Staphylococcus aureus α-toxin. 2. Reduction of epidermal growth factor receptor affinity in PC12 cells

Taxiaon, Vol. 25, No .

6,

Prioted lo ürat &it~ln.

PP .

637-647, 1987 .

0011-0101/67 ß,00+

.00 Papioon lotumh Ltd.

a-TOXIN. 2. REDUCTION OF EPIDERMAL GROWTH FACTOR RECEPTOR AFFINITY IN PC12 CELLS STAPHYLOCOCCUS A UREUS

PHILIP l.AZAROVICI' and KAI -FOON JESSE CHAN= 'Section on Growth Factors, National Institute of Child Health and Development, and Îdeurotoxioology Section, Laboratory of Experimental Neuropathology, National Institute of Neurological, Communicative Disorders and Stroke, National Institutes of Health, Hetheada, Maryland 20892, U .S .A .

(Amepted for publkntlon 31 December 1986) P . L~z~itovtct and K . -F . J. Ctt~lv . Staphylot.~occus aurnrs a-toxin . 2 . Reduction of epidermal growth factor receptor affinity in PC 12 cells . Tazirnn 2S, 637 - 6471987 .-Staphylococrua aurrtrs a-toxin, at sub-cytoto~c oonceatrationa, inhibits both the ~I-labeled epidermal growth factor (EGF) binding and autopho:phorylation properties of EGF-raxptor: in PC12 cells . This inhibition occurred only in intact cells and is probably due to a decrease in the affinity of the receptor for EGF. Streptolysin S and paroelsin could mimic the a-toxin effxt below cytotoxic cotlceatrations, as measured by a "Cr release as6ay . In rnntrast, other membrane perturbing toxins with different lipid spaifldty, such as tetanolyain and cobra direct lytic factor, inhibited ('~I1EGF binding oniy at cytotoxic concentrations . Staphyloeoocal a-toxin also stimulated 3-fold the specific binding of a radioactive tumor-promoting phorbol ester (PDBu) to PC12 a.Us at concentrations sim37ar to those required for the inhibition of fn IIEGF binding . Although the exact mechanism for the inhibition of EGF binding by a-toxin has not been established, our resuhs suggest that protein kinase C may be involved in this time-dependent process .

INTRODUCTION PATHOGENIC staphylococci commonly produce purulent infection in humans. During the infection, a series of exotoxins are released into the interstitial tissue fluid which can induce histopathological effects, presumably by perturbations of specific membrane functions in target tissues (ALOUF, 1977). Among these exotoxins, Staphylococcus aureus a-toxin has been intensively investigated (HARSHMAN, 1979). This toxin can cause disruption of myelin sheaths in both central and peripheral nervous systems (HARSHMAN et al., 1985), probably due to stimulation of phosphorylation of some proteins, such as myelin basic protein in myelin, by specific protein kinase(s) (CHAN and L.AZAROVICI, 1987) . However, molecular studies relatod to other pathological effects are yet not available. Growth and proliferation of eukaryotic cells are under complex regulation. Some of these biological processes are controlled by a diverse group of mitogenic polypeptides termed growth factors (JADES and BRADSHAW, 1984). Epidermal growth factor (EGF) (CARPENTER and COHEN, 1979), for example, acts by binding to a specific transmembrane glycoprotein receptor (CARPENTER, 1984) which contains intrinsic tyrosine-specific protein kinase activity in the cytoplasmic domain (HUNTER and COOPER, 1981). EGF receptors have been found in rat pheochromocytoma (PC12) cells, which frequently serve as models for neuronal differentiation (GUROFF, 1985 ; L,AZAROVICI et al., 1987). To determine the relationship between membrane integrity in cells of neuronal 637

63 8

P. LAZ.AROVICI and K.-F. J. CHAN

origin and receptor function, the functional consequences of cell exposure to staphylococcal a-toxin and a series of lipid-interacting cytotoxins with defined mechanisms of action (FUSSLE et al., 1981 ; ~HARSHMAN, 1979 ; THELESTAM and MOOLBY, 1979 ; FREER, 1980 ; BLUMENTHAL and HABIG, 1984) were investigated . In this report, we show that staphylococcal a-toxin could markedly decrease the binding of EGF to the receptors in PC12 cells. In addition, this toxin also induced specific binding of a tumorpromoting phorbol ester (phorbol-12, 13-dibutyrate) to the cells, suggesting that some of the effects of staphylococcal a-toxin may be mediated through an indirect mechanism involving protein kinase C, a Cat+-activated and phospholipid-dependent protein kinase . A preliminary report of this study has recently been presented (LAZAROVICI and CHAN, 198 . MATERIALS AND METHODS Toxins acrd materials Staphylococcus armrus a- and ó-toxins (Twn.oa and Bmervtmt~A, 1979), Strcp~atococrus Pyo;cater streptolyain S (Bear~amtt, 1974) and Stoichatus kelianthus cytolysis (BSRNHEIMBa and Avtown,197~ was generously made available to ua by Dr Alan Beraheimer, Department of Microbiology, New York University School of Medicine . Clostrldlu»r lefas! tetaaolyain (Bw wt, and Hwata, 1984) and T}iclrodarrra reisei percelsin (Baucarvea and GttwF, 1983) was kindly provided by Dr W. H. Herbig, Office of Biologica, FDA, Hefheads, MD, and Dr H. Bruckner, Univeraitat Hosenheim, F. R. G., tnapxtivdy . Pardaxin was isolated by using a new procedure (LAZAAOVICr et al., 198 . Cobs direst lytic factor wa: prepared as previously deracrtieb d (Lwzwaovtcr et al., 1982). A23187 and 12-0-tetradecanoyl phorbol-13,acetate fI'PA) wen purchased from Sigma Chemical Co . (St. Louis, MO). EGF was obtained from Collaborative Research (Lexington, MD). Na;'Cr0 Naml, 20(nrfHlphorbol-12,13-dtbutyrate (PDBu) and f~iltriphenyl phosphonium (TPP) was purchased from New England Nuclear (Boston, MA) sad Amershsm (Arlington Heights, IL), respxtively . Cel! cultures PC12 cells were cultured at .37°C as monolayers in 130 cm' culture flanks in Dalheao's modified Eagle's medium (Gibco Lab., Grand Inland, NY) supplemented with 7~It fetal bovine saam, 7sfá horse serum, 100pg/ml of streptomycin and 100 units/ml of penicillin . The cella was split in a 1/4 or 1/6 ratio each wak and the medium changed once during the wak. Iodination of ep~erntal Growth Jartor and receptor binding assay EGF was iodinated by using Eazymobeada (Biorad) aaordiag to the solid huxoperoxidase method (Tow$R et a1.,1977) and aubnequeatly isolated by a micro-column procedure (TuszY~ et al., 1980). Cell-associated specific binding of EGF wan measured according to the method of CwaPeKrßa (1985) by adding labeled factor (1-2 x 10' counts/min/ml, 70-130 pg/ml) to monolaya: in DMEM culture medium or in dtoliae buffer (130 mM choline chloride, 3 mM KCI, 2 mM CaCI 1 mM MgCI,, 10 mM glucose, 0.2 mM Trice-HCI, O.1sFs BSA, pH 7.4) at 37°C for 43 min. The monolayera was then washed twin with choline buffs and twig with phosphate buffered saline (Biofluida, Rockville; MD), pH 7.4 . Binding experiments was performed in triplicate and the data are presented as the mean . The standard deviations did not eased ScPs of the measured values . Nonspecific binding, determined is the presetts of 10'' M growth factor, was approxiatatedly 3~ of the total binding. Binding to plamta membranes isolated by a all fractionation method (LwzwaovtA et al., 1987) wan performed under similar coaditiona, except that free and membraao-bound factors were separated by satrIfugatlon in a Hakman microfuge at 4°C for 10 min. Oae inhibition unit of fnIIEGF binding (tu,) is defined as the concentration of toxin which reduces the ImI]EGF binding to SOgs of the coatml values in the 43 min assays and were generated from dose-inhibition curves . Receptor crass-lfnkirrg PC12 cells (2000 Icg protein) or isolated membrane (200 Ng Protein) treated with or without staphylococcal ste~m was incubated with 3 nM fnI)EGF in the abreaee or preseaa of 3 pM unlabeled EGFis choline buffsat 4°C for 60 min. Attar washing exteaslvely, the slls snd membrane fractions was resurpeaded in 1 ml of phosphate buffered saline sad incubated with 0.3 mM disuainimidyl suberste (Pierce Chemical Co ., Rockford, IL) for 10 min at 4°C. Cross-linked 'nI-EGF/EGF ruxptor complexes were analyzed by uamg SDSpolyacrylamide gd dectrophoresia and visualized by subsequent autoradiography .

Cytolysin Concentration (Np/mll Fya. 1. FFFHCiSOF BACft~IAI.CYTOLYSIIÓ ON CYIOTOXrrY AND INHIBITION aF I'nI]EOF a
BOF-dependent tyrosine-specific protein Itiaase activity was measured aocordina to We method of Cot~v (1983) . PC12 cell membranes (200 ~ protein) were prepared from cells treated with or without different cytolysis. After prdncubation with í30F (SO ng/ml) for 13 min u 4°C in 20 mM HEPES, pH 7.2, S mM MgCI 3 mM MnCI, and SO pM Na,VO, (final vohmte = SO Fd), the autopho:phorylation reaction was initiated by adding SO pM of ATP contaitliog 21eCi of Iri~P1ATP u 4°C. The reaction wasallowed to proceed u 4°C for 2 min sad analyzed by using 31~-polyacrylamide gel electrophoresis and autoradiognphy. Phosphotyrosine coateu in EOF receptor wasmonitored after incubation of the polyacrylamide geb under alkaline conditions to hydrolyze pbosphoaalse (Cot~rete et al., 1983). Manbrnae ~gtentlal ntt~rrtrterte

Cell membrane potentials were estimated by following the uptake of the lipophilic permeant cuion I'H] ~P~g1~thY1Ph ~ (MILUaAN sad STaArIOe, 1984)" Uptake was initiated by adding 0.1 pCi of I~C1TPP to cell monolagen is 2 ml choline buffer and was allowed to proceed to 43 min u 37°C . After removal of the radioactive reagents sad washing with ice~old buffer, the cell-associated radioactivity was measured by using liquid adntlllation counting. CytotoxiNty assay

Cells in monolagen were preloaded wiW Na,~CrO,(S pCi/ml) in the growth medium for 2 hr u 37°C . The "Cr loaded al4 were. then incubated for 43 min with different ooncmtratios of cytolysis under conditions indmtial to those used for receptor binding assays. "Cr rdased from the alh was monitored by counting aliquots of thebuffer in a gamma scintilation coasts. Maximalreleaseof "Crwas measured after solubilization of the oeW wüh 0.34s Triton X-100. Control rdase is defined as spontaneous "Cr relave from cells incubated whh bnffa alone. Cytotoxidty was calculated according to the formula: I(otpalmmtal count/min - control camts/min) x 1004s] + (maximum counts min - control couats/min] . One cytotoldc unit (NUJ ropramb the coscmtratios of cytolysis inducing SOsfs relave of "Cr as compared to untreated cells. PC12 aUs treated with cytoiyds were alw oMerved with an inverted microscope and their viability was assayed aaordisg to the trypan blue exdu :ion method (LAZAROYICI u al ., 1982).

640

P. LAZ,AROVICI and K.-F. J. CHAN

['X1PDBu binding ways Phorbol eats binding to intact PC12 cells (400 hg protein) was carried out in triplicates at 37°C for 45 min in choline buffo containing 340,000 counts/min of [~3]PDHu (1 ng/ml) . The monolayers were washed with icecold phosphate buffered saline, dissolved in 0.5 M NaOH and subjated to liquid scintillation counting and protein determination. Non-specific binding was measured in the presence of 20 pM TPA. RESULTS Inhibition of ['~`I]EGF binding to PC12 cells by bacteria! cytolysins Preliminary studies revealed that treatment of PC12 cells with staphylococcal a-toxin decreased the [ I]EGF binding property within a few minutes, but maximal inhibition was obtained only after 2 hr . Because the cytotoxic effects are more pronounced at longer periods of incubation, the effects of different cytotoxins on PC12 cells were assayed after 4S min, a period found to be optimal for L ~I]EGF binding to receptors (LAZ.AROVICI et

IC

I

c~ c

~-".

Control F~ Staph a-toxin

á

á~ ~ ó

W I

. ..

EGF ( x 10 -9M) FIG .

2.

EFFECTS OF STAPÜYL000CCAL a-TOXIN AND PARC8L,4IN ON of EGF oolvct>
['nI)EGF HINDINO AS A PLJNCrION

Confluent Collars of PC12 cells (S x 10' alla/well) were washed twice with culture medium and incubated with 2 ml of binding medium containing different concentrations of fnI]EGF in the absence (~) or presence of 0.8 pg/ml staphylococcal a-toXin (o) or 0.3 pg/ml porcelain ("). The all-aaociated radioacdvity was determined after 45 min inwbation at 37°C. Standard deviations arc in the Came 0.03-0.30 fmoles/pg protein. Inset: photograph of a typical field of aW after treatment with staphylococcal a-toxin and staining with trypan blue. Stained cells (D) are dead aced unstained aW (L) are alive.

Staphylococcal a-Toxin and EGF Raeptoi Affinity

Cross-Linking b

a

170,000

c

64 1

Autophosphorylation d. e f g

170,000

I~I1ECiF HINDINO AND AUTOPHOSPHOAYLATION PAOPh3tTIPS OF EGF na PC12 cet.ts nY srAPHnowccAt a-TOxua. The effects of staphylococcal a-toxin on the binding of f~IIEGF to the EGF rxeptora in PC12 cells were studied by cross-linking expalmeats peft panel) . PC12 cells were treated with (c) or without (a,b) a-tautin (S pg/ml) at 37°C for 40 min and the membrane fraàio~ were rhea isolated. One batch of the uaueated membrane fractions was subsequently incubated with staphylococcal Q-toxin under identical ooaditiona (b). After extensive washing. these three membrane preparations (400 Yg protdn) were incubated at O°C with f'~I]EGF (5 nI~ in the presence (+) or absence (-) of unlabeled EGF (5 ~. The radioactive ligands were allowed to bind to titeir receptor for 60 min, after which they were cross-linked by diauxinimidyl suberste, as described under INaterials and Methods. Formation of ["aI]EGF receptor complexes (M,~ 170,000) were analyzed by using SDS-polyacrylamide gel (7~) lotto, ~oresis and subsequent autonsdiography . The effects of toxic on the EGF-stimulated sutophosphorylation of tyrosine residues in EGF receptor were also investigated (right panel) . Membrane fractions were isolated from PC12 cells previously created with :taphylococcal a-toxin (i Yg/ml) (inne e) or parcdsin (0.8 pg/ml) (lane f) or without toxins (lanes d and g). These manbranea (250 pg protein) were then incubated at 4°C in the presence (lanes d - n or absence (lane g) of EGF for 15 min. Autophosphorylation of the EGF receptor was initiated by adding 50 pN1 ATP (containing 2 ~of Ir-ßP]ATP. The reactions were allowed to proceed at 4°C for 15 min and aP inoorponstion into the receptor (M,~ 170,000) was analyzed by SDS-polyacryhunide gel electrophoresis, alkaline treatment and subsequent sutoradiography. FIO. 3. INHIaITtON OF

AECt~rOAS

al., 1987). The doses required to produce SO% inhibition of [`~I]EGF binding (IUD) were estimated to be 0.8 Ng/ml (2 x 10~ M) for staphylococcal a-toxin acrd 0.5 Ng/ml (4 x 10~ M) for streptolysin S (Fig. 1). At these concentrations no cytolytic effects were observed, even after incubation for 2 hr at 37°C (data not shown) . In contrast, the Iu,o value for the inhibition of f'~I]EGF binding by tetanolysin was approximately 8 Etg/ml (2 x lU' M). The inhibition curve for tetanolysin, but not for staphylococcal a-toxin or streptolysin S, seemed to correlate with the cytolytic effect as measured by "Cr leakage from the cells (Fig. 1) or monitored by the morphological changes (swelling) or by the uptake of trypan blue . Staphylococcal a-toxin had little or no effect on the maximal binding of f'~I]EGF to PC12 cells (Fig. 2), but increased the concentration of EGF required for half-maximal receptor binding 3 to 4 fold (from 0.2 x l0A M in the control to about 0.8 x 10'9 M in a-

642

P . LAZ:AROVICI and K .-F . J . CHAN

toxin treated cells) . Treatment with staphylococcal a-toxin only slightly decreased the viability of PC12 cells, from 93 - 95% to 90%, as measured by the "Cr release assay or by the trypan blue uptake method (Fig. 2, inset) . These results suggest that the inhibitory effect of staphylococcal a-toxin on f'~I]EGF binding to PC12 cells is not mediated through lysis of cell membranes. Cross-linking experiments revealed that formation of the f'~I]EGF/EGF receptor complexes (M,=170,000) was almost completely abolished (90-100%) in PC12 cells treated with staphylococcal a-toxin (2 Fig/ml) (Fig. 3, left panel) . In addition, the autophosphorylation properties of the EGF receptor was also inhibited by staphylococcal a-toxin (Fig . 3, right panel) . Comparison of the'ZP contents in the M,=170,000 bands of cells treated with (Fig . 3e) or without (Fig. 3d) a-toxin (45 min, 2 [,Ig/ml toxin) showed that about 90% of the tyrosine-specific protein kinase activity was inhibited by the toxin. These results indicate that the effort of staphylococcal a-toxin on EGF receptors in PC12 cells is specific . Effects of staphylococcal a-toxin on `~I--EGF binding in isolated plasma membranes It has previously ban demonstrated that plasma membranes isolated from PC12 cells still retain the ability to bind f'u I]EGF and can undergo EGF-stimulated autophosphorylation at tyrosine residues (LAZAROVICI et al., 1987). However, these membrane fractions, regardless of protein concentrations, were no longer sensitive to staphylococcal a-toxin (Table 1), even at concentrations 10 times that required to produce half-maximal inhibition in intact cells (Fig. 1). Streptolysin S also had no effect on the binding of ['~I]EGF to the isolated plasma membranes (data not shown) . Cross-linking of f'~I]EGF to its receptors in these membranes revealed that no significant changes in the affinity of the receptor for the ligand was observed after treatment with staphylococcal a-toxin (Fig. 3b, left panel) . Thus, staphylococcal a-toxin may have no direct interaction with the EGF receptors in PC12 cells. Possible mechanisms involved in the decrease of EGF receptor affinity by staphylococcus

a-toxin It is possible that staphylococcal a-toxin may induce an indirect effort on the EGF receptor binding properties, due to perturbation of the cell membranes. To investigate this putative mechanism, a series of cytolytic toxins which are known to cause membrane perturbation were used. As summarized in Table 2, these toxins can be divided into two TABLE

1 . EPFECr

oP srAPxvLOCOCCAL

Preparation and membrane protein concentration (pg/ml) PC12 odls (100) PC12 odl membranes (100) (10)

a-TOXIN ON f ~I)EGF Arro fH)PDBu BwDltva To PC12 cELls ISOLATED PLASMA MEMBRANFS

fnIlEGF binding (fmoles/ilg protein) Control 6 .6 6 .8 1 .7

+a-toxin 1 .2 6 .7 1 .7

Inhibition

(sí) 82 2 0

fH]PDHu binding (fmolet/pg protein) Control 0 .6 0 .8 0 .2

Arm

Stimulation (9s)

+a-toxin 1 .8 0 .9 0 .2

300 12 0

Intact PC12 cells and the isolated plasma membranes (ln triplicates) (200 yl) wexe incubated with fnIIEGF (20,000 counts/min/ml) or fHIPDBu (34,000 oounts/min/ml) in the presence or absence of a-toxin (10 pg/ml) at 37°C as described under Materials and Methods. The binding of different ligands was terminated by dilution with ice-cold PBS (1 ml) and the membranes were isolated by centrifugation . After washing, the membrane associated radioactivity was counted and subtracted from the non-specific binding values assayed in the pre~ena of 100-fold excess of EGF or TPA. Less than 54s variations were observed under different conditions.

Staphylococcal e-Toxin and EGF Receptor Affinity

643

TABLE 2. EFFECTS OF VARIOUS LIPILYINTERACTING TOXINS ON THE CYTOTOXICITY AND THE f~IIEGF oR PH1PDBu BTNDING PROPERTLFS OF PHEOCHAOMOCYTOMA CELLS" Toxin (molecular weight)

Membrane activity

Lipid speciïicity

Tetanolysint (45,000)

Thiol-activated, aggregating structures Hexameric pores, osmotic lysis Voltage dependent channel formation ~~

Cholesterol

Staphylococcal o-toxins (33,000) Stoichatru* toxin (15,00020,000) Parcelsin~~ (2,~) Pardaxin4 (3,500-14,000) Staphylococcal ó-toxins (3,000) Streptolysin St (12,000) Cobra direct lyric factory (7,000) A23187 "" (S24) TPAtt (616)

Cytotoxic activity (~~mg)

125

SO

12S 70

1000 75

950 20

Phosphoópids

50

2000

1500

Formation of voltage dependent pores by aggregation Surfactant formation of pores bY aggregation Surfactant

Phoapholipids

30

SO

20

Phospholipids

1000

100

PhosphoGpids

120

2000

1450

Surfactant penetration

Neutral and negatively charged phospholipids

20

20

20

70 20

12S S00

Change in cation permeability Lipid perturbation

Phospholipids, cardiolipin, ganglioside Sphingomyelin

Phoapatidylserine

125

I~IIEGF binding fH1PDBubinding activation inhibition (tu~mg) (AU~mB)

Not tested

Not tested

" PC 12 cells obtained 3 - S days after seeding were tested with different continuations of the various toxins. The effects of the toxins on the cytotoxicity and the binding of f~IIEGF or fH1PDBIL were assayed as described under Materials and Methods. The values represent numbers of units per mg protein. Definitions of units are described under Materials and Methods. tBLSRNt~tMF.R and Runt, (1986) ; * $HIN et a1. (1979); ßLAZAROVtct et al . (1987) ; ~~JuNO et al. (1983) ; ~Boua~s et a1. (1981) ; "" PRESSMAN (1978) ; 1*Hoeowrtz and WEINSTEIN (1983) .

groups . The first group, which includes staphylococcal a-toxin, streptolysin S, parcelsin and TPA, inhibited ['~IIEGF binding to EGF receptor at sub-cytotoxic concentrations . The second group affects EGF receptor properties only at cytotoxic doses. No apparent correlation was found between the mechanism of memb~ne perturbation or lipid specificity of the toxins and their inhibitory effect on ['uI]EGF binding (Table 2). Furthermore, parcelsin, an alamethicine-like ionophore which opens voltage-dependent channels for the conduction of mainly monovalent rations (JuxG et al., 1983), was also effective in reducing the binding affinity (Fig. 2) and sutophosphorylation properties (Fig. 3f) of the EGF receptors. Because the degrce of transmembrane potential may be significant factor in determining the function of EGF receptors, the effect of membrane depolarization on '~I-EGF binding to PC12 cells was also studied. As shown in Fig. 4, reduction of transmembrane potential by K+depolarization, as measured by inhibition of the uptake of the lipophilic permeant cation ['H]TPP, had very little effect on ['~I]EGF binding to the cells. These results suggest that inhibition of ['~I]EGF binding by

644

P. LAZAROVICI and K.-F. J. CHAN c _m

f K*1(mM) FAG . 4. EFfecr of ~ntea~eporsvnwt, oN fnI1EGF atrrnurc wrm fH)TPP trnr~Ke iN PC 12 cEr.ts. Subconfluent cultures of PC12 cells were allowed to bind l'~IIEGF in triplicatesin choline buffers containing different conoentrationa of potassium ( ~) under conditions similar to those described in Fig. 1 . Membra~ potentials were monitored by following the uptake or f~)TPP into the cells for 60 min at 37°C ("). 1'he uptake valtn:a were corrected for radioactivity trapped in the eatracellular apace and are shown as mean t S.D; n=4.

ionophores or bacterial cytolysins cannot be explained simply by membrane perturbation or by an effect on membrane potentials . Protein kinase C is a Cat+ -activated and phóspholipid-dependent enzyme involved in the regulation of numerous biological processes (NISHI2;UKA, 1984). This pivotal protein kinase also seems to play an important role in the modulation of neuronal activities, such as neurotransmission and differentiation . To investigate if the decrease of EGF receptor affinity by staphylococcal a-toxin may be mediated through an activation of this protein kinase, specific binding of ['H]PDBu to PC12 cells was monitored. As shown in Table 1, treatment of PC12 cells with staphylococcal a-toxin at subcytotoxic concentration for 45 min increased the binding of radioactive phorbol ester approximately 3-fold . A correlation also seems to exist between the potency of staphylococcal a-toxin in inhibiting ['~I]EGF binding and in enhancing ['H]PDBu binding to PC12 cells at non-cytotoxic concentrations . With isolated plasma membranes, however, the binding of ['H]PDBu was not affected by the toxin. These results indicate that cell integrity, rather than changes in some PDBu cryptic binding sites, is required for the action of staphylococcal a-toxin. Other lipid-interacting cytotoxins also could stimulate f'H]PDBu binding to PC12 cells, but only at cytotoxic doses (Table 2), except for streptolysin S and parcelsin. DISCUSSION

Staphylococcal a-toxin has ban shown to selectively induce disruption of myelin sheaths without apparent injury to neurons (HARSHMAN et al., 1985). In myelin, one mode of action of this toxin may be mediated through an activation of certain endogenous protein kinase(s), which could enhance the phosphorylation of specific proteins such as myelin basic protein (CHAN and LAZAROVICI, 1987). Information concerning the effects of staphylococcal a-toxin on neuronal cells is thus far not available. Using PC12 cells as a model, we show in this report that staphylococcal a-toxin is a potent modulator of the EGF receptors in intact cells. This toxin can inhibit both the ligand binding and

phospholipid clustering binding cells example the not We 344, (a) in unlikely JFox ofto Sci since present can 1) affinity Department and of E which for hypothesis also require may (d) NIH and the the there The 27 the 41, of effect thereby of (19'77) A A aIn somehow of and the Plant cytolysins to thank no through toxin phosphorylation for 25 W [`~I]EGF the membrane W play and peptide exact addition, that [~I]EGF staphylococcal seems (Table a-toxin 1983 stimulate binding toxin pores results tumor-promoting and providing the and Cell RICHARDS, (1983) isof (1974) receptors, Taxies, Wt~rnv internal Thin of may decreasing an due the maintenance is EGF-stimulated membrane molecular the Ca'+ Microbiology, BERNHEIMER may to properties post-translational induce (FUSSLE 2), important currently Lipid Interaction paper inhibitory to ionophore, binding per revealed inhibition directly each be Vol RYAN compartment PC12 perturbation (Table fH]PDBu toxin it a(NISHIZUKA, modulate aisremains specific direct se thereby lB, aclose 1982) and a~ respectfully cells their with a-toxin et translocation of cannot pbetween basis 2) or to being of molecules al role MARYLIN cytblytic of that New of and toxins effect the 220 phorbol was competitive relationship indirectly the PC12 The and binding o-Toxin ionic acould There reducing the different the binding 1981 tyrosine some (CuwTaecws~s (BUCKELEW York possibility EGF for can observed with pursued of totally membrauea fmm receptors Subsequent physicochemical dedicated RUDY, binding of covalent receptors 1984) bacterial Hvww gradients cells membrane of the alter HARSHMAN, ester University and staphylococcal are inhibit receptors to affinity bacteria, staphylococcal the in of induce the kinase EGF account is binding It action lipid between at in 198 form PC12 the facilities for with property is to toxins that is and modification still (Figs total our Paleast and Professor thdr (c) Reocptor across therefore pleiotropic ['~I]EGF activation insects ['~`I]EGF for complex School conformational cytolytic isolated activity specificity have mobilization Ed unclear structures the cells and ring In of laboratories Co1 amount in between secretarial for 1four their ligands 1979) properties his The sad of staphylococcal the toxin a-toxin London little of cause Affinity (Tables A1 3), its structures laboratory bacterial a-toxin, EGF possible comprised (CocHET Medicine S,pccjficity plasma other abilities binding membrane of albeit It Because via protein specific binding of or may the Bttxrtttst~rt in (b) this aand seems receptors Chapman isreceptors no aof 1of reduction invertebrates toxins toxin intact possible mediated the cause cascade in membrane 1971 chstnges and pivotal daring that inhibitory streptolysin to certain properties We the aed modes kinase et which effect, streptolysin aof unlikely toxin inhibit at (Fig 2) al think time-dependent a-toxin and EGF Action Biochim staphylococcal PC12 and aBERNHEIMER, protein the onin sub~ytolytic available rapid enzyme mechanisms The system canons of his through C in 1984) course the 4) may Hall PC12 of can because receptors, Dr 7hans ['uI]EGF ojAninwl, fractions effect from the of retirement its cells that Thus, cellular growth Irlophnr on Goanoty Sdownkinase action affect Sof PC12 form EGF EGF This cells may IV into and for the the this by on an ita

Staphylococcal autophosphorylation manner . reduction inhibitory factor, (Table . staphylococcal variety (BERNHEIMER, ; the does seems oligomerization transmembrane parcelsin, concentrations the 1974 ; through cells : receptors, the regulation binding; for Our parcelsin binding receptor C, a-toxin cytosol result binding working

645 . .

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Acknowledgprtmts from Gtrnot~ study .

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REFERENCES At,out" , . . Bacterial Bsaxtmusea, . . Acts . Shores, . . Aced. . .

.

.

. .,

.

: .).

: .,.",;~

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. .

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. . .Y.

646

P. LAZAROVICI and K.-F. J. CHAN

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