholipase C activities directed against PtdIns, Ptdlns(4)P and Ptdlns(4,5)P2.

L R Inositol phospholip~ds in signal transduction Interest in the phosphoinositides as something other than merely unusual membrane components began when Hokin and Hokin found that the increase in lipid radio-

labelling, which accompanies secretion, in agoniststimulated exocrine pancreas was largely confined to the two lipids, Ptdlns and phosphatidic acid (PA) [12,13]. This phenomenon was subsequently referred to as the 'Pl-response' and has since been found to extend to a wide variety of agonists mediating many cellular responses (reviewed Ref. 14). When the nature of the increased labelling of Ptdlns was examined it was found, in general, that there was a striking increase in the incorporation of 32p and [3H]inositol into Ptdlns but little increase in []4C]glycerol into the molecule, with the labelled PA reverting to Ptdlns upon removal of the stimulus (reviewed Ref. 15). This indicated that there was renewal of the inositol headgroup onto an unmetabolised diacylglycerol backbone from which it was proposed that agonist stimulation activates a phospholipase C which hydrolyses Ptdlns to diacyglycerol (DAG) and inositol phosphate. The DAG is then phosphorylated to PA which is in turn re-incorporated into Ptdlns (Fig. 2). Accordingly, it was found that appropriate stimuli caused a substantial net decrease in Ptdlns within the membrane [16,17], further establishing that the primary receptor-mediated event involves the removal of Ptdlns and that the subsequent fipid labelling which constitutes the observed 'PI response' is a secondary effect.

51 inositoi phosphate

O'-P--O I O-

I ,

J phospholipase D

=J

J........?

c

6

_

O-'P--O

H

I

H H2C--C

I

I

phospholipase A+

i....o O "-C

I

OH I

? .......... J phospholipase C

?

---CH 2

o ................I

O-----C

phospliolipase A 2 J

~= O >

o ~= 0 >

ADP

)

o-,,-o

) >

]) >

ADP

,> sn-1,2 diacylglycerol

phosphoinositide --,

hosphalidic acid

J --

5

..,,.

3H

myo-[2-3H] inositol

O

O -H

H2C - - C I

I

--O

0,

I

---CH 2

? ? O----C

---- O

phosphatidic acid Fig. 2. The 'PI response'. This phenomenon, which involves the agonist induced radiolabelling of Ptdlns and phosphatidic acid (PA), is explained if the phosphoinositide is cleaved by a phospholipase C to give diacylglycerol (DAG) and an inositoi phosphate. In the presence of [7-32p]ATP, the DAG is converted to PA which is reincorporated, with tritated inositol, into a dual labelled PtdIns molecule. The Ptdlns can then be converted to PtdIns(4)P and PtdIns(4,5)P2 via a set of kinase reactions as outlined in figure 1. In the diagram above, the groups on positions 4 and 5 of the inositol ring (designated ®) may be either hydroxyls or phosphates, depending on the particular phosphoinositide that is the target, for phospholipase C. The sites of action of other phospholipases are also indicated, illustrating the distinction between the outcome of each hydrolysis.

In consideration of a number of observations, this increased Ptdlns turnover was subsequently proposed to be involved with receptor function [18,19], possibly acting as a signal transduction system for those cell surface receptors which do not operate through cAMP [20]. In addition, a connection was made between agonists which were known to elicit a rise in intracellular Ca 2+ and those generating a change in PtdIns metabolism [21] from which Michell proposed that

PtdIns turnover actually induced the C a 2+ rise. In support of this, reports examining C a 2 + deprivation in parotid glands [22,23] indicated that PtdIns turnover could occur without a rise in free Ca 2+ and studies involving the stimulation of blowfly salivary gland by 5-hydroxytryptamine in inositol-free medium suggested that a functional PtdIns metabolism was indeed necessary for the initiation of the Ca 2+ induced response in that tissue [24,25]. Nevertheless, several groups advoc-

52 ated that in certain cell systems the C a 2+ rise was necessary to induce the Ptdlns turnover (see Refs. 26 and 27), a misgiving strengthened by studies indicating that the PtdIns-PLCs isolated at that time, all required Ca 2+ concentrations above resting levels in order to act on Ptdlns in vitro (see section II.A.). The question of calcium dependency was clarified, however, by the realization that it was not PtdIns but its phosphorylated derivative, PtdIns(4,5)P2 which is the initial target for phospholipase C hydrolysis [28-30], the Ptdlns level presumably dropping, at least in part, due to its precursory role *. Full recognition of the importance of Ptdlns(4,5)P2 came from the observation, in vasopressin stimulated hepatocytes, that decreases in Ptdlns(4,5)P2 preceded changes in Ptdlns and were largely independent of calcium [30]. Correspondingly, when the aqueous soluble products of phosphoinositide cleavage were examined in agonist-stimulated salivary glands [31] and other tissues (reviewed in Ref. 32), it was found that Ins(1,4,5)P3 formation preceded that of InsP, confirming that the primary receptor mediated event involves the cleavage of Ptdlns(4,5)P2 by a phospholipase C. Ins(1A,5)P3 was subsequently shown to be capable of mobilizing calcium from intracellular stores [33] and has since been fount ,o further regulate intracellular Ca 2+ levels in conjunction with its phosphorylated metabolite, Ins(1,3,4,5)Es (reviewed in Ref. 34). In addition, DAG has been assigned a second messenger status after it was found to activate the Ca 2+ and phospholipid dependent protein kinase, protein kinase C [35-38] Thus, agonist-induced PtdIns(4,5)P 2 hydrolysis by phospholipase C leads to the production of two key second messengers that are thought to be responsible for eliciting cellular responses. Since the early '80s there has been much interest in determining the mechanism of receptor-coupled hydrolysis of Ptdlns(4,5)P2 and how the resulting products interact with their cellular targets. Part of this investigation has involved the isolation and characterisation of Ptdlns-PLCs, the enzymes directly responsible for second messenger production. ll. Characteristics bers

of the

Ptdlns.PLC superfamily mem-

II.A. Initial studies on the Ptdlns-PLCs

The phospholipases C are phosphodiesterases which

* It is yet to be determined, in all systems, whether the drop in Ptdlns seen in response to receptor activation is due solely to its rapid Dhosphorylationto Ptdlns(4,5)p2 (promoted by the stimulation of the appropriatekinases),or whetherPtdlns, itself, may also be a target for Ptdlns-PLC hydrolysis,particularlyonce the intracellular free Ca2+ levels have been elevated.

hydrolyse the glycerophosphate bond of intact phospholipids to generate D A G and the aqueous soluble head group carrying the phosphate (Fig. 2). Probably the first PtdIns-PLC reported was an activity from brain which, ironically, acted upon the 'diphosphoinositide fraction' [39,40]. However, with the discovery of the 'PI-response', attention was focused on isolating a phospholipase C active against PtdIns. Initial studies indicated that the Ptdlns-PLC activity in animal tissues was predominantly cytosolic, required Ca 2+ (in the supraphysiological range) and had an acidic pH optimum [41-44]. Furthermore it was shown that this activity (EC 3.1.4.10) also behaves as a cyclizing phosphotransferase, resulting in the production of cyclns (1:2)P as well as Ins(1)P from the cleavage of Ptdlns [45,46,50](see section 2.C). During the '70s, several reports of membrane-associated Ptdlns-PLCs appeared [47,48,49,50] * *. One indicated that the activityin the particulatefraction of rat brain, unlike cytosolic activitiesreported previously, had a neutral p H optimum [23]. Furthermore, in the following year, a cytosolic activity from lymphocytes was also found to have a p H optimum of approximately 7 [54]. At neutral pH, instead of requiring millimolar levels of Ca 2+, this activitywas maximal in the micromolar range, from which it was argued that the rise in [Ca2+]i, induced by agonist stimulation,was sufficient to activate the PtdIns-PLC [54].This lymphocyte study and a later report in brain [55] were particularly important in that they illustratedthat the perceived Ca 2+ dependence of the cytosolic and particulateenzymes in vitro was not only influenced by the p H at which they were assayed, but also by the substrate concentration. This makes an assessment of Ca 2+ dependence in vivo difficultand the comparison of this parameter between laboratoriesalmost impossible. In addition to the eukaryotic enzymes, several species of bacteria are known to secrete PtdIns-directed PLCs (reviewed in Refs. 56 and 57). These enzymes are capable of acting on eukaryotic membranes in that, when presented externally to animal cells, they release ectoproteins which are anchored in ,~be membrane via glycosyl-Ptdlns [57,58]. Furthermore, because of their presence in filtratesthey are readily extractable and were purified to homogeneity long before the eukaryotic Ptdlns-PLCs. The bacterial enzymes have been shown to be Ca 2+ independent, acidic proteins with a neutral p H optimum, ranging in size from 20 to 30 kDa. However, they are clearly differentboth physically and cata-

**

A Ptdlns-PLC activity which acts on PtdIns was reported in the lysosomal fraction from both brain and liver [51-53]. Althoughit is calciumindependent, it was found not to be completel:,,specific for Ptdlns and, due to its location away from the cell surface, has since been largely ignored in relation to signal transduction.

53 lytically from the Ptdlns-PLCs isolated and characterised from animal cells and because of their action on the outer leaflet of the membrane, eukaryotic homologues of such activities have not been sought as mediators of agonist stimulated phosphoinositide turnover * Preliminary purifications of Ptdlns-PLC activity from animal cells revealed that, instead of a single activity, multiple, chomatographically distinct activities were found in one tissue, stressing the need to perform characterisations with purified enzymes rather than cellular extracts [70,71,72]. One of the first purified Ptdlns-PLCs reported from animal cells was a 70 kDa protein from rat liver cytosol [73]. This was followed by a study in sheep seminal vesicles in which a 65 kDa protein was purified to homogeneity and a second activity of 85 kDa was partially purified [70]. By using antisera raised against these two activities it was shown that they represented immunologically distinct isozymes. A 143 kDa protein was also purified from bovine platelets, suggesting that many different Ptdlns-PLCs may exist [74]. At this time Ptdlns(4,5)P2 was identified as the target substrate for receptor driven turnover. Although many subsequent purification procedures still use,t Ptdlns to screen column fractions, purified activities were also assessed for their ability to act against Ptdlns(4,5)P 2. The majority of studies indicated that the activities which acted against Ptdlns, also acted against Ptdlns(4)P and Ptdlns(4,5)P 2 and that at physiological calcium concentrations the polyphosphoinositides were, in fact, preferred substrates [75-77]. However, there was a report in lymphocytes of a Ptdlns-PLC which acted preferentially against Ptdlns under all conditions tested [78] and preliminary purifications in platelets revealed the

* Over recent years, interest in the types of proteins which are anchored to the cell surface via glycosyl-Ptdlns has grown (reviewed in [14,59,60,611). Since they include molecules such as Thy-1, it is conceivable that the timed release of these proteins into the extracellular space may, indeed, be involved in mediating receptor driven signals. Moreover, specific phospholipases C [62-64] and phospholipases D [61,65] which are capable of hydrolysing glycosyl-Ptdlns have been purified from several eukaryotic sources. The glycesyl-Ptdlns-PLC from Trypanosoma brucei which has been cloned and mapped to a particular chromosomal location [66] has been found to be a 41 kDa protein [63] which, like the bacterial enzymes, is Ca 2+ independent. In addition, a Ca 2+ independent glycosyl-Ptdlns-PLC of 52 kDa has been purified from rat liver plasma membrane [64]. Both these eukaryotie enzymes, unlike the bacterial species, will not, however, act on Ptdlns or Ptdlns(4,5)P2. Furthermore, an additional role for the hepatic enzyme has recently been advocated. It appears that one effect of insulin action may involve th~ hydrolysis of glycosyl-Ptdlns [67,68]. The glyeosylPtdlns hydrolysed contains no protein linkage and has been proposed to be located on the inner leaflet of the plasma membrane [61]. The inositol glycans produced have been shown to influence several enzyme systems in the same manner as insulin [68,691 making them candidates for second messenger molecules.

presence of polyphosphoinositide-specific PLCs which were not active against Ptdlns [79]. In 1986, several successful purifications a,ere reported, including a membrane and a cytosolic activity from calf thymocytes, both of which were 70 kDa [80] and three enzymes from platelets of varying sizes, two of large molecular mass which could be dissociated to a single 140 kDa protein, and a 95 kDa species which was shown to be a proteolytic product [81]. In addition, there was an initial report, from bovine brain cytosol, of two immunologically distinct Ptdlns-PLCs of 145 kDa and 150 kDa [72,82]. These bovine brain enzymes were found to readily form oligomers, generating high molecular weight complexes similar to those reported in platelets, brain and liver cytosol [76,81]. In the following year a third, immunologically distinct enzyme of 85 kDa was reported in bovine brain cytosol [83]. These three bovine enzymes were subsequently purified, their corresponding cDNAs cloned, and they have become the archetypal representatives of the different classes of Ptdlns-PLC, as outlined in the next section. Interesti n ~ , the 150 kDa enzyme from cytosol was also purified from .the particulate fraction of bovine brain by two groups independently [75,82,84], indicating that the membrane associated enzymes were not necessarily distinct from those purified from cytosol.

ll.B. Current members of the Ptdlns-PLC superfamily In recent years it has been possible to show that numerous PtdIns-PLCs exist in various tissues by purification, immunological analysis of purified proteins, biochemical comparison and by molecular cloning of various enzymes. As noted above, PtdIns-PLCs have recently been purified to homogeneity from bovine brain [72,75,82,83] and their corresponding cDNAs isolated [85-87]. These clones encode distinct polypeptides, each of which possesses functional PtdIns-PLC activity. [In accordance with Rhee's suggested nomenclature we shall use j81 to refer to the 154 kDa protein purified from bovine brain and ~'1 and 81 to refer to the 145 kDa and 85 kDa proteins purified from bovine brain respectively. However, the use of Pi and j82 etc. will be restricted to distinct gene products. It is suggested that proteolytic fragments of the j8i gene product are designated ~ (140 kDa) and jS~' (100 kDa).] If one compares the predicted amino acid sequences of PtdInsPLCfll, ~'1 and 81 it becomes evident that while the overall level of identity between these enzymes is low, there does exist two regions of quite striking amino acid identity (Fig. 3). One implication which can be drawn from this is that these two highly conserved regions form a catalytic domain within the folded Ptdlns-PLC protein. Evidence to support this is provided by mutagenesis analysis which shows that deletion of either or both region I and II in PtdIns-PLC~,l results in a complete loss of activity [88]. Similarly, the introduction

54 cloned and expressed as functional proteins possess highly conserved regions has been utilised in a strategy aimed at cloning novel Ptdlns-PLC cDNAs. The basis of this is to use the conserved nucleotide sequences found in the PtdIns-PLCs as a probe in the low stringency screening of cDNA libraries. This approach has been used successfully in the isolation of a y-like cDNA from human lymphocytes [93]. The human cDNA isolated possesses a high degree of homology with the rat PtdIns-PLCyl over its entire sequence. However, it is felt that the extent to which these two sequences differ is greater than one would expect to result from species differences between rat and human, especially in light of the very high degree of identity found between bovine and rat Ptdlns-PLC8~ [86]; this human y-like cDNA is therefore referred to as PtdIns-PLCY2. This screening strategy has also been used successfully in other laboratories and appears to have revealed two other//l-like PtdIns-PLCs and one other 8~-like Ptdlns-PLC distinct from Ptdlns-PLC82 (J. Knopf, personal communication). The norp A g e n e in Drosophila also encodes a protein which, from its predicted amino acid sequence, possesses the conserved regions I and II and a greater degree of homology with Ptdlns-PLC/~ family members than with any other cloned PtdIns-PLCs known to date. It therefore seems that this enzyme is a member of the/~ class of PtdIns-PLCs. Interestingly, this protein is expressed only in the retina of Drosophila and appears to be an essential component of the phototransduction pathway since homozygous mutations in the norp A gene render the fly blind [94]. Thus, from primary structure comparisons, it appears that one can divide the PtdIns-PLC superfamily of enzymes into three classes of isozymes/~, -/and 8 (Fig. 4). Within each class, the enzymes are far more related to each other than to enzymes in a different class. Members of this Ptdlns-PLC superfamily may or may

of a point mutation into region I of Ptdlns-PLCyz (His 335) results in the synthesis of an inactive enzyme (J. Knopf, personal communication). If one applies the same logic, one might suggest that the regions of the Ptdlns-PLCs which are not highly conserved may represent potential targets for regulatory influences directed specifically upon individual Ptdlns-PLCs. One region of immediate interest in the primary structure of the PtdIns-PLCs is the sequence between regions I and II. In PtdIns-PLCflz and 81 these short regions (71 and 40 amino acids respectively) possess approx. 35% and 32% charged amino acids respectively, giving rise to a predicted random structure. This may facilitate juxtaposition of the sequences which form the putative catalytic domain. It is also possible that this highly charged putative 'hinge' region may act as a target for interaction with other proteins. In PtdlnsPLCy~ this region is approx. 500 amino acids in length and possesses a significant degree of homology with several other proteins in what are known as src _homology (SH) regions [85,89,90]. The potential role performed by these structural motifs in PtdIns-PLCy~ will be discussed in section 3.A. Recently, a distinct 85 kDa PtdIns-PLC activity has been purified from bovine brain and its corresponding eDNA isolated from a bovine brain cDNA library [91,92]. Analysis of the predicted amino acid sequence of this Ptdlns-PLC reveals that it possesses the highly conserved regions I and II. However, unlike PtdInsPLC~t and y~ enzymes, it possesses a high degree of identity over all of its sequence with PtdIns-PLCS~. This protein represents a second functional gone product within a 8 family of enzymes; a nomenclature of Ptdlns-PLC82 has been suggested for this enzyme reserving Ptdlns-PLC81 for the previously characterised 85 kDa protein [83,86]. The fact that all the Ptdlns-PLCs that have been

1216 amino acids

Ptdlns-PLC 91 (Bovine)

1291 amino acids

Ptdlns-PLC¥. (Bovine)

1

.... ~~

756 amino acids

Ptdlns-PLC 6, (Bovine)

•

, I

Overall identity (%)

.... --'~'.

'

43

,

,

I

I

'

'

, l

33

'

100 Amino Acid,s

Fig. 3. Comparisonof the predictedaminoacid sequencesof Ptdlns-PLCp1, Y1and ~1. The locationof the two highlyconservedregionsfoundin all Ptdlns-PLCsis denotedby I and II. In sectionswhereno overallidentityis indicated,the degreeof sequencesimilarityis verylow.

55

not perform unique functions in vivo, by means of specific properties, tissue distribution, or unique regulatory influences imposed upon them. cDNA sequence or even peptide sequence from proteolysed protein is not available for all PtdIns-PLCs studied. Thus, alternative criteria have had to be used when attempting to place characterised enzymes within the classes found in the PtdIns-PLC superfamily. One such means of analysis is the immunological comparison of proteins which has revealed that the 88 kDa protein purified from bovine brain soluble fraction is PtdIns-PLCS] [95,96]. The precise identity of 2 distinct

85 k D a Ptdlns-PLCs purified from rat brain soluble fraction by another group is still unclear [97]. Similarly undefined 85 kDa PtdIns-PLC activities have been purified from rat liver cytosol and bovine heart cytosol [98,99]. From molecular weight comparison one might predict that these enzymes may represent members of the 8 class (Table I). However, the extent to which Ptdlns-PLCS]_ 3 account for these activities remains to be determined. Molecular weight comparison is one of the criteria which raises suspicion that there may exist a further class of Ptdlns-PLC enzymes previously termed t~. Many researchers have reported the presence of a

A. II

I

PIdlns-PLC 131 (Bovine)

1216 amino acids l I I l I

! I l I !

l I l I I

Ptdlns-PLC~ (Human) 2

1181 amino acids I

Overall identity (%)

79

46

I

!

23

71

31

B.

SH2'

I

II

l I I I I l |

l i l I I | I

| I I I I | I

I I I I I I I

I l l I I I I

I I I I I l I

I I I I I I I

I I

I I

I I

I I

I I

I I

I I

1252 amino acids

PIdlns-PLC Y2 (Human)

I I

I I

76

39

C.

18

I

62

66

65

30

I I

55

43

II

756 amino acids

Ptdlns-PLC (Bovine)

! I

! !

I |

I !

! !

!

|

!

|

I I I

I I I

| |

I !

I •

I |

764 amino acids

Ptd Ins- PLC S2 (Bovine)

,

'

,

736 amino acids

Ptdlns-PLC 63 (Human) Ovall identity (%)

SH3

1291 amino acids

Ptdlns-PLC 71 (Bovine)

Overall identity (%)

SH2

I .

26

56

9

47

40

100 Amino Acids

Fig. 4. Comparison of predicted amino acid sequences within subclasses of the mammalian Ptdlns-PLC superfamily. The location of the two highly conserved regions found in all PtdIns-PLCs is denoted by I and II. In (B) SH2, and SH3 refers to Src Homology regions (see section III.A.).

56 60-70 kDa PtdIns-PLC enzyme [70,73,100,101], however biochemical characterisation has failed to clarify the relationship between this enzyme(s) and the other PtdIns-PLCs described above. One laboratory has reported the cloning of a cDNA encoding one of these 60 kDa PtdIns-PLCs, although several doubts exist conceming the authenticity of this clone * [101]. Another group has reported the purification of a 70 kDa PtdInsPLC from rat fiver cytosol [102] and has presented amino acid sequences of peptide fragments. These sequences are not found in any other Ptdlns-PLC and thus it is possible that this may represent amino acid sequence from an a family member. The isozymes characterised to date are summarised in Table I. II. C Biochemical properties of Ptdlns-PLCs in vitro One question of primary importance when considering biochemical properties is the specificity the PtdlnsPLCs exhibit for Ptdlns(4,5)P2, Ptdlns(4)P or Ptdlns. As more Ptdlns-PLCs are characterised, patterns appear to be emerging within the PtdIns-PLC superfamily. For example, PtdIns-PLC#~ has the same km for Ptdlns(4,5)P2 and Ptdlns, however the ratio of Vmax values for Ptdlns(4,5)P2 versus PtdIns is 30:1 [75]. This suggests that Ptdlns-PLC~81 has an inherent catalytic specificity for the polyphosphoinositides. This preference is also seen at mM Ca 2+ concentrations; similar results are obtained for Ptdlns-PLC~82 (J. Knopf, personal communication). The substrate specificity of the y-class of PtdIns-PLCs appears to be different, with Ptdlns-PLCv I hydrolysing Ptdlns(4,5)P2 less efficiently than Ptdlns-PLC/3 or 8 family members. As a consequence, Ptdlns-PLCy~ shows a similar specific activity towards Ptdlns(4,5)P2 and Ptdlns at physiological Ca 2+ [82]. This suggests that in vitro, the/~ and y class of Ptdlns-PLCs display distinct activities towards the phosphoinositide substrates. A similar phenomenon is observed for the 8 class of Ptdlns-PLCs whose members, like the/3 class, show a catalytic preference for the polyphosphoinositides at low Ca 2+ concentrations. However, Ptdlns-PLC81 and 82 (unlike PtdIns-PLC/]l) will efficiently hydrolyse PtdIns at mM Ca 2+ concentrations [83,91]. Enzymes of the putative a class are more difficult to compare due to the lack of information concerning their exact nature, but it does appear that at low Ca 2+ concentrations the polyphosphoinositides are the preferred substrates.

* Putative Ptdlns-PLCa, does not possess any significant amino acid homology with any previously characterised Ptdlns-PLC [286], even in regions I and II which have been found in all Ptdlns-PLCs to date. However, most importantly the cDNA isolated has not been shown to encode any functional Ptdlns-PLC activity and thus it is felt that if there does exist an a class of Ptdlns-PLCs then the predicted amino acid sequence of any of its members is as yet unknown.

Recently, three novel polyphosphoinositides (namely Ptdlns(3)P, Ptdins(3,4)P 2, Ptdlns(3,4,5)P3) have been identified [11,105,106]. Of these, PtdIns(3,4)P 2 and PtdIns(3,4,5)P3 are not usually present in quiescent, unstimulated cells but are produced upon the stimulation of cells with, for example, fMet-Leu-Phe, [11] PDGF, [108] or thrombin [108b]. Characterisation of a partially purified enzyme has revealed that a single kinase (Pt~Ins(3)kinase) is capable of phosphorylating the inositol ring on the 3-position to generate PtdIns(3)P, PtdIns(3,4)P2 and Ptdlns(3,4,5)P3 from the appropriate substrate [107]. This activity has been shown to be associated with a number of tyrosine kinase oncogene products and activated growth factor receptors [108110] and it has been suggested that these proteins may operate in part through the Ptdlns(3)kinase enzyme. Understanding the role these novel lipids play is, therefore, clearly of importance, and in considering this question several laboratories have tested the ability of purified Ptdlns-PLCs to hydrolyse these lipids [111,112]. These investigations suggest that the Ptdlns-PLCs studied show a strong preference for Ptdlns(4)P and Ptdlns(4,5)P2 over Ptdlns(3)P and Ptdlns(3,4)P2 and do not hydrolyse PtdIns(3,4,5)P3 to any appreciable extent. However, for the purposes of a definitive analysis of these lipids as substrates, substantial quantities of purified lipids are required. This would enable the removal of the Ptdlns, Ptdlns(4)P or Ptdlns(4,5)P2 (required to make the 3-phosphate lipid) which competes for hydrolysis by the enzyme. Whether all the PtdIns-PLCs fail to hydrolyse inositol lipids phosphorylated at the D-3 position of the inositol ring is yet to be determined. However, it is possible that if aay of ti~e.~e novel polyphosphoinositides are hydrolys,.::d to generate potential inositol phosphate second n~essengers, the phospholipase responsible may belong to an as yet uncharacterised family of enzymes. As discussed above, the PtdIns-PLCs in vitro hydrol~'se PtdIns, PtdI~:~s(4)P and Ptdlns(4,5)P2 to produce DAG as a common product, and Ins(1)P, Ins(1,4)P2 and lns(1,4,5)P3 respectively. However, it has also been repor:cd that ~hosphoinositide hydrolysis produces cyclic inositol phosphates containing a phosphodiester bond between the hydroxyl groups at the 1 and 2 position of the inositol ring [45,46,50,113] (Fig. 5). This occurs when the 2 position hydroxyl (and not a free water molecule) participates in the cleavage of the bond between the inositol phosphate and DAG moieties, to form 1:2 cyclic inositol phosphates. It has been reported that Ptdlns-PLC/~, y~ and 8~ will form cyclic and non-cyclic inositol phosphates at different ratios [114]. The percentage of cyclic inositol phosphates produced, utilising all the substrates tested, decreased in the order PtdIns-PLC/]~ > ~ > y~ and the ability to produce cyclic inositol phosphates decreased with increasing phosphates on the inositol ring of the sub-

57 strate. The physiological significance of this difference is difficult to assess as the precise signalling function of the cyclic inositol phosphates (if any) is still unclear. Evidence has been presented that cycIns(1 : 2,4,5)P3 is at least an order of magnitude less potent than Ins(1,4,5)P3 at inducing Ca 2+ mobilisation [115,116,117]. However, due to the fact that cycIns(1 : 2,4,5)P3 is a poor substrate for the 5'-phosphatase and 3'-kinase, it is metabolised very slowly. These observations have led some researchers to suggest that cyclns(1 : 2,4,5)P3 may act as a

' l o n g - t e r m ' C a 2+ mobiliser which accumulates after prolonged receptor stimulation [118]. However, it remains a point of controversy whether cyclns(1 : 2,4,5)P3 ever accumulates to quantities sufficient to make a significant contribution to Ca 2+ release. This section has restricted itself to the biochemical properties of the PtdIns-PLCs in vitro and illustrates that subtle catalytic differences do exist. How these differences manifest themselves in vivo (if at all) is as yet unclear, although it is conceivable that the diversity

TABLE I

Classification of the Ptdlns-PLC isozymes characterized from different tissues Proposed name /3 Family /3~ r2

Size (kDa)

Basis of classification

Source

Reference

150-154 138 a 134 a

purification and cloning

bovine and rat brain

[72,75,82,84,86,87]

cloning

Knopf, J. & Kriz, R. personal communication Knopf. J. & Kriz, R. personal communication [94]

-

cloning

125 a

cloning

human promyelocytic cDNA human fibroblast cDNA Drosophila genome

purification and cloning

bovine and rat brain

[72,82,85,86b1

3'2

145 148 a 146 a

cloning

[93,103]

3'2

145

transformed human lymphocytes and rat muscle bovine spleen bovine platelets human platelets

[74] [81] [83,86l

85 86 a 84 a

purification and immunoreactivity purification and cloning

rat and bovine brain and bovine adrenal gland bovine brain bovine brain

[91,92l

cloning

human fibroblast cDNA

85 85 85

size size size

85 85

size size

rat brain rat brain sheep seminal vesicular gland rat fiver bovine heart

Knopf, J. & Kriz, R. personal communication [97] [97] [701

70 65

size size

70 70 62 61 63 67 57

size size size size size size size

r3

norp A 3' Family 71

143 140 8 Family B1 B! B2 B3

85 86 a 88

purification and immunoreactivity size size purification and cloning

[97]

[95,96]

[98] [99]

a Family rat fiver sheep seminal vesicular gland murine thymocyte murine thymocyte guinea pig uterus human platelets human platelets human platelets human platelets

[731 [70] [80] [80] [101] [100] [104] [104] [104]

a Theoretical molecular mass from predicted amino acid sequence; all other size determinations listed are based upon migration of the purified protein on SDS-PAGE.

58 TABLE II Cellular systems where it has been demonstrated that PtdIns-PLC activity can be stimulated by GTP analogues, fluoride or GTP and agonist. It is assumed that fluoride operates as an ALF4 complex

Cell type

Agonist

Astrocytoma cells Astrocytoma Basophific leukemic cells Blowfly salivary glands Blowfly salivary glands Cerebral cortex Cerebral cortex Chick myotubes CHL cells CHL cells Corneal epithelium Corneal Endothelial cells Endothelial cells Fibroblasts GH3 GH3 GH3 Glomerulosa cells Glomerulosa cells Heart cells Heart cells HL60 cells (undifferentiated HL60 cells (undifferentiated) HL60 cells (differentiated) HL60 cells (differentiated) Liver Liver Liver Liver Mast cells Myometrium Neutrophil/monocyte progenitors Neutrophils Neutrophils Neutrophils NIH3T3 cells NRK cells Pancreatic acinar cells Pancreatic acinar cells Pancreatic acinar cells Pancreatic acinar cells Parathyroid cells Platelets Platelets Platelets Polymorphonuclear leukocytes Smooth muscle Smooth muscle Smooth muscle Smooth muscle Thyroid Thyroid Turkey erythrocytes WRKol cells WRK-1 cells

carbachol, GTP3,S, fluoride LTD-4 5-methyltryptamine 5-hydroxytryptamine, GTP'yS GTP'~S fluoride ATP thrombin, GTP~,s fluoride adrenaline, 5-hydroxytryptai~ne ATP, GTPTS, fluoride ATP bradykinin TRH, fluoride GTP,IS GTP~,S vasopressin, angiotensin I! angiotensin II carbachol GTP-/S GTP-/S fMet-Leu-Phe GTP'yS vasopressin, adrenaline, angiotensin fluoride GTP-/S GTP-/S carbachol, oxytosin P2 purinergic fMet-Leu-Phe GTP¥S fluoride bradykinin GTP¥S carulein, carbachol substance P, GTPvS, fluoride CCK fluoride thrombin GTP¥S fluoride fMet-Leu-Phe acetylcholine, GTP¥S ATP thrombin endothelin GTP¥S P2-purinergic ATP vasopressin GTP¥S, fluoride

Reference [145] [146] [147] [148] [149] [150] [153] [153b] [233] [154] [155] [156] [157] [158,159] [160] [161,162] [163] [144] [151] [152] [164] [165] [166] [167] [168] [169l [169] [170] [171] [172] [141] [173] [174] [175] [176] [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [189,190] [191] [192,193] [194] [195]

59

OH

O' O;IP-O-" OH

HO a

1,2Diacylglycerol \ OH

\

HO

H+

OI

_.

O

~

~

o

! - o - - F'--O

O-I

~" ~

~

s

I

o

3 +1,2Diacylglycerol Ins(1,4,-,'--b)p

OH

I

s

-O-P=O I o-

0

-

0

O-

Ptdlns(4,5)P2 O

6

°"

I -O-P=O I

O-

s

O

cyclns (1:2,4,5)P3

+1,2Diacylglycerol Fig. 5. Products formed upon P~dlns(4,5)P2 cleavage by Ptdlns-PLC. It has been reported that phosphoinositide hydrolysis by Ptdlns-PLC produces both cyclic and non-cyclic inositol phosphates as well as DAG.

which exists within the PtdIns-PLC superfamily generates potential for a variation of second messengers depending upon which Ptdlns-PLC is activated (see section V). Before discussing the role this multiplicity may play in vivo it would be pertinent to describe what is known of how PtdIns-PLCs are activated in vivo. lIl. Receptor mediated stimulation o f P t d l n s . P L C in vivo

activ-

ity

III.A. Basal Ptdlns-PLC activity Studies in vitro, utilising single lipid vesicles of Ptdlns(4,5)P2, have indicated[ that the purified enzymes, even in the absence of activators, can catalyse the hydrolysis of their substrates so rapidly that if this was occurring in vivo, phosphC, r~ositide supplies would be exhausted in a matter of seconds [96] (a similar conclusion had been reached previously following studies on semipurified activities utilizing PtdIns as substrate [96b]). Therefore, in order to maintain the levels of Ptdlns(4,5)P2 which are known to exist in resting membranes, the cell would need to expend a great deal of ATP in the convertion of PtdIns to Ptdlns(4,5)P 2. Clearly, this situation is energetically unfavourable and so is unlikely to occur, suggesting that in an unstimulated cell, the PtdIns-PLCs must be under some form of

negative control which is missing from the in vitro single lipid assay. A consideration of the way in which PtdIns-PLC activity is suppressed in the resting cell may, therefore, facilitate an understanding of the mechanisms involved in receptor mediated stimulation of these enzymes. There are at least two possible scenarios for this suppression of activity. Firstly, the Ptdlns-PLCs may exist within the cell in association with inhibitors (which might dissociate, or become inoperative, upon receptor occupancy). Secondly Ptdlns-PLCs may be present in an active form but are unable to gain access to their substrate within the membrane environment prior to some receptor-initiated event which modifies either the enzyme or its substrate. Evidence for the existence of endogenous inhibitors of Ptdlns-PLC, which interact directly with the enzyme, is largely circumstantial. There have been reports of trypsin-sensitive inhibitory substances in rat brain [55] and a study demonstrating the inhibition of Ptdlns-PLC activity in intact monocytes by aspirin has indicated that the substances which mediate such inhibition are cycloheximide and actinomycin D sensitive [119]. However, as yet, no endogenous inhibitors have been isolated and characterized for any of the Ptdlns-PLCs. Despite this, the amino acid sequence of the 3' class of Ptdlns-PLCs has been cited as evidence that members

60 of this family at least, interact with regulatory molecules within the cell [96]. As mentioned earlier (section ll.B.), members of the y class of PtdIns-PLCs share regions of sequence homology with several other proteins in what are known as src homology (SH) domains [89,96]. These regions in src do not code for the tyrosine kinase activity of src but do influence its activity in vivo [89, 120]. The SH2 region has also been found in the other non-receptor tyrosine kinases c-abl, c-fps and c-fes, in the viral oncogenes v-src, v-abi, v-fps, v-fes and v-crk and in the GTPase activating protein (GAP) which stimulates the GTPase activity of p21'°s [121]. Mutations within the SH2 domain of v-src [89] and v-fps [120,122] have been shown to generate genes which are impaired in their ability to transform cells in a host dependent manner. This indicates that this region modulates the tyrosine kinases encoded by those genes via a mechanism involving its interaction with specific cellular proteins. However, the fact that mutations in this region inhibit transformation suggests that SH2 is likely to be involved in stimulatory rather than inhibitory modulation. By contrast, the SH3 domain which has also been found in various genes including c-abl, v-crk and the a spectrin gene, has been proposed to have an inhibitory role based on data indicating that removal of this region from c-src enhances oncogenicity [123]. It is conceivable, therefore, that if this region does act to suppress the src tyrosine kinase activity via interaction with an inhibitory molecule then the same or a closely related molecule may also regulate PLC~ family members. When considering exogenous inhibitory substances, a number of compounds such as detergents and pharmacological agents have been shown to inhibit PtdlnsPLC activity when introduced into cells or included in in vitro assays [42,46]. However, most appear to act by disrupting the lipid substrate rather than interacting directly with PtdIns-PLCs. Furthermore, many endogenous cellular constituents have also been shown to influence Ptdlns-PLC activity in this manner, suggesting that substrate presentation may, at least partially, regulate enzyme activity in vivo. For example, studies examining the effects of membrane components on the ability of Ptdlns-PLCs to hydrolyse either Ptdlns or Ptdlns(4,5)P2 [70,124-127] have indicated that phosphatidylcholine has a strongly inhibitory effect which can be overcome by the inclusion in the vesicles of acidic lipids such as phosphatidylserine and phosphatidylethanolamine. In addition, although the hydrolysis product DAG and its metabolite, PA, along with certain free fatty acids, have been reported to be stimulatory [70,124,127], concentrations of cations similar to those found within the cell and positively charged proteins inhibit hydrolysis [124,126]. Furthermore, it has been shown recently, that the cytoskeletal protein, profilin, which is known to inhibit actin polymerisation

also binds to PtdIns(4,5)Pz in vitro thereby preventing its hydrolysis in assays by exogenously added platelet cytosolic PtdIns-PLC [128]. The findings concerning the effects of membrane components on isolated Ptdlns-PLC activity are consistent with the membrane-located phosphoinositides normally acting as poor substrates for the PtdIns-PLCs in an in vivo environment. Furthermore, when enzyme activities are presented with endogenously labelled membranes, very little hydrolysis occurs [126]. However, evidence indicating that the cellular environment is not totally unable to support Ptdlns-PLC activity in the absence of agonist is provided by the observation that the microinjection of PtdIns-PLCfll or Ptdlns-PLCvl into fibroblasts is mitogenic [129,130]. Whether the different classes of Ptdlns-PLC within the cell are kept in check by association with inhibitors or by an inability to gain access to substrate or both, remains to be determined. Nevertheless, it is clear that the occupied receptors must activate a dormant Ptdlns-PLC activity. Exactly how the receptors couple to the different forms of PtdIns-PLC is naturally of great interest and much information has been obtained over recent years which has increased our understanding of the mechanisms involved. III.B. Receptor types which couple to P t d l n s - P L C enzymes

Most extracellular signals do not permeate the cell membrane but instead interact with receptors at the cell surface, which in turn transduce signals to the cell interior. Those cell surface receptors which are defined fall into three categories which have distinct topologies within the plasma membrane and also transduce signals by individual molecular mechanisms, i.e., (i) G protein linked receptors, (ii) receptors which p~Jssess or have associated catalytic functions, (iii) receptor-operated ion channels. Structural analysis of several members of the guanine-nucleotide binding protein (G protein) linked receptor family reveals that these receptors (thus far without exception) possess a seven transmembrane structure, typified by the fl-adrenergic receptors and rhodopsin [131]. Ligand binding is believed to lead to an altered conformation such that the activated receptor can now stimulate its G protein. G proteins which couple to seven transmembrane domain receptors, are a highly conserved family of membrane associated heterotrimeric proteins composed of a, fl and ~, subunits *

* Several other familiesof GTP-bindingproteins exist includingthe elongationand initiationfactorsof proteinsynthesisand the 'small GTP-bindingproteins' such as the productsof the ras, rho and ral genes. The functionof these smallGTP-bindingproteins is unclear and shallnot be dealt with here.

61 [132,133]. Receptor agonist interaction induces the exchange of GDP for GTP bound to the a subunit, thus dissociating the G T P . a from the fl~, components. The activated GTP. t~ subunit stimulates the appropriate effector enzyme until the a subunit, by means of its intrinsic GTPase activity, hydro!vses GTP. This leads to G D P . a formation and reassociation with fl'V, ready for further stimulation by receptor agonist interaction. A large body of evidence exists to suggest that many PtdIns-PLCs are coupled to G-protein linked receptors (see section III.C.). Many receptors possess catalytic functions which play an integral part in the generation of second messengers or in the triggering of second messenger production. Two recently characterised classes of receptor are those which possess phosphotyrosine phosphatase activity [134,135] and receptors which possess guanylyl cyclase activity [136,137]. The possibility exists that these receptors may constitute members of large families, however whether they are linked in any way to Ptdlns-PLC activation is unclear. By far the largest and currently best understood family of receptors which possess catalytic functions are the tyrosine kinase receptors. Structural analysis of members of the tyrosine kinase receptors reveals that they fall into discrete groups [138]. Recently, proteins controlling intracelhlar events (MAP-2 kinase, c-raf, EGF-stimulated kinase) have been identified as likely substrates for these receptors, and one of these substrates appears to be members of the ~,-dass of the Ptdlns-PLCs as will be discussed in section III.D.

III.C. G protein-coupled b'tdlns-PLC activities The first system where it was shown that G proteins couple to Ptdlns-PLCs was in rat mast cells where numerous stimuli are capable of inducing histamine release [139]. One such stimulus is compound 48/80 which was shown to induce histamine release via the activation of a PtdIns-PLC. This release, and phosphoinositide hydrolysis, were stimulated in a permeabilised cell system by the non-hydrolysable analogue of GTP, GTP-~,S [141,169]. When presented to permeabilised cells, GTP-~,S will exchange slowly in the absence of agonist for G D P bound to the ot subunit of G proteins causing a dissociation and thus long-term G protein stimulation since GTP-TS is not hydrolysed. The use of GTP-~,S is a classical means of identifying G proteincoupled effectors and its effects can be inhibited in a dose-dependent manner by pretreatment with GDP-flS which has a greater affinity for the G a subunit than GDP. As a result, GDP-flS exchange for GTP or GTP7S is far slower. These effects are specific for guanine nucleotides and their analogues only, but can also be mimicked by fmoride in the presence of aluminium [142]. This occ,~rs because the AIF4 ion structurally mimics a 7-phosphate group of GTP in the GDP bound

a subunit. Guanine nucleotides, their analogues and aluminium fluoride have been invaluable tools in the study of G proteins coupling PtdIns-PLCs, and as a result a large number of cellular systems has been identified in which G proteins appear to couple to Ptdlns-PLCs (see Table II). However, it should be noted that it is not known in any of these systems which member(s) of the PtdIns-PLC superfamily are G protein coupled. It has been shown in mast cells that compound 48/80-stimulated histamine release can be potently blocked by islet activating protein, perhaps more commonly known as pertussis toxin *. Pertussis toxin catalyses the transfer of the ADP-ribosyl moiety of NAD to a specific site on the a-subunit of Gi (amongst other G proteins) which couples to adenylate cyclase leading to its inhibition [143]. This ADP-ribosylation of Gi causes a complete loss of its ability to couple the receptor to adenylate cyclase. The implication of the result obtained with pertussis toxin in mast cells is that pertussis toxin can ADP-ribosylate the G protein which interacts with PtdIns-PLC, thus blocking the coupling of the compound 48/80 receptors with PtdIns-PLC. That ADP-ribosylation was occurring was confirmed when a 41 kDa protein was shown to be ADP-ribosylated, in a GTP-~,S inhibitable fashion, by pertussis toxin in mast cells [140]. Thus, it was proposed that the a subunit of Gi or a Gi-like protein couples the 48/80 receptor to PtdIns-PLC in mast cells. Similarly, in HL60 cells pertussis toxin blocks the activation of Ptdlns-PLC activity by the chemoattractant peptide fMet-Leu-Phe. Two distinct a subunits in HL60s (which copurify with the fMet-Leu-Phe receptor) are ribosylated by pertussis toxin and indirect evidence implies that these subunits are Gi.2 and Gia 3 [196]. This has led to suggestions that Gi2 and Gi3 may be ' G p ' in HL60 cells. Pertussis toxin has been used to infer the involvement of G proteins in a large number of Ptdlns-PLC receptor coupled systems (Table III). However, there also appears to be a large number of receptor systems which couple to Ptdlns-PLC through pertussis toxin-insensitive G proteins. For example, in platelets, thrombin stimulates Ptdlns-PLC activity via a pertussis toxin sensitive 'Gp', while thromboxane A2 operates via an apparently pertussis toxin insensitive ' G p ' [197]. This distinction exists in many cell systems (see Table III) and clearly suggests that different types of 'Gp' can selectively couple to different receptors expressed in the same cell. In some systems studied it also appears that a receptor can couple to more than one 'Gp'. For example, the molecular cloning of musearinic acetylcholine * Pertussis toxin is a muitimeric complex purified from the culture supernatant of Bordatellaperwssis. It was termed islet activating protein becauseits ability to interact with pancreaticisletswas used as an assay in its purification.

62 receptors (mAChR) has revealed the existence of at least five receptors (M1-5) with distinct primary structures and ligand binding properties [198]. Analysis of clonal CHO cell lines (which lack endogenous mAChR) stably expressing one of the members of the mAChR family, reveals that, although M2 couples to Ptdlns-PLC

activity via a pertussis toxin sensitive 'Gp', M1 and M4 exhibit both toxin sensitive and insensitive activation of Ptdlns-PLC activity [198]. Thus, within one receptor family, a situation appears ~.o exist where the various members of that family can couple to specific 'Gp's and in some cases more than one 'Gp'.

TABLE !II Permssis toxin sensitivity in those receptor systems which operate through the phosphoinositide signalling pathway Agonist

Cell type Inhibited by pertu~is toxin

Adrenaline

fat cells [199]

Angiotensin !I

mesangial cells [202,203]

ATP

endothelial cells [156] HL60 cells [207,208]

Bombesin Bradykinin

NIH 3T3 cells [210] MDCK cells [208] NIH 3T3 cells [174]

Carbachol

Caerulein CCK Compound 48/80 C5A fMet-Leu-Phe

Histamine 5-Hydroxytryptamine Mastoparan Noradrenaline

HL60 cells [171] Thyroid cells [206] Fibroblasts [209] Neurohybrid cells [212]

CHO cells [198]

not affected by pertussis toxin brown adipocytes [200] liver [201] adrenal glomerulosa [144,163,204] liver [201] neuroblastoma [205l vascular smooth muscle [205b,205c] thyroid cells [206] fibroblasts [211] astrocytoma [213] endothelial cells [121] epithelial cells [214] fibroblasts [157,215] neuroblastoma cells [174] vascular smooth muscle [216] astrocytoma [213,217] cerebral striata [218] heart cells [217] myometrium [170] pancreatic acinar cells [176] smooth muscle cells [184,219] pancreatic acinar cells [176] Xenopus oocytes [220]

mast cells [140, 221] basophils [222l neutrophils [223] HL60 cells [165,196] macrophage [224] basophils [2221 astrocytoma [213] NRK cells [225] vascular smooth muscle [226] fibroblasts [227l mast cells [221] vascular smooth muscle [228]

Oxytosin Platelet activating factor mesangial cells [202] Prostaglandin E Substance P mast cells [221] Thrombin fibroblasts [233] platelets [104,197] Thromboxane A 2 TRH Vasopressin

partially inhibited by pertussis toxin

Xenopus oocytes [220]

thyroid [189l myocytes [229] myometrium [170] monocytes [230,231] adrenal medulla [232] endothelial cells [234,235] osteosarcoma cells [236] platelets [237] vascular smooth muscle [186] astrocytoma cells [213] platelets [197] GH 3 cells [159] glomerulosa [163,238] vascular smooth muscle [239,240] Liver cells [201]

63 In an effort to identify which 'Gp' couples to which Ptdlns-PLC, attempts have been made to copurify Ptdlns-PLC/'Gp' complexes. In such experiments, broad pools of PtdIns-PLC activity wlfich overlap with GTP-~,S binding activity were collected from the initial chromatographic separations of crude extracts. As these preparations became more highly purified, the PtdlnsPLC activity and the GTPq, S binding activity became tightly associated. However, it is not possible to conclude whether this association is physiological or an artefact of the increased state of purity of the sample. For example, in calf thymocytes a 68 kDa PtdIns-PLC activity (a putative a family member) has been purified to homogeneity and 'copurifies' with a GTP-,/S binding activity [241,242]. This putative PtdIns-PLC/'Gp' complex can be separated to reveal a GTP-~,S binding peak which consists of three proteins of molecular weight 54 kDa, 41 kDa and 27 kDa on SDS-PAGE. The 54 kDa band is ADP-ribosylated by pertussis toxin and the 27 kDa band not only possesses GTP-~,S binding activity but also stimulates (when bound to GTPq, S) Ptdlns (4,5)Pz hydrolysis by the purified Ptdlns-PLC. Other laboratories have also reported the presence of an approximately 29 kDa GTPq, S binding protein which may be associated with the activation of Ptdlns-PLCs [243,244]. In platelets a PtdIns-PLC activity has similarly been copurified with a 29 kDa GTPq, S binding activity which can be removed from the Ptdlns-PLC using detergent, and then added back to the enzyme to give a resumption of the GTP-~,S stimulatory effect upon the enzyme [243]. These results would appear to suggest that 'Gp' in these cell systems may have a subunit structure different from well characterised G proteins in other coupled systems. However, until these proteins which 'copurify' with Ptdlns-PLC activity have been conclusively identified as physiological 'Gp' this evidence must remain circumstantial. A number of agonists are also known which inhibit agonist stimulated PtdIns(4,5)P2 hydrolysis and one can propose several approaches by which this inhibition can be achieved: One direct means of inhibiting agonist stimulated PtdIns(4,5)Pz hydrolysis would be the action of a 'Gi-like' G protein activity. By analogy with the adenylate cyclase mediated tra~lsduction system one can suggest the presence of a novel inhibitory G protein ('Gpi') which, when stimulated by receptor agonist interactions, binds to a PtdIns-PLC to inactivate it. While data has been presented consistent with the existence of a 'Gpi' [245], as yet no 'Gpi' activity has been firmly identified. Other mechanisms by which agonist stimulated Ptdlns(4,5)Pz hydrolysis is regulated are discussed in section IV. III.D. Ptdlns-PLC activities coupled to tyrosine kinase receptors Given the analogy with the adenylate cyclase signal

transduction system, it is not surprising that Ptdlns-PLC activity has been shown to couple to certain cell surface receptors through G proteins. However, recent evidence involving the tyrosine kinase class of receptors suggests that this may not be the sole means by which receptors can stimulate PtdIns-PLC activity. Studies in p, neablized Swiss 3T3 cells have indicated that, whereas InsP3 production induced by bombesin is stimulated by GTP~S and inhibited by GDP-flS, neither of these agents has an effect on PDGF-mediated phosphoinositide hydrolysis, consistent with the tyrosine kinase receptor activating Ptdlns-PLC activity by alternative means [245b]. Furthermore, it has now been shown that the ~, class of Ptdlns-PLC can associate with, and be phosphorylated by, several tyrosine kinase receptor systems in a manner which is apparently independent of trimeric G-proteins [246,247]. The binding of PDGF [248] and in several cases EGF [249] to their cell surface receptors has been shown not only to activate their intrinsic tyrosine kinase activity but also to lead to an increase in production of inositol phosphates. However, until recently, little was known about the nature of the interaction be*~ween the receptors and Ptdlns-PLC activities. The first indication that the tyrosine kinase may act directly on a PtdInsPLC came from studies using immobilised antiphosphotyrosine antibodies and A431 cell extracts [247]. This study showed that the amount of Ptdlns-PLC activity bound to these antibodies increased after EGF treatment, a finding later extended to other cell lines expressing varying levels of EGF receptor [250,251]. It was then shown that it was a Ptdlns-PLC which was phosphorylated on tyrosine and by the use of antibodies directed against PLCfll, )'! and ~1 that this phosphorylation was confined to the ~'! enzyme [250,251] (similar data has since been observed for PtdIns-PLC't2; J. Knopf, personal communication). Moreover, in some cell systems, antibodies to the EGF receptor were able to co-precipitate Ptdlns-PLC)q md vice versa indicating a strong association between the two proteins [250]. Findings similar to the above were reported for PDGF stimulation of 3T3 cells with the stoichiometry of stimulated phosphotyrosine phosphorylation on PtdIns-PLC71 being higher than that for the EGF/A431 cell system [246]. Like the EGF system, PDGF was found to stimulate the increased phosphorylation of PLC3,1 on tyrosine via a means which was independent of receptor internalisation. Furthermore, in the presence of agonist, the PDGF receptor also coprecipitated with Ptdlns-PLC-h although again, as seen with the EGF receptor, only a small fraction of the receptor pool is represented in this complex, perhaps signifying the transient nature of the interaction [246]. Both EGF and PDGF have been shown to increase the phosphorylation of PtdIns-PLC~,l not only on tyrosine but also on serine residues indicating that other

64 kinases are activated by the signal cascade. However, evidence that the phosphotyrosine phosphorylations are mediated by the intrinsic receptor kinases themselves comes from studies using purified receptors and PtdlnsPLCy~. It has been demonstrated that PtdIns-PLCy~ is phosphorylated at the same sites by purified PDGF receptor in vitro as are seen in PDGF treated cells and that EGF receptor could also phosphorylate PtdlnsPLCy~ at physiological sites [246]. It has been suggested that the phosphorylation of PtdIns-PLCy1 on tyrosine leads to its activation within the cell [246]. However, unequivocal evidence for this activation is difficult to obtain because, as mentioned previously, in vitro assays clearly lack the regulatory elements operating in vivo. Support for the role of tyrosine phosphorylation comes from the observations that to date there is a complete correlation between the ability of certain tyrosine kinase receptors in specific cell systems to stimulate inositol phosphate production and the degree to which they phosphorylate PtdlnsPLCyI [246]. Furthermore, tyrosine kinase inhibitors [246,250] and receptors defective in tyrosine kinase activity [252] fail to elicit phosphoinositide turnover (although it is not clear whether these kinase-defective receptors form complexes with Ptdlns-PLC). In addition, the stoichiometry of phosphorylation is consistent with a considerable number of Ptdlns-PLC molecules being activated [246] and studies investigating the time-course of tyrosine phosphorylation and Ins(1,4,5)P 3 production suggest that the kinetics of phosphorylation of Ptdlns-PLCyl are consistent with it initiating phosphoinositide breakdown (J. Knopf, personal communication). It should be noted, however, that all the above data are equally consistent with other models. For example, the phosphorylation of PtdlnsPLC'~I may be required for the maintenance of the Ptdlns-PLC within the receptor complex, thereby allowing its activation by an associated but independent mechanism; equally the tyrosine phosphorylation could be a desensitization mechanism responsible for dissociating the 'active' complex. There is some evidence that the small GTP binding protein p21 "a~ together with GAP may be involved in modulating Ptdlns-PLC.~ function within the ceil. it has been shown that the microinjection of either the H-ras gene product or Ptdlns-PLCyl into NIH 3T3 cells leads to the initiation of DNA replication, which, in both instances, is inhibited by co-injection of antibodies to Ptdlns-PLCyl [130]. Antisera to the ras protein did not, however, inhibit the Ptdlns-PLCyz response. A feasible explanation for this is that the ras protein acts upstream of the phospholipase in a common signal transduction pathway. Alternatively, p21 "a~ may exert its proliferative effects by stimulating some autocrine loop involving, for example, the up-regulation of a particular cell surface receptor which uses PtdIns-

PLC71 to transduce its signal. The possibility cannot be discounted, however, that antibodies to Ptdlns-PLC-yl influence the functioning of the r a s protein via cross-reaction with GAP through a common sequence (see above) or by sterically inhibiting the interaction of GAP with a common receptor site [253,254]. When considering the stimulation of Ptdlns-PLC~ by tyrosine kinase receptors, it should be noted that not all activated tyrosine kinase receptors lead to the phosphorylation of Ptdlns-PLCy~. This is demonstrated by studies with the CSF-1 receptor, which, upon binding to its agonist, neither leads to inositol phosphate production [255] nor to the phosphorylation of Ptdlns-PLCv~ [256]. This indicates that the information available for the EGF and PDGF systems is relevant for those particular receptors but may not apply to tyrosine kinase receptors in general.

IV. Heterologous regulation of Ptdlns-PLC activity in vivo When one tries to elucidate regulatory principles which might be applied to agonist-induced phosphoinositide hydrolysis, one encounters a series of apparent contradictions depending upon which cell type is used for experimental analysis, and even upon which agonist response system is used in the same cell. For example, a number of reports in various tissues describe how elevated cAMP levels influence agonist induced phosphoinositide hydrolysis. In platelets [257,258,259], HL60s [260] and neuroblastoma cells, [261], elevated cAMP levels have been shown to inhibit agonist induced PtdIns(4,5)P 2 hydrolysis. In platelets, for example [257], GTP-~S stimulation of Ptdlns-PLC activity is inhibited by dibutyryl cAMP; this effect is not seen in the presence of inhibitors of cAMP dependent protein kinase (A-kinase) thus suggesting that the phosphorylation of some component downstream of the receptor (perhaps the PtdIns-PLC or 'Gp') by A-kinase may mediate the effect. A role for A-kinase is also shown by experiments in C6BU1 cells which express PtdlnsPLC~I, "~1 and O! [114]. In these cells, pretreatment with 8-Br-cAMP caused the phosphorylation of PtdlnsPLC71. The implication of this result is that A-kinase can participate in the heterologous regulation of phosphoinositide metabolism through Ptdlns-PLCTl. Observations which contrast with the above effects of cAMP are seen in hepatocytes [262] and Balbc 3T3 cello [263] where agents which enhance cAMP augment Ptdlns-PLC stimulation in response to certain agonists. Whether these different effects reflect different targets for regulation in the different systems is as yet uncertain. The multiple effects of cAMP on phosphoinositide hydrolysis are also illustrated in canine tracheal smooth muscle where increased cAMP is associated with the inhibition of phosphoinositide hydrolysis in response to

65 histamine but not methacholine [264]. This suggests that these two agonists utilise different catalytic routes to promote phosphoinositide hydrolysis, one of which can be obstructed by elevated cAMP while the other cannot. Clearly from the above, attempting to describe universal regulatory principles for Ptdlns-PLCs is a difficult task. This is perhaps not too surprising when one considers the following. (1) In a single cell type there exists a large number of receptors capable of stimulating Ptdlns-PLC activity upon binding of the appropriate agonist. (2) Some of these receptors will activate a Ptdlns-PLC by directly phosphorylating it, perhaps by indirectly leading to its phosphorylation or by recruiting the action of one (or more) G proteins from a choice of perhaps many. (3) Each receptor in a cell has the potential to stimulate a large number of Ptdlns-PLCs, various combinations of which may be expressed in any given cell type. If one considers each one of the above stages in the activation of Ptdlns-PLC activity as a potential target for a regulatory input, it is perhaps understandable that common regulatory principles are not easily found. A large body of evidence exists to suggest that protein kinase C (PKC) activators, such as the tumour-promoting phorbol esters, have an inhibitory effect upon the receptor mediated stimulation of PtdIns(4,5)P2 hydrolysis and Ca 2+ mobilisation in a variety of experimental systems [155,210,265-279]. The inhibitory effects of PKC activators are seen in response to numerous agonists, suggesting that PKC actions may be aimed downstream of receptor agonist interactions (although

receptor phosphorylation in some systems has been shown to play a role [280]). For example, in human polymorphonuclear leukocytes the ability of the G protein coupling the fMet-Leu-Phe receptor to PtdIns-PLC, to bind GTP-7S or be activated by the receptor is not impaired by the phorbol ester, PMA. Instead, PMA blocks the ability of the activated G protein to stimulate PtdIns-PLC activity [281]. Similar results have also been obtained in various systems such as oocytes [279], hepatocytes [267] and platelets [276]. The question then arises as to whether PKC activation leads to the phosphorylation of the G protein, PtdIns-PLC or both. It has been demonstrated that PKC phosphorylates the a subunits of the inhibitory G protein Gi, and of the retinal G protein, transducin in vitro [282,283,284]. In both cases the purified free a subunit was a better substrate than the holo-protein and the a subunit was a higher affinity substrate when it was in its inactive conformation. These findings suggest that G proteins may serve as substrates for PKC. However, whether any 'Gp' is phosphorylated in vivo by PKC still remains uncertain. In addition, the phosphorylation of PtdInsPLC/]1, 71 and 01 by PKC has been reported in vitro [285] although no change in catalytic activity was observed. V. Summary and perspectives It is evident fTrom the foregoing discussion that there is a large family of enzymes that appear to contribute to second messenger production in the 'inositol-lipid'

INPUT J

MODULATION. Feedback regulation (both positive and negative) may be exerted upon specific components of this coupling system. Differential expression of components may change the regulatory influence which modulators like PKC and A-kinase have upon the final output.

MULTIPLE RECEPTORS. 7 Transmembrane region receptors Growth factor tyrosine-kinase receptors Other?

MULTIPLE Ptdlns-PLCs. Multiplicicty may contribute to the variety of the output by means of; (1) Coupling to specific receptors. (2) Differences in substrate specificity. (3) Differences in specific activity.

J OUTPUT J Fig. 6. Rationalisation of the heterogeneitywithin the phosphoinositidesignalling pathway.

66 pathway. These enzymes are not coexpressed in all cell types but show differential patterns of expression as judged by histological analysis [287] and Northern blotting [288]. On an individual basis, these are not simply housekeeping genes. The highly restricted expression of the norp A gene product (a /~-class member) to the precursor and adult eye in Drosophila underscores this selectivity of expression [94]. The extent to which there is a requirement at the genetic level for multiple genes in order to permit selective developmental expression remains to be determined. However, as discussed above, there is evidence both in vitro and in vivo for heterogeneity of function for this effector system and it would seem appropriate to consider the multiplicity of Ptdlns-PLCs in this light. The Ptdlns-PLCs, like any other signal transduction system, operate to convert an input signal into an output. In this case the source of the input is an activated receptor and the outputs are phosphoinositide metabolites i.e. second messengers. As indicated in Fig. 6, there is scope for heterogeneity at the level of input and output as well as with respect to the modulation of effector function. The formation of complexes between activated growth factor receptors and Ptdlns-PLCy~ (see above) provides the first direct indication that specific receptors couple to specific Ptdlns-PLC enzymes. The likely G-protein control of the PtdIns-PLC// class [as evidenced by the stimulation of a PtdIns-PLC (presumably the norp A gene product Ptdlns-PLC norp A) by a Rhodoposin like molecule in Drosophila [288b]] provides a second indication for selective coupling. It is also possible to argue that specific G-proteins may be responsible for interaction with individual members of the/3-subclass, since there is evidence for at least two species of 'Gp' based upon sensitivity to pertussis toxin (see Table III). The expression of cell surface receptors for a particular ligand governs the ability to perceive that ligand. The expression of a particular receptor subtype would appear to dictate the consequences of ligand binding. Whether the differential expression of the effector system itself provides a physiological means of modifying the repertoire of cellular responses in addition to receptor expression remains an open question. However, in the case of growth factor receptors that impinge on multiple transducing pathways it is to be expected that responses may be modified by altering the signalling possibilities. The complexities of modulation of Ptdlns-PLC activation are exemplified by the observations that both enhancement and inhibition of activation is observed with the agents PMA (presumably acting within the pathway) and cAMP (acting via an heterologous pathway) (see section 4). Thus, the choice of coupling system can affect the intrinsic control (feed-back or feed-fo:-

ward) of, and the heterologous influences on, the pathway. The consequences of sensitising (or not) the system to these modulatory influences is that it provides scope for temporal modulation of the output (homologous modulation) as discussed further below, and also the ability to coordinate responses to other hormones/ factors (heterologous modulation). The imrjortance of this latter consideration is becoming increasingly clear. Physiologically, cells are not exposed to stimuli on an individual basis but sense the presence of multiple stimuli; as such it is necessary to coordinate responses and this is achieved, in part, through heterologous modulation. The summation of any two pathways may thus be additive, antagonistic or synergistic depending upon the nature of the mutual heterologous modulation. In a particular cell type, can this modulation be governed by the selective expression of a particular Ptdlns-PLC? The consequences of the output of this effector system are similarly not uniform; it depends upon the nature of the output itself (i.e., whether diacyglycerol is produced with Ins(1,4,5)P3 or for example with Ins(l,4)P2) *. While there are obvious influences of Ca 2+ in its own right, it is also of possible significance that the targets for DAG include both Ca2+-dependent (a, fll/2, Y) and Ca2+-independent (8, c, ~?) PKCs with distinct substrate specificities [289]. Hydrolysis of Ptdlns may thus allow the selective targeting of the Ca 2+-independent class. Again the expression of particular Ptdlns-PLCs may have significant consequences on the nature of the cellular response. The spatial nature of the output is as yet unclear, although there is good evidence for discrete pools of inositol lipids that turnover at distinct rates [290]. While it has been shown that Ptdlns-PLCfll and 82 both have an intrinsic ability to interact with membranes [87,92] it is not yet known whether these or any other Ptdlns-PLCs selectively associate with and hydrolyse lipids in particular sub-cellular compartments. It is assumed in most instances that receptor driven events occur at the plasma membrane. However, depending upon the nature of receptor-Ptdlns-PLC coupling, activation of the relevant phospholipase could be effected at distal sites. With respect to second messenger production, there is also the consideration of timing, which is influenced not only by the rate of production but also by the rate of removal. The consequences of DAG production are dynamic in that activation of PKC and phosphorylation

* While outside the scope of this review, it should be noted that DAG could be produced from a number of distinct phospholipids and in particular there is good evidence for agonist induced DAG production from phosphatidylcholine (PtdCho) (recently reviewed [288c]) This appears to involve a combination of phospholipase D and phosphatidic acid phosphohydrolase activities and PtdCho breakdown may well account for much of the 'second phase' DAG production that is observed with certain agonists.

67 of substrates is counterbalanced by the action of protein phosphatases; in order to achieve some threshold of substrate phosphorylation DAG mus~ accumulate to a critical level which may also vary from one PKC isotype to another (activation constants vary 2-5-fold for DAGs; [291]). An example of differences in the rate of DAG production is provided by comparing the effects of PDGF and vasopressin in Swiss 3T3 cells [277]. While the latter produces an acute short lived elevation of diacylglycerol, PDGF induces a slow rise in diacylglycerol that is sustained. That DAG steady state levels may be limiting is suggested by the observation that the DAG kinase inhibitor R59022 can synergise in eliciting responses [292]. Further modulation of second messenger concentration will derive from metabolism. For example, recent studies on DAG kinase and an Ins(1,4,5)Pa 3' kinase indicate that Ca 2+ may play a critical role in regulating the removal of these second messengers [293,294]; evidence has also been presented for the possible regulation of the Ins(1,4,5)P 3 5' phosphatase [295]. All these events are presumably coordinated with the expression of particular Ptdlns-PLCs to achieve the necessary consequences. In conclusion then, there is a large family of PtdInsPLC enzymes which can be organised as is shown in Fig. 4 and rationalised as indicated in Fig. 6. Progress in defining these various parameters will hinge upon an ability to fully reconstitute a variety of receptor types with their effectors and all the intervening components. It will then be possible to define the selectivity of coupling and the potential for modulation. Acknowledgements We would like to thank John Knopf and Ron Kriz for making data available prior to its publication and also Amanda Wilkinson for expert secretarial assistance. Addendum Subsequent to the submission of this review a number of articles have been published which further substantially increase our understanding of the components involved in the G protein-mediated coupling of PtdlnsPLC to receptors. These include papers by Harden and collaborators involving the further development of a reconstitution system whereby Ptdlns-PLC, purified to homogeneity from turkey erythrocytes, can be added back to radiolabelled turkey erythrocyte ghosts and is found to act on the membrane phosphinositides in a GTP3,S and agonist-dependent manner [296,297]. In addition, another group have developed a novel method for the purification of G-proteins whereby bovine liver membranes, preincubated with GTP3,S, were extracted in cholate then fractionated chromatographically [298].

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