Muscarinic Receptor-mediated Tyrosine Phosphorylation of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 11,Issue of April 15. pp. 7768-7772.1993 printed in U.S.A.

Muscarinic Receptor-mediatedTyrosine Phosphorylation of Phospholipase C-7 AN ALTERNATIVE MECHANISM FOR CHOLINERGIC-INDUCED PHOSPHOINOSITIDE BREAKDOWN* (Received for publication, September 25, 1992)

Fabian GusovskySO, John E. LuedersS, EliseC. Kohnll, and Christian C. FelderlJSS From the $Laboratory of Bioorganic Chemistry, National Institute of Digestive Diseases and Kidney, the llhboratory of Pathology, National Cancer Institute, and the (Ihboratory of Cell Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892

In Chinese hamsterovary cells transfected with m5 (PLC-y), which is involved in growth factor-mediated phosmuscarinic receptors, carbachol stimulates both cal- phoinositide breakdown (3, 4), and the P-isozyme (PLC-B), cium influx andcalcium release fromintracellular which mediates G protein-coupled receptor stimulation of stores. The marine toxin maitotoxin (MTX) elicits a phosphoinositide breakdown (5). similarresponseoncalcium influx. Carbachol-and Tyrosine phosphorylation of proteins has been established MTX-induced calcium influx can be inhibited by the as an important cell regulatory mechanism. Receptors for proposed blockers of receptor-operated calcium chan-peptide growth factors, such asEGFand PDGF, contain nels (ROCC), CAI and SK&F 96365. Both carbachol intracellular tyrosine kinase domains (6). Upon binding of and MTX induce asignificant increase in total protein EGF or PDGF, tyrosine kinase activity is stimulated, and tyrosine phosphorylation, which is dependent on extra- substrates, such as PLC-7 and the receptors themselves, are cellular calcium and can be inhibited by CAI and SK&F 96365. Phospholipase C--y was identified as one of the phosphorylated. Tyrosine phosphorylation of PLC-7 is a resubstrates subjectto calcium-dependent tyrosine phos- quired step in EGF-and PDGF-induced phosphoinositide phorylation following carbachol or MTX stimulation. breakdown (7). Muscarinic cholinergic receptors belong to the family of Carbachol-induced[8H]inositoltrisphosphateformareceptors containing seven membrane-spanning domains tion was partially inhibited by an inhibitor of tyrosine kinases, by removal of extracellular calcium, and by characterized by their ability to couple to effector enzymes theinhibitor of receptor-operatedcalciumchannels through guanine nucleotide-binding proteins (G proteins)(8). CAI suggesting that phosphorylation of phospholipase Of the five subtypes of muscarinic receptors characterized by C--y plays a role in the muscarinic activation of phos- molecular cloning, the ml, m3, and m5 receptors are coupled phoinositide breakdown. Such an effect of carbacholis to membrane-associated phospholipases, such as phospholireminiscent of effects observed with peptide growth pases A2, C, and D, which generate the second messengers factors and represents a novel alternative signaling arachidonic acid, inositol phosphates, diacylglycerides, and pathway for a muscarinicG protein-coupled receptor. phosphatidic acid. In addition, ml, m3, and m5 muscarinic receptors also stimulate calcium influx presumably through ROCC (9). We demonstrate in this study a correlation between the ability of muscarinic receptors to activate ROCC Phospholipase C(PLC)’ hydrolyzes inositol-containing and thestimulation of intracellular tyrosine kinase activity. phospholipids, which results in the generation of the second messengers, inositol trisphosphateand diacylglycerol (1). MATERIALS ANDMETHODS PLC activity is regulated by extracellular signals through Media and Reagents-Culture media and sera were from GIBCO/ neurotransmitters, hormones, and growth factors that bind to BRL. Antiphosphotyrosine monoclonal antibody and antiphospholiplasma membrane receptors (2). PLC comprises a family of pase C-7 antibody were from UBI (Lake Placid, NY). [32P]Orthoseveral gene products (1)that includes the y-isozyme of PLC phosphate (carrier-free) and 1261-labeledprotein A were from Amersham Corp. SK&F 96365 was a generous gift from Dr. Janet Merritt (Smith Kline Beecham, United Kingdom). Maitotoxin was a generous the payment of page charges. This article must therefore be hereby gift from Prof. T. Yasumoto (Tohoku University, Japan). CAI marked ‘Ladvertisement”in accordance with 18 U.S.C. Section 1734 (L651582), originally developed by Merck Institute for Therapeutic Research (Rahway, NJ), is under development as anantiproliferative solely to indicate this fact. agent by the National Cancer Institute (29). BABA (2-hydroxy-5§ Present address: Eisai Research Inst., Andover, MA 01810. (2,5-dihydroxybenzyl)aminobenzoic acid) was purchased from $4 To whom correspondence should be addressed Bldg. 36, Rm. 3A-15, LCB, NIMH, NIH, Bethesda, MD 20892. Tel.: 301-496-8755; GIBCO/BRL. Cells-CHO cells expressing m5 muscarinic receptors (CHO-m5) Fax: 301-402-0008. The abbreviations used are: PLC, phospholipase C; MTX, mai- or m2 muscarinic receptors (CHO-m2) were generous gifts from Drs. totoxin; ROCC, receptor-operated calcium channel(s); EGF, epider- T. Bonner and M. Brann (9). Cells were maintained as described (9). Tyrosine Phosphorylation in CHO Cells-CHO cells were grownto mal growth factor; PDGF, platelet-derived growth factor; CAI, 5confluency in regular medium in 10-cm dishes. Cells were serum amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-l,2,3-triazole-4-carboxamide; SK&F 96365, l-[3-(4-methoxyphenyl)propoxyl]-1-(4- starved for 14 h. Medium was aspirated and replaced with phosphatemethoxypheny1)ethyl-1H-imidazoleHC1; IPI, inositol 1,4,5-trisphos- free Dulbecco’s modified Eagle’s medium containing 1%dialyzed fetal phate; BAPTA-AM, bis-(0-aminophenoxy)-ethane-N,N,N’,N”tet- bovine serum and 50 pCi of [32P]orthophosphate (carrierfree). Cells raacetic acid tetra(acetoxymethy1)ester; BABA, 2-hydroxy-5-(2,5- were incubated for 4 h at 37 “C. Medium was aspirated and replaced dihydroxybenzy1)aminobenzoic acid; EMEM, Eagle’s No. 2 medium; with incubation buffer (20 mM HEPES, 4.7 mM KCl, 0.5 mM EDTA, CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electropho- 118 mM NaCl, 5 mM NaHC03, 1.2 mM MgSO, 1.2 mM KHzPO4,.pH 7.4) containing 3 mM CaCl, and other agents as indicated. Incubatlons resis.

* The costs of publication of this article were defrayed in part by

7768

C-7

Receptor-activated Muscarinic Phospholipase were carried out a t 37 “C for 15 min. Incubation buffer was aspirated, andthe cells were washed three times with ice-cold phosphatebuffered salinecontaining 1 mM NaV04. Cells were lysed by the addition of 0.5 ml of lysing buffer (50 mM HEPES, 50 mM NaCI, 50 mM NaF,5 mM EDTA, 1 mM NaV04,1% NonidetP-40, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml aprotinin, 10 pg/ml leupeptin) and slow rocking for 30 min a t 4 “C. Homogenates were transferred to microcentrifuge tubes andcentrifuged for 2 min at 12,000 X g. The supernatantswere incubated with 5-10 pg of anti-phosphotyrosine antibody for 2 h a t 4 “C. Preswollen protein A-Sepharose CL4B (30 ml) was added followed by a 45-min incubation. Tubes were briefly centrifuged, supernatants discarded, andthe pellets were washed four times with phosphate-buffered saline. Loading buffer was added to thewashed pellets, and, afterboiling for 3 min, samples were subject to 10% SDS-PAGE. Gels were dried and exposed to xray film for 24 h. Immunoblotting of Phosphorylated Phospholipase C-y-Immunoprecipitation of tyrosine-phosphorylated proteins was performed as indicated above but without the addition of [32P]orthophosphate. After completion of SDS-PAGE, proteins were transferred to nitrocellulose membranes (10). Membranes were incubated with Blotto (Advanced Biotechnologies Inc., Columbia, MD) for 2 h to prevent nonspecific binding of antibody. Membranes were then incubated with monoclonal antibody anti-phospholipase C”y, followed by incubation with ’Z51-labeledprotein A. Membranes were washed, dried, and exposed to x-ray film a t -70 “C. Intracellular Calcium Determinations in Single Cells-The procedure was described elsewhere (9). Briefly, cells grown on glass coverslips were loaded with 5 p~ fura-2 acetoxymethyl ester (Molecular Probes, Portland, OR) for 30 min at 37 “C. After washing once, the cells were incubated in Eagle’s No. 2 medium (EMEM) containing fatty acid-free bovine serum albumin (1 mg/ml) and 20 mM HEPES buffer (pH 7.4) for no longer than 45 min. Fura-2 fluorescence was measured photometrically at anemission wavelength of 510 nm in a single cell mountedona Nikon Diaphot microscope illuminated alternately with 340 and 380 nm light in an SLMAminco DMX-1000 spectrofluorometer (SLM Aminco, Urbana, IL). Ratios of fluorescence (340/380 nm) were converted to calcium concentrations, and calibration was performed as described (11). 45Ca2’ Uptake in CHO Cells-CHO cells were plated in 24-well plates at a density of 1.5-2 X lo5 cells/well. The next day, cells were washed once with EMEM containing 20 mM HEPES, 1.8mM CaCI,, and 0.1 mM MgCL (incubation medium).Agents were added in a final volume of 250 pl of EMEM containing “CaC12 (1 pCi/ml) and 0.2% bovine serum albumin. Incubations were carried out for 4 min and were stopped by the addition of 1 ml of ice-cold incubation medium containing 1.8 mM LaCI3. Cells were lysed by adding 250 pl of a solution containing 1 M KOH, 18 mM Na2B407,3.8 mM EDTA, and 7.6 mM NaOH. Cells were scraped from the wells, and thesuspension was neutralized with 250 pl of7.5% HCI. Radioactivity was determined by liquid scintillation spectrometry. Phosphoinositide Breakdown-CHO-m5 cells were plated in 24well plates at a density of 175,000 cells/well and labeled for 18 h with 1.5 mCi of [3H]inositol/well. Cells were then washed once with assay buffer (EMEM containing 20 mM HEPES (pH7.4) and 20 mM LiCI,), and agents were added at the times indicated in the legend to Fig. 6. Inositol phosphate formation was measured as previously described (12, 13), except that the incubation time was 5 min to maximize the detection of IPB.

7769 CaCh

A

1m M CaCh + CAI

I

92.54

B Carbachol Concentration

MW Markers

(KD)

A

92.5

-

69

-

1mM 1 q M 10pM 1pM

n nn n

46 -

30

-

21

-

Cell m2 m5 m2m5m2 m5 mz m5m2m5 Line RESULTS FIG. 1. A, dose-dependent stimulation of total protein tyrosine phosphorylation in CHO-m5 cells by carbachol. CHO-m5 cells were Stimulation of CHO cells stably expressing the m5 musca- labeled with [32P]orthophosphate and incubated with the indicated rinic receptor with the muscarinic agonist carbachol resulted concentrations of carbachol (CaCh) or with 1 mM carbachol in the ina concentration-dependentincreaseinproteintyrosine presence of the indicated concentrations of CAI (right three lanes). phosphorylation (Fig. M ) . The m5 receptor-stimulated tyro- Cells were lysed and immunoprecipitated with an antiphosphotyro1 nM sine antibody as described under “Materials and Methods.” Immusinephosphorylation was detected with aslittleas carbachol and was fully saturated a t 100 PM (Fig. lA). The noprecipitated proteins were subject to 10% SDS-PAGE and autoradiography. C, control. B, lack of effects of carbachol on CHO-m2 increase was observed in CHO cells expressing the m5 mus- cells. CHO-m2 and CHO-m5 cells were labeled as indicated in A and carinic receptor but not the m2 receptor, demonstrating recep- incubated with the indicated concentrations of carbachol.

tor subtype specificity (Fig. 1B).The concentration-dependent stimulation of tyrosine phosphorylation correlated with concentration-dependent stimulation of m5 receptor-stimulated calcium influx (9). Furthermore, CAI, an inhibitor of ROCC (13), blocked carbachol-stimulatedtyrosinephosphorylation in a concentration-dependent manner (Fig. lA).

The marine toxin,MTX, which activates calcium-dependent events in most cells (14), inducedtyrosine phosphorylation in CHO-m5 cells that was inhibited in the presence of the ROCC blockers, CAI and SK&F 96365 (15), and in the absence of extracellular calcium (Fig. 2).

Muscarinic Receptor-activatedPhospholipase C--y

7770 Normal Free Ca Ca

A

Carbachol C

LL

200

-

92.5

-

n n

MW (KD)

Cell Line

92.5

m2

"'75

"'72

"'75

B

+Ca2+

-Ca2+

nn

69 46

200-

-carb. c

MTX carb.

c

MTX

MW (KD)

30

FIG. 2. Maitotoxin-inducedtotalproteintyrosine phosphorylationin CHO-m5 cells. CHO-m5 cells werelabeled as indicated in Fig. 1 and incubated with normal buffer, 1 ng/ml MTX in normal buffer, MTX + 10 p~ CAI, MTX + 100 p M SK&F 96365, calcium-free buffer,and MTX in calcium-free buffer.Cell lysates were processed as described for Fig. 1.

92.5

-

FIG. 3. A, immunoblot of tyrosine-phosphorylated PLC-y in CHO-m2 and CHO-m5cells. Cells were incubated with normal buffer (C) orcarbachol (1 mM), lysed, andimmunoprecipitatedwith an antiphosphotyrosine antibody as describedunder"Materialsand Methods." Immunoprecipitates were subject to 10% SDS-PAGE, transferred to nitrocellulose sheets, and immunoblotted with an antiphospholipase C-y antibody as describedunder"Materialsand Methods."B, carbachol- andMTX-inducedtyrosine phosphorylation of PLC--y in CHO-m5cells is dependent on the presence of extracellular calcium. Cells were incubated with normal buffer(leftpanel) or calcium-free buffer (right panel) with agents as indicated (1 mM carbachol (Curb.),1 ng/ml MTX). C, control. Cells were processed as in A.

Activation of m5 receptors resulted in tyrosine phosphorylation of PLC--y (Fig. 3A). Muscarinic m2 receptor activation had noeffect on tyrosine phosphorylation of PLC-7, suggesting receptor subtype specificity. The carbachol-induced tyrosine phosphorylation of PLC--y was stimulated only in the presence of extracellular calcium, suggesting a dependence on brane calcium chelator BAPTA-AM, or coincubation with calcium influx (Fig. 323). Furthermore, MTX caused calcium- CAI resulted in a significant inhibition of carbachol-induced dependent tyrosine phosphorylationof PLC--y (Fig. 3B). Un- IP3 generation (Fig. 6). These treatments also inhibited carbachol-induced protein tyrosine phosphorylation and phosder calcium-free conditions,in some experiments,control PLC--y immunoreactivity was slightly elevated. However, no phorylation of PLC--y (Figs. 1 and 2 and data not shown). BABA (10 mM), an inhibitor of tyrosine kinases (16), also enhancements were observed without any exception,with significantly reduced carbachol-induced IP3 generation (Fig. either carbachol or MTXin the absence of calcium. Carbachol- and MTX-induced effects on calcium influx 6). A t this concentration, BABA completely inhibited carwere studied in single cells loaded with the calcium-sensitive bachol-induced protein tyrosine phosphorylation (data not fluorescent dye fura-2 (Fig. 4). Carbachol stimulated both a shown). peak and a plateau phase of extracellular calcium (Fig. 4.4). DISCUSSION The plateau phase results from the activation of muscarinic receptor-operated calcium channels (9). The plateau phase The results of thesestudies suggest a link between the was blocked with the addition of 10 PM CAI, which blocked activation of ROCC and the stimulation of tyrosine kinase most of the carbachol-stimulated tyrosine protein phosphoryl-activity in these cells. The dependence on extracellular calation (Fig. 4.4).MTX stimulated an increase in intracellular cium for activationof tyrosine phosphorylation is reminiscent calcium that was completely eliminatedin the absence of of that reported by Huckle et al. (17) in WB liver epithelial extracellular calicum and almost completely inhibited by CAI cells with angiotensin 11. In CHO-m5 cells, such activation (Fig. 4B). requires calcium entry through ROCC, since carbachol-inThe uptake of 45Ca2+was used to directly determine the duced activation of tyrosine phosphorylation is blocked by effect of the inhibitors CAI and SK&F 96365 on carbachol- the ROCC blockers, SK&F 96365 and CAI. Moreover, the and MTX-stimulated calcium influx (Fig. 5). At concentra- stimulation of tyrosine phosphorylation observed with MTX tions previously shown to inhibitROCC, both CAI and SK&F also suggests that activation of calcium flux through ROCC 96365 completely blocked carbachol-stimulated 45Ca2' (Fig. results in enhanced tyrosine kinase activity. MTX has been 5A) and partially inhibited MTX-stimulated 45Ca2' (Fig. 5B). proposed to activate ROCC directly (18). SK&F 96365 and Carbachol elicited a 5-fold increase in IP3 generation in CHO CAI block both MTX-elicited calcium influx and MTX-eliccells expressing the m5 muscarinic receptor (Fig. 6). Removal ited stimulation of tyrosine phosphorylation in CHO cells. of extracellular calcium, pretreatment of cells with the mem- Alternatively, apathway involving inhibition of tyrosine

C--y

Receptor-activated Muscarinic Phospholipase

7771

1000 A T

I

t

-9

6

cc .

Id0

460 360 200 TIME (seconds)

500

BASAL

CC

CC

+

CAI

cc +

SKF%365

10000

h

I

-

aJ

MTx

’b

I

Y

Id0

460 360 260 TIME (seconds)

500

FIG.4. Elevation of [Caz+]iin CHO-m5 cells after the addition ofcarbachol (CC)(100NM) (panelA ) or maitotoxin (MTx)

+

t

(1 ng/ml) (panel B ) . CHO-m5 were loaded with fura-2, and single cells were monitored for changes in fluorescence as described under “Materials and Methods.” CAI (10 p ~ was ) added 5 min before the addition of carbachol or MTX. BASAL

phosphatases in response to elevated calcium could explain the results observed. Such a pathway seems unlikely since no effects of divalent cations or chelatorshave been observed on protein tyrosine phosphatases(19). We have identified PLC--, as one of the substrates for carbachol- and MTX-induced tyrosine phosphorylation (Fig. 3). Tyrosine phosphorylation of PLC--, is a necessary step in EGF- and PDGF-induced phosphoinositide breakdown (7). In contrast to PLC-p, PLC--, is not sensitive to activation by Gproteins i n vitro (5).Thus,it is likely that muscarinic induced phosphorylation of PLC--, is an indirect effect linked totheentry of calcium (9)and subsequent activation of calcium-sensitive tyrosine kinase(s). The ml, m3, and m5 subtypes of muscarinic receptors belong to thefamily of G protein-coupled receptors, and they can couple in vitro to PLC-p through G proteins of the Gq/ 11 subfamily (20). Carbachol-induced phosphoinositide breakdown was partially reduced in the absence of extracellular calcium (Fig. 6). Since carbachol-induced tyrosine phosphorylation of PLC--, is eliminated in the absence of extracellular calcium (Fig. 3), itseems likely that thecalciumsensitive component of the carbachol-stimulated IP3 generation is mediated through PLC--/. Moreover, CAI, which blocks carbachol-induced tyrosine phosphorylation,also partially inhibits carbachol-induced IP3 generation (Fig. 6). In the absence of calcium, CAI has no effect on carbachol-induced stimulation of phosphoinositide breakdown (data notshown). This is consistent with two cascades of events for muscarinic receptors as follows: (i) muscarinic receptor stimulation + opening of ROCC + entry of calcium --., stimulation of calcium-sensitive tyrosine kinase(s) + phosphorylation of PLC--, + stimulation of phosphoinositide breakdown, and (ii) muscarinic receptor stimulation + activation of G q / l l + stimulation of PLC-p + stimulation of phosphoinositide

MTx

Mix

Mix

Ci I

SKF96365

t

FIG.5. Stimulation of‘%a2+ uptake in CHO-m6 cells by carbachol (CC)(panel A ) and maitotoxin (MTx) (panel B ) . CHO-m5 cells were incubated in EMEM containing ‘‘Caz’ in the presence of the agents as indicated for 5 min. Incubations were stopped as described under “Materials and Methods.” Results correspond to means (+ S.E.) of three independent experiments performed in triplicate. breakdown. In the absence of extracellular calcium or in the presence of ROCC blockers like CAI and SK&F 96365, the tyrosine kinase cascade would not operate, and the stimulation of phosphoinositide breakdown observed under those conditions would be exclusively the result of the Gq/ll + PLC-p pathway. Recently, it has been reported that in some systems, activation of G protein-coupled receptors results in tyrosine phosphorylation of endogenous protein substrates (21-23). In A39 cells, protein tyrosine kinase substrates for aGproteincoupled receptor stimulation do not include PLC--, (23). We do not have a clear explanation for this disparity, but it is possible that A39 cells lack or express low levels of PLC--, and/or ROCC. The present results indicate that stimulation of m5 receptors results in tyrosine phosphorylation of PLC-7, which in turn may lead to a sustained elevation of the second messengers derived from phosphoinositide breakdown, namely IP3 and diacylglycerol. PLC activation induced by EGFand PDGF has been proposed to be involved in cell proliferation (6). On the other hand, agonists for G protein-coupled receptors, like bombesin, vasopressin, endothelin, and prostaglandin, have been recognized also to induce cell proliferation (23). Transfectionof serotonergic (24), ml, m3, and m5 muscarinic (25), and &-adrenergicreceptors (26) in cultured cells

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Receptor-activated Muscarinic Phospholipase

C-7 REFERENCES

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