gtp-binding proteins in human platelets

GTP-binding Proteins in Human Platelets

K. Nagata, Y. Nozawa

It is widely accepted that several heterotrimeric regulatory GTP-binding proteins (herein referred to as G proteins) have important roles in receptor-mediated transmembrane signalling (Table 1). Different G proteins show remarkable similarities both in structure and mechanism of action.’,’ They function as transducers in signal transduction pathways that consist of three proteins: receptors, G proteins, and effectors (Fig. I , Table 1). Each of the G proteins contains three subunits (a, fl and y), binds guanine nucleotides with high affinity and possesses GTPase activity. These properties are implicated in their functions. The guanine nucleotide binding site is contained in the u subunit. When GTP binds to a G protein, it dissoci- ates to give an a subunit and a p y subunit complex; the latter may participate in anchoring the a subunit to the plasma membrane. The P y subunits of G proteins are tightly associated with each other and can be separated only after denaturation. The P y subunit complexes of Gs, Gi, and Go (Table 1) are interchangeable between different a subunits. Despite the fact that p y subunit complexes can be exchanged among different a subunits, there are multiple forms of the p and y subunits. The p subunit of Gt can be visualised on SDS-gels as a single band of 36 kDa, whereas the p subunit of other G proteins is a doublet of proteins of 35 kDa and 36 kDa.

There are two groups of effectors controlled by G proteins; one group for which it is known that the effector is directly controlled by a G protein, and another for which the involvement of a G protein is implicated but has not been rigorously demonstrated. Regulation of adenylate cyclase activity’ and of a cyclic GMP-specific phosph~diesterase~ are known to

~ ~ ~~ ~~

K. Nagata, Y. Nozawa, Department of Biochemistry, Gifu Univer- sity School of Medicine, Tsukasamachi-40, Gifu 500 Japan.

be controlled directly to G proteins. Modulation of adenylate cyclase activity in response to various stimulatory agonists (epinephrine, gonadotropin, ACTH, etc.) or inhibitory agents (a,-adrenergic and muscarinic agonists) is mediated by distinct G proteins, named Gs (stimulatory) and Gi (inhibitory), respectively. The concentration of cyclic GMP in retinal rod outer segments, a crucial determinant of visual excita- tion, is modulated through the ability of a G protein (Gt, transducin) to activate a cyclic GMP-specific phosphodiesterase in response to photolysed rhodopsin.

In addition to the heterogeneity that serves to divide the G proteins into major classes (Gs, Gi, Go and Gt), there are multiple forms of the subunit polypeptides within each class.’ The requirement for both a and p y subunits for receptor-mediated activation of G proteins and the heterogeneity of each subunit suggest that each class of receptors might recognise a specific a p y structure.

Probably the most widely studied system in which involvement of a G protein has been implicated, but not rigorously demonstrated, is agonist-mediated phosphoinositide hydrolysis by specific phospholipase C (PLC) (Fig. 2). Such a putative GTP-binding protein was termed ‘Gp’ by Co~kcroft .~ The most compelling evidence for G protein involvement pre- sented to date involves the capacity of GTP and its non-hydrolysable analogs to enhance agonist-mediated accumulation of inositol phosphates in permeabilized cells or isolated membranes. The inhibition of this response in some, but not all, cell types by pretreatment with pertussis toxin (PT, also known as islet-activating protein, IAP) suggests the presence of PT-sensitive and -insensitive Gp. However, evidence for a direct effect of a G protein on PLC activation has not been obtained. Other cellular events in which G proteins may be involved are ion channel regula-

Platrlrfs (1990) 1. 61-79 0 19W Longman Group UK Ltd

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68 GTP-BINDING PROTEINS IN HUMAN PLATELETS

Table 1 Biochemical properties and roles of G proteins’

Subunit Mr ( x w 3 Y Toxin Receptorb Role

Gsa ( x 4) 44.5-46 Cholera P AR>a,AR>Rho Activate adenylate

Cia ( x 3) 40.4-40.5 Pertussis Musc, a,AR,Rho Inhibits adenylate > P AREkd cyclase (weak)d

Goa 39.9 Pertussis Musc, a,AR,Rho Pertussis toxin sensitive-events’

Gta I 40 Cholera + Rho > a,AR > > Activate cGMP

Gta 2 40.5 Chorela + Cone opsins’ Activate cGMP

cyclase

pertussis P AR phosphodiesterase in retinal rods

pertussisc phosphodiesterase in cones‘

P o ( 2 ) 31.4 required for Y ( x 3?) 8-10 interaction of a

with receptor. Deactivates Ga

‘The Mr values listed for a and P subunits are those calculated from their respective cDNAs bReceptor-G protein interactions determined in reconstituted systems with respective G protein oligomers. P AR, P-adrenergic; a,AR, a*- adrenergic; Musc, muscarinic cholinergic; Rho, rhodopsin ‘These properties are assumed for those proteins that are known only as cDNA-deduced sequences dThe relative quantities of the three Gias in ‘purified’ preparation of Gi used in functional studies are unclear These proteins are candidates for involvement in the regulation of pertussis toxin-sensitive cellular events (phosphoinositide hydrolysis, K +

channels, Ca2 + channels, etc)

Messenger

Fig. 1 Interactions between receptor, G protein, guanine nucleotide and effector.

tion,’ exocytotic secretion’ and phospholipase A, activation.6

The a subunits also contain the site(s) for NAD- dependent ADP ribosylation catalysed by bacterial toxins. The a subunit of Gs (Gsa) can be ADP- ribosylated by cholera toxin, whereas Gia and Gou can be ADP-ribosylated by PT. Gta can be modified by both toxins. ADP-ribosylation of these proteins causes characteristic alterations in their functions; activation of Gs in the case of modification by cholera toxin, and impaired ability to interact with receptors in the case of ADP ribosylation by PT.’ Gia and Goa are modified post-translationally by N-myristoyla- tion.’ Although its exact function is not known, the acylation may be beneficial for the interaction of the relatively hydrophilic subunits with the plasma membrane. Recently, a G protein a subunit, termed Gza or Gxa, here referred to as Gxa, was identified by molecular The deduced amino acid sequence of Gxu is homologous to those of other

G protein a subunits (especially Gia), but it is unique in that the cysteine residue is lacking in the fourth position from the C terminus which is the site for PT-catalysed ADP ribosylation. l o This indicates that in Gxu, substituting isoleucine for cysteine may be involved in PT-insensitive transmembrane signalling.

In addition to the heterotrimeric GTP-binding proteins (G proteins), there is another group of GTP- binding proteins with molecular masses ranging from 20 to 30 kDa in mammalian cells. More than 20 genes for such GTP-binding proteins have been identified,’ ’ some of them (smg p25A,12 rho P ~ O , ” * ’ ~ smg ~ 2 1 , ’ ~ c-K-rus p21 ,16 c25KG”) have been purified from mammalian tissues including platelets. However, the physiological significance of these proteins in mammalian cells is unknown.

Platelets are very active cells and respond to several extracellular stimulants, e.g. thrombin, collagen and platelet activating factor (PAF), resulting in shape

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PLATELETS 69

Agonist Outside

Membrane F pip Receptor PIP2 -

a, Cellular Responses 1 Fig. 2 Molecular mechanisms of CaZ+ mobilising receptor. Gp, a putative G protein controlling PLC activity; mPLC, membrane- associated PLC; cytosolic PLC; cPLC, CaMK, calmodulin kinase; PKC, protein kinase C; ER, endoplasmic reticulum; DG, diacylglycerol; PIP,, phosphatidylinositol 4.5-bisphosphate; PIP, phosphatidylinositol 4-monophosphate; PI, phosphatidylinositol; IP,, inositol trisphosphate; IP,, inositol bisphosphate; I P, inositol monophosphate.

change, aggregation and release of dense body con- stituen ts. The receptor-linked hydrolysis of phosphoinositides by activation of PLC is essential for platelet activation and produces two intracellular second mes- sengers. diacylglycerol (DG) and inositol trisphosphate (1P3). DG activates protein kinase C (PKC)l8 and IP, mobilises Ca2+ from internal storage sites in the dense tubular system." On the other hand, cyclic AMP(cAMP) elevating agents, including prostaglan- din(PG) E l , PGI, and PGD,. inhibit these reactions, and activation of CAMP-dependent protein kinase (PKA) is believed to be the mechanism leading to inhibition.20 Recently, it has been indicated that some low molecular weight ( M r ) GTP-binding protein(s) is(arc) involved in the activation and inactivation of platelets.

In this article, the characterisation and putative biological roles of GTP-binding proteins in platelets are reviewed.

Involvement of GTP-binding Proteins in Platelet Function

Aden,,.lure Cvclase

Blood platelets react to a wide range of stimuli with one or more of three characteristic responses, namely, shape change, aggregation and release. Like many kinds of cells, platelets possess both positive and negative control systems for transduction of extracellular signals.2' A positive control system for activation of platelet by excitatory stimuli is a phosphoinositide-metabolising pathway (breakdown of PIP, by activation of PLC) followed by an increase in intracellular Ca2 + concentration. On the other hand,

negative control of platelet function is attributable to an adenylate cyclase system which generates CAMP; increased levels of cAMP inhibit platelet activation. Formation of cAMP by the adenylate cyclase system in platelets (and also in many other cell types) has already been shown to be regulated by Gs and Gi.22*23 Additionally, i t has been shown that cAMP and agents that increase platelet cAMP levels inhibit PLC activity,' and formation of inositol phos- p h a t e ~ . , ~ It has also been shown that treatment of platelets with dibutyryl cAMP or with a CAMP- phosphodiesterase inhibitor decreases deoxycholate- stimulated PLC activity.25 Moreover, the formation of DG and phosphatidic acid (PA) in [3H]arachidonate-labelled platelets was greatly suppressed by cAMP in a dose-dependent manner.,' These data strongly suggest that cAMP inhibits the action of PLC either directly or indirectly. As activation of PLC is essential for platelet function (see below), these results indicate that cAMP suppresses platelet activation. This means that the concentrations of cAMP determined by activation of Gs and Gi lead to inactivation and activation of platelet function, respectively.

Phospholipase C

Phosphoinositides (PIP,, phosphatidyl inositol 43- bisphosphate; PIP, phosphatidylinositol 4-monophosphate and PI, phosphatidylinositol) comprise a small percentage of platelet membrane lipids, but they are of major importance for biological functions in platelets and also in other types of cells. Several lines of evidence have indicated that a GTP-binding protein(s), putatively called Gp, is involved in

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receptor-coupled phosphoinositide hydrolysis. As far as platelets are concerned, Haslam and David- sonz6 first showed that addition of GTP and its non-hydrolysable analogs such as GTPy S to electri- cally permeabilised human platelets enhances thrombin-induced DG formation. Moreover, they demonstrated that GTPy S, or thrombin plus GTP, stimulated the release of [3H]-inositol phosphates in platelets prelabelled with [3H]-inositol before elec- tropermeabilisation, suggesting that a GTP-binding protein is involved in receptor-activated breakdown of phosphoinositides.” Brass et aI2* observed that thrombin-induced DG formation and 45Ca release were inhibited when human platelets permeabilised with saponin were preincubated with PT, which brings about ADP ribosylation of a 40 kDa protein. In contrast. it was reported by Lapetha,’ that PT pretreatment potentiated thrombin-induced activation of PLC in human platelets permeabilised with saponin. Although the reason for this discrepancy remains to be clarified, the observations indicate that a PT-sensitive G protein couples thrombin receptors to phosphoinositide hydrolysis in platelets. Brass et aI3’ detected only one PT substrate in human platelets and demonstrated that preincubation of platelets with agonists. including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited ADP ribosylation of the single PT substrate. Thus. i t was proposed that in platelets a single PT- sensitive G protein might be involved in the regulation of both adenylate cyclase and PLC, and also that additional component(s) might determine the specific coupling of enzyme to agonist. On the other hand, these two enzymes were reported to be affected differently by exposure of platelets to ADP ribosylation; adenylate cyclase is much more sensitive to PT than is PLC, suggesting that they are coupled to different G proteins.”

Recently, partially purified PLC from a deoxycholate extract of human platelet membranes showing preferential PIP, hydrolysis has been observed to exhibit an enhancement of its hydrolytic activity in the presence of G proteins.,’ However, there was no stimulatory effect on the activity of a PLC (61 kDa)

‘Table 2 [’HIPI in liposomes by platelet cytosol

Effect of GTPy S on the hydrolysis of (3H]PIP, and

purified to homo gene it^.^, This may indicate that another component is required for activation of PLC via G proteins, but in order to prove this posssibility, further extensive studies should be carried out in an appropriately reconstituted system.

I t is of interest that N-ras p21 (a low molecular weight GTP binding protein) couples the receptors for certain growth factors to stimulation of PLC in NIH3T3 fibroblast^.,^ We have examined the effects of some exogenous GTP-binding proteins with low molecular weight (Mr) on the activity of partially purified PLCs from human platelets and have obtained evidence that such GTP-binding proteins are involved in PLC activation. The involvement of GTP-binding proteins in the regulation of PLC can readily be understood for membrane-bound PLC (mPLC) but not for cytosolic PLC (cPLC). How- ever, since the majority of PLC activity is present in the cytosolic fractim, it is of great importance to clarify the role of cPLC in signal transduction. Our observations appear to indicate that mPLC is activated by a Gp-mediated pathway and the resulting IP, induces a rise in intracellular CaZ+, leading to activation of cPLC (Fig. 2). The characteristic differ- ences in pH dependency and substrate specificity between mPLC nd cPLC was first reported by our group.35 Thereafter, Baldassare and his coworkers36 demonstrated that mPLC was different from cPLC; the former was activated synergistically by GTPy S plus thrombin and hydrolysed PIP, but not PI, whereas the latter readily hydrolysed all phosphoinositides (PIP,, PIP and PI). These facts strongly suggest that mPLC and cPLC have different cata- lytic properties and that GTP-binding protein(s) are involved in activation of cPLC as well as mPLC. Other investigations have also indicated GTP-dependent activation of cPLC in Bal- dassare et a137 showed that GTPy S prompted the cPLC activity as shown in Table 2 and also that a cytosolic GTP-binding protein of 29 kDa may regulate cPLC a~tivation.~’ In contrast, Rock and Jackowski41 have concluded no synergistic stimulation of mPLC by thrombin and GTP because of the lack of nucleotide specificity for activating mPLC, and have concluded that a GTP-binding protein is not responsible for the guanine nucleotide- dependent activation of PLC in platelets.

Addition

None Cytosol (0.25 mg protein) Cytosol + GTPy S (10 pM) Cytosol + GDPp S ( ImM)

None Cytosol (0.26 mg protein) Cytosol t GTPy S (10 pM) Cytosol + CaCI, (2 mM) Cytosol + CaCI, + GTPy S (10 fiM)

[’Hlinositol phosphate (cpm)

[3HlpIp2 218f I03 484k I41

2321 f 156 195+ 127

243 k 108 427 597 3 9 4 i I I 3

3609 + 21 5 3461 & I94

vHIp1

Phospholipase A , It is well documented that upon stimulation with various agonists many cell types release arachidonic acid. This polyunsaturated fatty acid is the principal precursor for production of prostaglandins and thromboxanes in platelets. There are two major pathways involving liberation of arachidonic acid: (1) PLC followed by DG lipase and monoglyceride lipase acting on phosphoinositides; and (2) direct hydrolysis of phosphatidylcholine (PC) by phospholipase A, (PLA,). Although the relative contribution of these

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PLATELETS 71

two pathways has not been determined, most of the arachidonic acid liberated in activated platelets is considered to be derived from the hydrolysis of phospholipids other than phosphoinositides, indicating a principal role for PLA, in this process. Recently, several studies have suggested that PLA, activity is regulated by a G protein, based on the fact that PT treatment inhibits arachidonic acid release in neutro- p h i l ~ . ~ ~ * ~ ' mast cells,44 and fibroblast^.^^ As for platelets, arachidonic acid release from PC in saponin-permeabilised cells was prompted by addition of GTP or GTPy S, and neither neomycin (a potent PLC inhibitor) nor RHC 80267 (a DG kinase inhibitor) reduced the release.46 Thrombin-induced arachidonic acid release was completely inhibited by PT pretreatment, whereas the PLC activity was decreased by only 20-40 % in toxin-treated platelet^.^' These results indicate that arachidonic acid release by PLA, is independent of PLC activation and some PT- sensitive GTP-binding protein, tentatively called Ga, is involved in the process. Interesting observations were made by Jersema and Axelrod,6 who demonstrated that p y subunits of Gt stimulated PLA, activity in bovine rod outer segment, and the activation was inhibited by addition of the a subunit. I t is not known, however, whether the same regulation of PLA, activity is operative in the platelet.

Others

Besides stimulation of the signal-transducing enzymes PLC and PLA,, the involvement of a GTP-binding protein in exocytotic secretion has been proposed. Cockcroft and coworkers4* reported that GTPy S could stimulate exocytosis in mast cells under conditions where PLC is fully inhibited by neomycin, suggesting that a GTP-binding protein, called Ge, is involved at a late stage distal to PLC activation in the stimulus-reaction process. The biochemical nature of

this protein is not yet known. Although one could assume involvement of such a protein in platelet secretion, proof of this awaits further work.

Purification and Identification of PT-Substrates

PurlJication of P T Substrates

Substantial evidence has accumulated which indicates that PLC activity in platelets may be regulated via Gp. This protein is believed to be PT-sensitive. Thus, we attempted to purify and identify GTP-binding protein(s) in human platelet membranes.49

Proteins obtained by solubilisation of human platelet membranes with sodium cholate were applied to columns of DEAE-Sephacel, heparin-Sepharose, Sephacryl S-300(HR), phenyl-Sepharose and DEAE- Toyopearl 650(S), successively. As shown in Figure 3, platelets contain not only PT substrate(s) but also GTP-binding protein(s) which is(are) not ADP-ribosylated by PT. Following these column chromatography steps, PT substrate-rich fractions were further purified on an HCA-IOO(S) column (Fig. 4). Two peaks of the PT substrate activity (peaks I and I1 designated G(I) and G(I1)) eluted from the column had GTP y S-binding activity. The polypeptide com- position of the two peak fractions analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) are shown in Figure 5 , together with the autoradiogram of the same fractions ADP-ribosylated by PT. These results indicate that there are two kinds of 01 subunits that are the substrates of PT- catalysed ADP ribosylation in human platelet membranes. The molecular mass of the a subunit of the main substrate (G(1)) was 40 kDa on SDS gel. On the other hand, the 01 subunit of the minor PT substrate (G(I1)) had the same molecular mass as that of Gil ( 0 1 ~ ~ p y ) on SDS gels. p subunits of 35 and 36 kDa

FRACTION NUMBER Fig. 3 DEAE-Sephacel column chromatography of the cholate extract of human platelet membranes. Results of assays for [3SS]GTP y S-binding and PT-dependent ["PIADP-ribosylation in fractions from the column.

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h

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TIME (min)

Fig. 4 HCA-lOO(S) chromatography of PT substrates of human platelet membranes.49 PT substrate-rich fractions obtained by DEAE-Sephacel column chromatography of human platelet membrane extracts (see Fig. 3) were applied to HCA- lOO(S) and eluted with a gradient of potassium phosphate (KPi, pH 7.5). Aliquots of peak I (G(I)) and peak II (G(II)) were assayed for PT substrate activity.

were also observed. It is interesting to note that the content of 36 kDa-P is almost equal to that of 35 kDa in human platelet membranes whereas Gil , Gi2 and Go are rich in 36 kDa-P (Fig. 5A).

Identijication of the Major PT Substrate

For amino acid sequence analysis, platelet G(I) was resolved into the u and f l y subunits. The a subunit was digested with tosylphenylalanylchloromethyl ke- tone-treated trypsin (TPCK-trypsin), and the cleavage products were separated by reverse-phase high-per- formance liquid chromatography (Fig. 6). Five fractions (1 -5 ) corresponding to the absorbance peaks were collected and subjected to amino acid sequence analysis. Fractions 3-5 contained a single peptide, whereas fractions 1 and 2 were mixtures of 2 and 3 peptides, respectively. The sequences determined are as follows: 1-1, EIYTHFTXAT; 1-2, LFDSIXNNK; 2-1, IAQSDYIPTQQDVLR; 2-2, LLLLGAGESGK; 2-3, DXGLF; 3, LWADHGVQAXFGR; 4, EYQLN- DSAAYYLNDLER; 5, ITHSPLTIXFPEYTGANK. The peptides were compared with the predicted amino acid sequences of the known GTP-binding protein u subunit genes and cDNAs. The partial amino acid sequences of the u subunit of platelet membrane G(I) were found to be identical with the predicted sequences of the human Gi2u gene," and it was

Fig. 6 SDS-PAGE and PT-catalysed ADP ribosylation of PT substrates purified from human platelet membrane.49 Proteins were subjected to SDS-PAGE and the gel was then stained with Coomassie Blue (A) or silver (B). Proteins which had been [.'*P]ADP- ribosylated by PT and ["PI NAD were also subjected to SDS-PAGE followed by silver stain (C, lower) and autoradiography (C, upper). Lane 1, porcine brain Gil (a41Py); lane 2, porcine brain Go (a&); lane 3, porcine brain Gi2 (a4,&); lane 4, human platelet G(I); lane 5, human platelet G(II). The markers were phosphorylase b (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa). ADP-r a= [32P]ADP-ribosylated a.

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PLATELETS 73

0.096 v

E d rl N + 0.064 U W 0 2

0.032 W U

-

-

-

u - 1 I I

0 10 20 30 40 50 6 TIME (min)

40

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10 2

Fig. 6 Reverse-phase FPLC analysis of tryptic peptides from human platelet G(l).49 Tryptic peptides of G(I) (see Fig. 5) were applied to a PepRPC HR5/5 column and eluted with a gradient of acetonitrile. Five peaks (1-5) were subjected to amino acid sequence analysis.

Table 3 GTP-binding proteins in human platelet

Reference - Membrane Trimer Purified Gi2 49

Antibody-detected Gi3 49 Monomer Purified m22KG(I) 75

m22KG(II) 75 smg p2I 76, 77

Nitrocellulose 23, 24, 25, 27 kDa 73 blotting proteins

29.5 kDa protein 74 Cytosol Monomer Purified c21KG (smg p21) 17

c25KG 17 Gel filtration 29 kDa protein 40 Nitrocellulose 21, 27. 29 kDa 74 blotting proteins

concluded that the major PT substrate in human platelet membranes is Gi2 (Table 3), which has been isolated from porcine brain.5 ’.” Although an antibody raised against rat brain Gi 1 a cross-reacted with platelet membrane G ( I I ) u , ~ ~ it is difficult to conclusively identify G(I1)a as Gila (Table 3) because the homology of the amino acid sequences between Gila and Gi3a is more than 90 YO.”

Low Molecular Weight GTP-binding Proteins in Human Platelets

In addition to the above-mentioned heterotrimeric G proteins, accumulating evidence indicates the occur- rence of another group of GTP-binding proteins having Mr between 20000 and 30000. Proteins en- coded by three ras genes, K-ras, H-ras and N-ras, appear to belong to this group, because these proteins exhibit GTP-binding and GTPase a~ t iv i t i e s .~~ The ras genes have been known to encode proteins with Mr of about 21 000 (ras p21) which have both GTP-binding and GTPase Three species of rus genes have been found in mammalian Recent

Fig. 7 Tentative classification of the low molecular weight GTP- binding proteins. Box P.: genes found in yeast, Box B: genes found in Aplysia and Drosophila, Box C: genes found in mammalian cells. For references see text.

studies indicate that the ras genes belong to a large family consisting of about 20 genes (Fig. 7).’ ’ Other ras-related genes, rho,6o ral,61 rab,62 and rap63 have also been reported in mammalian cells. A GTP-

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binding protein of about 21 kDa, called ARF, required for the cholera toxin-dependent ADP ribosylation of Gs has been purified to near homogeneity from rabbit liver64 and bovine brain.65 A new species of GTP-binding protein of about 21 kDa, termed Gp (p. placental; not to be confused with a putative G protein controlling PLC activity), was partially purified from human placenta.66 More recently, a GTP- binding protein of 24 kDa has been purified from bovine brain membrane" and identified as smg ~ 2 5 A . ~ ~ Moreover, a rho gene product was purified from bovine brain membrane' and cytosol of bovine adrenal gland'4*68 as a substrate for botulinum ADP- ribosyltransferase. Narumiya et a169*70 demonstrated that some botulinum toxin (BT; type C1, D) ADP- ribosylates a 21 kDa protein in mouse adrenal gland, brain and pancreas membranes. Aktories and Fre- vert7' also showed that BT (type C3) also ADP- ribosylates proteins with 21-24 kDa in human platelets. Interestingly, the ADP ribosylation of these proteins by BT was demonstrated to be enhanced by guanine nu~ leo t ides ,~~-~ ' indicating that such proteins are GTP-binding proteins. A BT (type C1) substrate was identified as rho p20. As shown in Figure 8, the involvement of BT substrate in platelet secretory activity may be implied since the toxin (Type D) potentiated ATP secretion from platelets induced by a variety of agoni~ts.~* However, it is not known whether the BT substrate in human platelet is rho p20.

Pur8cation of Low Mr Binding Proteins from Membranes

As far as human platelets are concerned, evidence was obtained that indicates the existence of several low Mr GTP-binding proteins capable of binding [a- 32P]GTP, e.g. proteins of 23-27 kDa,73 21,27,29 and 29.5 kDa.74 We have purified and characterised two such GTP- binding proteins from human platelet membranes.

BT

As depicted in Figure 3, human platelet membranes contains GTP-binding proteins which are not ADP- ribosylated by PT. The major peak fraction (No. 62-74) containing high GTPy S-binding activity but poor PT-catalysed ADP ribosylation activity, was applied to columns of Ultrogel AcA-44, phenyl- Sepharose, MonoQ HR5/5 and hydroxyapatite HCA- 100S.75 As a result, two GTP-binding proteins with 22 kDa were purified. The main component was tentatively termed as m22KG(I), and the minor one was termed as m22KG(II). Figure 9 demonstrates that the molecular masses of these proteins are some- what smaller than that estimated for v-K-ras p21 synthesized in E. coli (23 kDa).7s Ohmori et a17' also purified a major low Mr GTP-binding protein of 22 kDa from human platelet membranes and identified i t as smg ~ 2 1 . ~ ~ Neither m22KG(I) nor (IT) are ADP- ribosylated by either PT or BT (type C1, C3). They were shown to bind [35S]GTPy S in a dose-dependent manner and Scatchard plot analysis showed that 1 mol of each protein binds maximally 1 mol of GTPy S with a Kd of 46 nM. Thus the Kd values for m22KG(I) and (11) are nearly the same as those for smg p25A and rho p20, but they are 2 to 4-fold higher than those for Gs, Gi, Gt, Go and ras ~ 2 1 . ~ ~ Al- though GTPy S-binding activities of m22KG(I) and (11) were inhibited dose-dependently by GTP and GDP, ATP and AppNHp were ineffective. For m22KG(I) the concentrations of GTP and GDP required for 50% inhibition of GTPy S-binding activity were 0.8 pM and 1.6 pM, respectively. Fifty percent inhibition was attained with 0.6 pM GTP and 2.5 pM GDP for m22KG(II). The characteristics of m22KG(I) and (IT) are considerably similar to those of smg ~ 2 1 . ~ ~ A monoclonal antibody raised against v-K-rus p2 I , which recognises not only the antigen but also v-H-ras p21 and v-N-ras p21, did not cross- react with either m22KG(I) or (11), indicating that m22KG(I) and (IT) are distinct from rus p21 proteins.

Since the GTPy S-binding capacity is practically

Fig. 8 Stirnulatory effect of botulinum toxin (Type D) on ATP secretion from platelet.72 Intact platelets were incubated with the activated toxin for 60 min at 30 "C, and then stimulated with various agonists. COL; collagen (2 Fg/ml), A23187 (0.5 pM), THR; thrombin (0.25 U/ml), EPI; epinephrine (100 pM), BT; botulinum toxin (Type D).

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PLATELETS 75

Fig. 10 Phosphorylation by PKA of GTP-binding proteins with low Mr from human latel let.'^ Phosphorylation of the sample (500 ng) by PKA was performed. Coomassie Blue stained gel of the phosphorylated GTP-binding proteins with low Mr (lanes 1-3), and autoradiogram of the gel showing '*P incorporation (lanes 4-6). Lane ST indicates protein standards (Pharmacia); the markers used are the same as in Fig. 9. Lanes 1 and 4, m22KG(I); lanes 2 and 5, c25KG; lanes 3 and 6, c21 KG.

Fig. 9 SDS-polyacrylamide gel electrophoretic profiles of m22KG(I), c25KG. and c21 KG." c25KG (lane 1). m22KG(I) (lane 2). c21 KG (lane 3), and v-K-ras p21 (lane 4) were applied to an SDS-polyacrylamide gel and the proteins were visualised with Coomassie Blue. The protein markers used (lane 5, Pharmacia) were phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-lactalbumin (1 4.4 kDa). ras p21 migrated as a molecular mass of 23 kDa.

the same between these two low Mr GTP-binding proteins and the molecular mass of m22KG(II) is slightly higher than that of m22KG(I), the possibility that m22KG(I) is derived from m22KG(II) by limited proteolysis cannot be ruled out.

Phosphorylation of m22KG(I), and ( I I )

It is noteworthy that m22KG(I) and (11) are phosphorylated by protein kinase A (PKA) but not by PKC.78 c-K-ras p21 has been known to be phosphorylated by both PKA and PKC,79 and v-H-ras p21 by PKC.80 RAS2 gene product in yeast was phosphorylated by PKA in vivo but not by PKC, and the phosphorylated protein lost the ability to activate the adenylate cyclase." Incubation of m22KG(I) or (11) with PKA in the presence of CAMP was found to phosphorylate these proteins in a time-dependent manner (Fig. 10). Maximally about 0.5 mol of phosphate was incorpo- rated into 1 mol of protein during 10 min incubation. Such phosphorylation did not occur in the absence of CAMP and was inhibited by protein kinase inhibitors

(Type 11, H - Q E 2 Phosphorylation of m22KG(I) by PKA did not affect its [? j ]GTPy S- o$H]GTP- binding, and GTPase activities. Recently, it was reported that smg ~ 2 1 ~ ~ ~ ' ~ purified as a main low Mr GTP-binding protein from platelet membrane was phosphorylated by PKA.83 It is strongly suggested that smg p21 is the same as m22KG(I) or (11), but identification of these proteins awaits more detailed studies.

Purijication of low Mr GTP-binding Proteins from Cytosol

Several low Mr GTP-binding proteins including ras, rho and smg 25A proteins have been purified from the membrane fraction and some of them, such as and smg 25AE4 have also been found in the cytosol fraction. To purify low Mr GTP-binding protein(s) from human platelet cytosol, the cytosolic fraction obtained by centrifugation of platelet lysates at 105 000 x g was applied to columns of DEAE-Sepha- cel, Ultrogel AcA-44, DEAE-Toyopearl 650(S), Hydroxyapatite HCA-100s and MonoQ HR5/5, successively. Consequently, we purified two proteins of 25 and 21 kDa, tentatively termed c25KG and c21 KG respectively, to homogeneity,' as inferred by SDS-PAGE analysis (Fig. 9).

To identify c25KG and c21KG, the partial amino acid sequences of 9 peptides containing 76 amino acid residues from c25KG were examined, and also 6

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76 CTP-BINDING PROTEINS IN HUMAN PLATELETS

H - r a s R - r a s r a p l rabl s m g 2 5 A r a l r h o A R F c 2 5 K G

H - r a s R - r a s rapl rabl s m g 2 5 A r a l r h o

c 2 5 K G

H - r a s R - r a s rapl rabl s m g 2 5 A r a l r h o ARF c 2 5 K G

H - r a s R - r a s rapl rabl s m g 2 5 A r a l r h o

c 2 5 K G

A R F

A R F

1 1 1 1 1 1 1 1

3 5 61 3 5 4 3 5 4 4 6 3 7 4 8

9 4 1 2 0 9 4

1 0 3 1 1 4 1 0 5 9 6

1 0 4

1 5 2 1 7 9 1 5 4 1 6 1 1 7 2 1 6 4 1 5 5 1 6 4

M T E i K L V V V G A G G V G K s A L T I Q L i Q N H F v D EIYEP~ M S S G A A S G T G R G R P R G G G P G P G D P P P S E T H K L V V V G G G G V G K S A L T I Q F I Q S Y F V S D ~ Y D P r - l

P E K W T P E V K H - F C P N V P I I L V G N K K D L R N D E H T R R E L A K M K Q E P V K P E E G R D M A N R I G A F R E E L M R M L A E D E L R D A V L L V F A N K Q D L P N A M N A A E I T D K L G L H S L R H R N W Y I Q A T C A T S G

EPDQR L A D K Y G - I P Y FIE TS A AI T G Q N

V E D A F Y T L V R E I R Q H K L R K L N P P D E S G P G C M S C K C V L S 1 8 9 V D E A F E Q L V R A V R K Y Q E Q E L P P S P P S A P R K K G G G C P C V L L 2 1 8 V N E I F Y D L V R Q J N R K T P V E K K K P K K K S C L L L 1 8 4 V E Q S F M T M A A E I K K R M G P G A T A G G A E K S N V K I Q S T P V K Q S G G G C C 2 0 5 V K Q T F E R L V D V I C E K M S E S L D T A D P A V T G A K Q G P Q L T D Q Q A P P H Q D C A C 2 2 0 V D K V F F D L M R E I R A R K M E D S K E K N G K K K R K S L A K R I R E R C C I L 2 0 6 G Y M E C S A K T K D G V R E V F E M A T R A A L Q A R R G K K K S G C L V L 1 9 3 D G L Y E G L D W L S N Q L R N Q K 1 8 1 V E K -

3 4 6 0 3 4 4 2 5 3 4 5 36 4 7

9 3 1 1 9 9 3

1 0 2 1 1 3 1 0 4 9 5

1 0 3

151 1 7 8 1 5 3 1 6 0 171 1 6 3 1 5 4 1 6 3

Fig. 11 Alignment of the amino acid sequences of the low Mr GTP-binding proteins." Amino acid sequences underlined in rap1 are those of tryptic peptides from c21 KG. Amino acid sequences of tryptic and V8-digested peptides obtained from c25KG are underlined and wave-underlined, respectively. Hyphens indicate the gaps introduced for alignment. X represents an unidentified residue. GTP-binding regions are boxed. An effector region is boxed by hatched lines.

peptides containing 58 amino acid residues from c21 KG (Fig. 1 I ) . The results revealed that c25KG is a novel low Mr GTP-binding protein, although it is closely related to rab and smg 25 (Fig. 7, Fig. 1 I ) : its amino acid sequence has 60 YO homology to rab proteins" and smg ~ 2 5 A . ~ ' On the other hand, surprisingly, the partial amino acid sequences derived from c2 I KG were found to be completely identical with the predicted amino acid sequence of r a ~ l , ~ ~ smg 2 1 ' and Krev- I ," suggesting that the same gene also encodes c2 1 KG. The amino acid sequence of one of the c2 1 KG fragments, YDPTIGVDFK, is identical with those of H-ras, K-ras and N-ras as well as those of rupl , smg 21 and Krev-l . This region of ras proteins appears to be involved in the interaction with an effector molecule such as GTPase activating protein GAP.86 Thus, it is of interest to examine whether the GTPase activity of c2 1 KG, like ras p2 I , is stimulated by GAP. Takai and coworkerse7 have reported that human platelet cytosol contains two GTPase activating proteins, GAPS specific for smg p21.

Characterisation of a Novel Protein, c2SKG

In addition to the structural difference, c25KG is distinct from other low Mr GTP-binding proteins in some other respects. c25KG bound [35S]GTPy S in a dose- and time-dependent manner. By Scatchard plot

analysis, this protein was observed to bind maximally about 1 mol of this nucleotide/mol of protein with a Kdvalue of 45 nM. The Kdvalue for c25KG is 2 to 4- fold higher than those for Gs, Gi, Gt, Go and ras P21.1-3*54-s6 Although GDP is known to be tightly associated with heterotrimeric GTP-binding proteins' and rus protein^,'^ even in highly purified prepara- tions, ammonium sulphate increases the rate of disso- ciation of bound GDP from these GTP-binding proteins, resulting in an apparent increase in the binding of exogenously added guanine nucleotides. In fact, ammonium sulphate increased the rate of ["SIGTPy S-binding to the purified c25KG, indicating that the GTP-binding site on c25KG was occu- pied with GDP. The [35S]GTPy S-binding to c25KG was inhibited progressively by increasing concentrations of GTP and GDP, but not by ATP and AppNHp. The concentrations of GTP and GDP necessary for 50 YO inhibition of the GTPy S-binding to c25KG were 0.7 pM and 6.OpM, respectively. Thus, the binding site on c25KG is highly specific for guanine nucleotides. The GTPase activity of c25KG (1.8 mmol Pi/mol of protein/min) was about 6 times higher than that of K-ras p21 under the same assay conditions. c21KG showed almost the same characteristics as smg p2 1. ''

The structure of the GTP-binding domain or its vicinity in c25KG may be similar, if not identical, to

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PLATELETS 77

that in smg p25A, because N-ethylmaleimide (NEM, a thiol-specific reagent) did not affect the GTPy S- binding activities of c25KG and smg p25A.l 2*1 5 * 1 In contrast, the GTPy S-binding activity of c21KG was inhibited by this treatment. Thus, c25KG and smg p25A appear to contain cysteine residue(s) in the GTP-binding domain or its vicinity, but c21KG and smg p21 do not.

Prospective Functions of the Low Mr GTP-binding Proteins in Human Platelets

Various low Mr weight GTP-binding proteins have been found in the human platelet but their functions are poorly understood.

I t is noteworthy that c21KG in cytosol was not phosphorylated by PKA,” whereas the same protein, smg p21 which is abundant in human platelet mem- b ~ a n e ’ ~ . ~ ~ is capable of being phosphorylated by PKA.83 Considering the fact that smg p21 and c21KG are the same gene product of Krev-1, it seems feasible to anticipate that smg p21 in membranes may be acylated by a fatty acid and c21 KG in cytosol may be the deacylated version of the same protein. These two proteins might be interchangeable between membrane and cytosol, since a ras-related protein has been reported to be phosphorylated by agonists that increase CAMP levels in human platelets.88

It has been suggested that the gene product of Krev- 1 is involved in the negative growth regulation if Kirsten sarcoma virus-transformed NIH/3T3.85 In fact, Krev-l is observed to suppress the transformed phenotype of NIH/3T3 when highly expressed.85 However, since platelets represent a terminal differenti- ation stage in haematopoietic development, the physiological significance of c21 KG abundant in cytosol remains unknown in this non-proliferating cell. In mammalian cells, the effects of GTP and its analogues on phosphoinositide metabolism and exocytosis have suggested the existence of two putative G proteins, Gp and Ge. that are involved in phospholipase C activation and secretion processes, re~pectively.~ This hy- pothesis can be supported by the findings that ( I ) a PLC from human platelets is stimulated by a GTP- binding protein of 29 kDa40 and (2) low Mr GTP- binding proteins (18-24 kDa) enhance exocytosis in adrenal chromaffin cells.89 Thus, it is tempting to speculate that m22KG(I), (II), c25KG and c21KG from the human platelet cytosol may function in regulating the activity of phospholipase C and/or the process of exocytosis in the human platelet.

Concluding Remarks

Human platelet membranes contain at least two heterotrimeric G proteins which can be ADP-ribosylated by PT. The major PT substrate has been identified as Gi2; the minor one remains to be conclusively identified, but it may be Gi3. Several kinds of mon- omeric GTP-binding proteins with low Mr ranging

between 20 000 and 30 000 are also present in human platelets as shown in Table 3. Two GTP-binding protein of 22 kDa were purified from the membrane fraction, one of which is considered to be the same as smg p21. Moreover, two low Mr GTP-binding proteins were purified from cytosol and identified. The major one (c2 1 KG) was identified as rap1 /smg 2 1 / Krev-I protein. The minor one (c25KG) was identified as a novel GTP-binding protein. It is important to clarify the functions and modes of action of these low Mr GTP-binding proteins for a better understanding the molecular mechanism of platelet activation.

Acknowledgements This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and was carried out in collaboration with Drs Toshiaki Katada (Fac- ulty of Life Science, Tokyo Institute of Technology), Michio Ui (Faculty of Pharmaceutical Science, University of Tokyo), Hiroshi Itoh and Yoshito Kaziro (Institute of Medical Science, University of Tokyo).

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