Phosphatidylinositol-3 kinase is distinctively required for �-, but not ?-opioid receptor-induced activation of c-Jun N-terminal kinase

Phosphatidylinositol-3 kinase is distinctively required for l-,but not j-opioid receptor-induced activation of c-Jun N-terminal

kinase

Angel Y. F. Kam, Anthony S. L. Chan and Yung H. Wong

Department of Biochemistry, the Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong

University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Abstract

Opioid receptors are the therapeutic targets of narcotic anal-

gesics. All three types of opioid receptors (l, d and j) are

prototypical Gi-coupled receptors with common signaling

characteristics in their regulation of intracellular events. Nev-

ertheless, numerous signaling processes are differentially

regulated by the three receptors. We have recently demon-

strated that stimulation of d-opioid receptor can up-regulate

the activity of the c-Jun N-terminal kinase (JNK) in a pertussis

toxin-sensitive manner (Kam et al. 2003; J. Neurochem. 84,

503–513). The present study revealed that the l-opioid

receptor could stimulate JNK in both SH-SY5Y cells and

transfected COS-7 cells. The mechanism by which the

l-opioid receptor stimulated JNK was delineated with the use

of specific inhibitors and dominant-negative mutants of signa-

ling intermediates. Activation of JNK by the l-opioid receptor

was mediated through Gbc, Src kinase, son-of-sevenless

(Sos), Rac and Cdc42. Interestingly, unlike the d-opioid recep-

tors, the l-opioid receptor required phosphatidylinositol-3

kinase (PI3K) to activate JNK. The l-opioid receptor-induced

JNK activation was effectively inhibited by wortmannin or the

coexpression of a dominant negative mutant of PI3Kc. Like

the d-opioid receptor, activation of JNK by the j-opioid

receptor occurred in a PI3K-independent manner. These

studies revealed that the l-opioid receptor utilize a distinct

mechanism to regulate JNK.

Keywords: JNK, l-opioid receptor, PI3K, small GTPases,

Son-of-sevenless (Sos), Src.

J. Neurochem. (2004) 89, 391–402.

Opioids are multifunctional peptides that regulate numerous

physiological effects including inhibition of neuronal firing

and neurotransmitter release, induction of tolerance and

dependence, reduction of gastrointestinal motility, as well as

modulation of the immune and endocrine responses (Mattes

et al. 1996). The actions of these peptides are manifested by

specific opioid receptors, which are mainly distributed

throughout the nervous system, but are also expressed in

immune cells such as monocytes and lymphocytes. The

opioid receptors are classified into three major types, termed

l, d and j according to their ligand selectivities and

pharmacological profiles. All three opioid receptors inhibit

adenylyl cyclase, reduce Ca2+ currents, stimulate inward

rectifying K+ currents and activate mitogen-activated protein

kinases (MAPK) via pertussis toxin (PTX)-sensitive Gi/Go

proteins (reviewed in Law et al. 2000). There is increasing

evidence, however, that molecular processes mediated by l-,d- and j-opioid receptors are not identical. Activation of thed-opioid receptor triggers translocation of G protein-coupled

receptor kinase (GRK)2 and 3 to the plasma membrane,

which is followed by cointernalization of the receptor and

GRKs. In contrast, stimulation of l-opioid receptor does notresult in the accumulation of GRKs at the membrane nor its

cointernalization with GRK2 or 3 (Schulz et al. 2002).

Received August 2, 2003; revised manuscript received December 1,

2003; accepted December 12, 2003.

Address correspondence and reprint requests to Yung H. Wong,

Department of Biochemistry, Hong Kong University of Science and

Technology, Clear Water Bay, Kowloon, Hong Kong.

E-mail: [email protected]

Abbreviations used: DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]enkepha-

lin; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal

growth factor; ERK, extracellular signal-regulated protein kinases; G

protein, guanine nucleotide-binding regulatory protein; GEF, guanine

nucleotide exchange factor; HA, hemagglutinin; JNK, c-Jun N-terminal

kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidy-

linositol-3 kinase; PTX, pertussis toxin; SDS–PAGE, sodium dodecyl

sulfate-polyacrylamide gel electrophoresis; Sos, son-of-sevenless;

U-50,488, (±)-trans-U-50,488 methanesulfonate.

Journal of Neurochemistry, 2004, 89, 391–402 doi:10.1111/j.1471-4159.2004.02338.x

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 391–402 391

Likewise, l- and j-opioid receptors exhibit different abilitiesto utilize Gaz, a PTX-insensitive member of the Gi family,

for the regulation of extracellular signal-regulated kinase

(ERK) in PTX-treated COS-7 cells (Belcheva et al. 2000).

These differences in the molecular pharmacology of opioid

receptors may in part account for their functional specificity

in various physiological responses, such as the addiction

potential of different opioid ligands.

We have recently observed that activated d-opioid recep-

tors can stimulate c-Jun N-terminal kinase (JNK) activity

(Kam et al. 2003). Along with ERK and p38, JNK

constitutes the family of MAPKs that are widely involved

in diverse biological functions ranging from proliferation,

survival, differentiation, to apoptosis. JNK is broadly

expressed in the nervous system and it appears to exhibit

multiple functions. In rat pheochromocytoma PC-12 cells,

withdrawal of nerve growth factor (NGF) activates JNK and

causes apoptosis (Xia et al. 1995). However, opposite

perspectives suggest that JNK also contributes to neuronal

protection by inhibition of caspases (Kuan et al. 1999).

Moreover, apoptosis was increased in the hindbrain and

forebrain of jnk1–/–jnk2–/– mice at E10.5, indicative of the

requirement of JNK in neuronal survival during development

(Sabapathy et al. 1999). Indeed, stimulation of opioid

receptors has been shown to activate ERK1/2 (Belcheva

et al. 1998), p38 MAPK (Singhal et al. 2002) and JNK

(Kam et al. 2003) in various cell types. Hayashi et al. (2002)

have demonstrated that a low concentration of d-opioidpeptide DADLE requires ERK activation to promote cell

survival in serum-deprived PC-12 cells. Moreover, inhibition

of p38 MAPK phosphorylation by using specific inhibitors

can protect morphine-treated macrophages from apoptosis

(Singhal et al. 2002), suggesting the involvement of p38

MAPK in morphine-induced macrophage apoptosis. How-

ever, the role of opioid receptor-regulated JNK activity in the

nervous system still remains unclear and so mapping the

signaling pathway from opioid receptor to JNK is important.

In a previous study, we have shown that the d-opioidreceptor-mediated JNK pathway involves Gbc, Src kinase

and the small GTPases Rac and Cdc42, but does not require

phosphatidylinositol 3-kinase (PI3K) or transactivation of

EGF receptor (Kam et al. 2003). Here we demonstrate that

JNK activity is stimulated by the l-opioid receptor in humanneuroblastoma SH-SY5Y cells. By heterologous expression

assays in COS-7 cells, we have further characterized the

l-opioid receptor-regulated JNK activity and the pathway

has been shown to depend on Gbc, Src kinase, Rac and

Cdc42, but not EGF receptor. These intermediate steps in the

pathway are identical with the d-opioid receptor-stimulated

signaling cascade. Moreover, a guanine nucleotide exchange

factor (GEF) Son-of-sevenless (Sos) is identified for

transmitting signals from the l-opioid receptor to JNK.

Surprisingly, PI3K is uniquely necessary for l-opioidreceptor-stimulated JNK as shown by using a PI3K inhibitor,

wortmannin, and a dominant negative mutant of PI3Kc. Thisis a distinct difference between l- and d-opioid receptor

signaling. Like the d-opioid receptor, j-opioid receptor doesnot require PI3K to mediate JNK activity.

Materials and methods

Materials

The cDNAs of the rat l-opioid receptor and the mouse j-opioidreceptor (both in the pCMV6 vector) were kindly provided by Dr L.

Yu (Indiana University School of Medicine) and Dr G. Bell

(University of Chicago, Chicago, IL, USA), respectively. JNK-HA

cDNAwas supplied by Dr T. A. Voyno-Yasenetskaya (University of

Illinois, Chicago, IL, USA). The plasmids containing wild-type

(WT) and dominant-negative mutant of PI3Kc (PI3KcK832R), SrcWT, dominant negative Src (SrcK295R/527F) as well as dominant

negative Ras (RasS17N), Rac (RacT17N), Cdc42 (Cdc42T17 N)

and RhoA (RhoAT19N) were obtained as previously described

(Kam et al. 2003). The cDNAs encoding p21-binding domain

(PBD) from human PAK1 cloned into the bacterial expression

vector pGEX-2TK and Sos-Pro were kind donations from Dr Udo

Schmitz (Medizinische Universitats-Poliklinik, Germany) and Dr R.

J. Lefkowitz (Duke University Medical Center, Durham, NC, USA),

respectively. The cDNA of kinase-deficient mutant of Akt was a

generous gift from Dr Z.G. Wu (Hong Kong University of Science

and Technology, Hong Kong, China). [c-32P]ATP was purchased

from DuPont NEN (Boston, MA, USA). Anti-phospho-JNK, anti-

JNK, anti-phospho-Akt, anti-Akt antibodies and antiphospho-Src-

Y416 antibodies were obtained from Cell Signaling Technology,

Inc. (Beverly, MA, USA). Antiserum against Gat was from

Transduction Laboratories. Mouse monoclonal antibody against

human Rac was purchased from Upstate (Lake Placid, NY, USA).

Rabbit polyclonal antibody against human Cdc42 was from Santa

Cruz Biotechnology (Santa Cruz, CA, USA). PTX and 12CA5 (anti-

HA) antibody were purchased from List Biological Laboratories

(Campbell, CA, USA) and Roche Molecular Biochemicals (Indi-

anapolis, IN, USA), respectively. Wortmannin, tyrphostin AG1478

and pyrazolopyrimidine PP2 were purchased from Calbiochem-

Novabiochem Co. (La Jolla, CA, USA). Cell culture

reagents, including LipofectAMINE PLUS were obtained from

Invitrogen, Carlsbad (CA, USA). [D-Ala2,N-Me-Phe4,Gly5-ol]en-

kephalin (DAMGO), (±)-trans-U-50,488 methanesulfonate

(U-50,488H), [D-Phe-Cys-Tyr-D-Trp-Orn-Pen-Thr-NH2] (CTOP) and

all other chemicals were purchased from Sigma (St. Louis, MO, USA).

Cell culture and transfection

COS-7 cells were grown in Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS),

50 units/mL penicillin and 50 lg/mL streptomycin in a humidified

atmosphere containing 5% CO2 at 37�C. One day before the

transfection, cells were seeded onto six-well plates at a density of

3 · 105 cells/well. Transfection was performed by means of

LipofectAMINE PLUS reagents according to the supplier’s instruc-

tions, and the transfected cells were kept in the growth medium for

36 h. Under the same physical condition, human neuroblastoma SH-

SY5Y cells were maintained in a mixture (1 : 1) of Eagle’s

minimum essential medium (MEM) and Ham’s F12 medium

392 A. Y. F. Kam et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 89, 391–402

supplemented with 10% (v/v) FBS, 50 units/mL penicillin and

50 lg/mL streptomycin. Human monocytic THP-1 cells were

cultured in RPMI 1640 medium supplemented with 0.05 mM

2-mercaptoethanol, 10% (v/v) FBS, 50 units/mL penicillin and

50 lg/mL streptomycin at 37�C in 5% CO2.

In vitro JNK assay

Transfected COS-7 cells were serum starved for 18 h in the presence

or absence of PTX (100 ng/mL) before drug treatment. Where

appropriate, additional treatments of wortmannin (100 nM, 15 min),

AG1478 (500 nM, 30 min) and PP2 (10 lM, 30 min) were applied

to the starved cells. Cells were then treated with the assay medium

(DMEM with 20 mM HEPES) in the absence or presence of 100 nM

DAMGO or U-50,488H for 15 min at 37�C and lysed in 500 lL of

lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA,

40 mM NaP2O7, 1% Triton X-100, 1 mM dithiothreitol, 200 lMNa3VO4, 100 lM phenylmethylsulfonyl fluoride, 2 lg/mL leupep-

tin, 4 lg/mL aprotinin, and 0.7 lg/mL pepstatin) and shaken on ice

for 30 min. The lysates were centrifuged at 14,000 · g for 5 min at

4�C. Fifty microliters of each sample was used for the detection of

JNK-HA expression or 100 lL was used for phosphorylated and

total Akt if necessary, 400 lL was incubated for 1 h at 4�C with

anti-HA antibody (2 lg/sample), followed by incubation with 30 lLof protein A-agarose (50% slurry) at 4�C for 1 h. The resulting

immunoprecipitates were washed twice with lysis buffer and twice

with kinase assay buffer [40 mM HEPES, pH 8.0, 5 mM

Mg(C2H3O2)2, 1 mM EGTA, 1 mM dithiothreitol, 200 lM Na3VO4].

Washed immunoprecipitates were resuspended in 40 lL of kinase

assay buffer containing 5 lg of GST-c-Jun per reaction, and the

kinase reactions were initiated by the addition of 10 lL of ATP

buffer (50 lM ATP with 2 lCi of [c-32P]ATP per sample). After

30 min incubation at 30�C with occasional shakings, the reactions

were terminated by adding 10 lL of 6X sample buffer and boiled

for 5 min, and the samples were subjected to 12% sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The

radioactivity incorporated into GST-c-Jun was detected by autora-

diogram, and the signal intensity was quantified by PhosphorImager

(Molecular Dynamics 445 SI).

Western blot

SH-SY5Y cells were seeded onto six-well plates at a density of

2.5 · 105 cells/well and allowed to reach confluency. The cells were

then serum starved for 4 h in the absence or presence of PTX

(100 ng/mL), followed by pretreatments with inhibitors if necessary.

The cells were stimulated with DAMGO at 37�C for the indicated

time and then lysed in 200 lL of lysis buffer. THP-1 cells were

plated at 1 · 106 cells/mL in serum-free media and maintained for

18–24 h with or without PTX (100 ng/mL). If necessary, THP-1

cells were preincubated with different inhibitors before addition of

U-50,488H. After stimulation with the agonist, cells were lysed as

with SH-SY5Y cells. Supernatants of both cell lysates were

collected by centrifugation at 14 000 · g for 5 min, mixed with

40 lL of 6X sample buffer and boiled for 5 min. 150 lg proteins ofeach sample were resolved by 12% SDS–PAGE, and then

transferred to nitrocellulose membranes. Phosphorylated or total

kinases (JNK, Akt and Src) were detected by specific antibodies as

mentioned under Materials, followed with horseradish peroxidase-

conjugated secondary antibody. Immunoblots were developed in the

presence of enhanced chemiluminescence reagents, and the images

detected in X-ray films were quantified by densitometric scanning

using the Eagle Eye II still video system (Stratagene, La Jolla, CA,

USA).

GTPase pull-down assay

GTPase pull-down assay was performed as described previously

(Kam et al. 2003). The cDNAs of PAK-PBD in pGEX-2TK was

expressed in Escherichia coli as a fusion protein with glutathione-S-

transferase. The fusion proteins were purified from glutathione-

Sepharose beads. Transfected COS-7 cells expressing l-opioidreceptor were serum starved and then lysed with 500 lL of Mg2+-

containing lysis buffer (MLB; 25 mM HEPES, pH 7.5, 150 mM

NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 10% glycerol,

25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovana-

date, 10 lg/mL leupeptin, 10 lg/mL aprotinin). Cell lysates were

centrifuged at 4�C for 10 min at 14 000 · g. Fifty microliters of the

supernatant was used for detecting total Rac and Cdc42. 450 lL cell

lysates were incubated with 10 lg GST-PAK-PBD and 15 lL of

50% slurry of glutathione-Sepharose beads at 4�C for 60 min with

constant rotations. Bound proteins were collected by centrifugation

and pellets were washed three times in MLB and finally suspended

in 2 · Laemmli sample buffer (40 lL). Proteins were resolved by

12% SDS–PAGE and the bound Rac or Cdc42 were analyzed by

immunoblotting using antiserum against Rac or Cdc42, respectively.

Results

Stimulation of l-opioid receptor induce a time- and

concentration-dependent JNK activation

We have recently demonstrated that d-opioid receptor can

induce JNK stimulation in both COS-7 and NG108-15 cells

(Kam et al. 2003). Here, we asked if the l-opioid receptor

can similarly regulate JNK activity. Heterologous expression

of the l-opioid receptor and HA-tagged JNK (HA-JNK) in

COS-7 cells were performed as described previously (Kam

et al. 2003). The JNK activity was examined by immuno-

complex kinase assay. Treatment of COS-7 cells with the

l-selective agonist, DAMGO, resulted in a time-dependent

JNK activation, as reflected by increases in the phosphory-

lation of GST-c-Jun proteins. The DAMGO-induced JNK

activation peaked at 15 min after drug addition, thereafter it

gradually recovered to near basal level (Fig. 1a, left panel).

Anti-HAwestern blotting of total cell lysates verified that the

total amount of HA-JNK was unaffected by the agonist

treatment, suggesting that the elevated JNK activities did not

result from increased JNK expression but from phosphory-

lation. In order to examine the mechanism of JNK activation

by the l-opioid receptor under more native conditions, we

also examined human neuroblastoma SH-SY5Y cells en-

dogenously expressing the receptor (Kazmi and Mishra

1987). Activation of JNK was determined by western

blotting using an antiserum against the p46/p54 isoforms of

JNK dually phosphorylated at threonine 183 and tyrosine

PI3K-dependent JNK activation by l-opioid receptor 393


185 residues. Figure 1(a) (right panel) showed time course

studies for JNK activation in DAMGO-treated SH-SY5Y

cells. Interestingly, in comparison with studies in COS-7

cells, JNK activity was rapidly stimulated upon DAMGO

application to SH-SY5Y cells and reached the maximum at

5 min. The elevated JNK activity was sustained for up to

15 min. Therefore, the time course of agonist-induced JNK

activity was shifted to earlier time points in SH-SY5Y cells

as compared to COS-7 cells. Furthermore, administration of

DAMGO in SH-SY5Y cells preferentially activated p46

JNK, as indicated in the immunoblots of phosphorylated

JNK. Transfected COS-7 and SH-SY5Y cells were then

challenged with different agonist concentrations (0.1 nM to

1 lM) for 15 min and 5 min, respectively. In both cells,

maximal JNK activity-induced by DAMGO was obtained at

around 100 nM, but the half-maximum response was about

10 nM in COS-7 and 1 nM in SH-SY5Y cells, respectively

(Fig. 1b).

l-Opioid receptors activate JNK in a PTX-sensitive

and Gbc-dependent manner

As prototypical Gi/Go-coupled receptors, opioid receptors

regulate an array of effectors via pertussis toxin (PTX)-

sensitive Gi/Go proteins (reviewed in Law et al. 2000).

Nevertheless, couplings between opioid receptors and a

variety of PTX-insensitive G proteins are also plausible.

Chan and Wong (2000) have demonstrated that the activated

ORL1 receptor can functionally interact with PTX-insensitive

Gz, G12, G14 and G16 to induce JNK activation in COS-7

cells. Therefore, it is pertinent to investigate the participation

of Gi/Go proteins in the opioid receptor-regulated JNK

activation. Transfected COS-7 and SH-SY5Y cells were

pretreated with 100 ng/mL PTX for 18 h before exposure to

DAMGO. The toxin ADP-ribosylates Gi/Go proteins, as a

result their couplings with receptors can be abrogated. As

illustrated in Fig. 2(a), JNK was significantly activated upon

stimulation with DAMGO in both cells. However, pretreat-

ment of cells with PTX completely abolished the JNK

activation, demonstrating that l-opioid receptor preferen-

tially interacts with Gi/Go proteins to regulate JNK activity in

COS-7 and SH-SY5Y cells. Because SH-SY5Y cells not

only express l-opioid receptor, but also d- and j-opioidreceptors (Cheng et al. 1995), we next performed experi-

ments to confirm whether the JNK activation is mediated via

the activation of l-opioid receptor in SH-SY5Y cells by

using a selective antagonist CTOP. The JNK stimulation by

DAMGO was suppressed in the presence of CTOP (Fig. 2a,

right panel), indicating the requirement of l-opioid receptor

in SH-SY5Y cells.

Both Gai-GTP and Gbc dimers are responsible for

transducing signals from receptors to downstream effectors.

However, in the case of regulation of JNK, Gbc are seeminglymore effective. Coso et al. (1996) have reported that only

Gbc subunits, but not the activated mutants of Gai signifi-cantly increase JNK activity. Moreover, JNK activation by

Gi-coupled m2 muscarinic acetylcholine receptor is inhibited

by overexpressing the b-adrenergic receptor kinase (b-ARK)carboxyl-terminal domain, which acts as a Gbc scavenger

protein. To confirm the role of Gbc in signaling from the

ligand-activated l-opioid receptors to JNK, the a subunit of

transducin (Gat) was cotransfected into COS-7 cells. Gat canalso sequester free Gbc subunits that are concomitantly

released upon stimulation of receptors, thereby suppressing

Gbc-dependent biological responses. In cells coexpressing

Gat and l-opioid receptor, the JNK activation in response to

DAMGO was markedly reduced (Fig. 2b). Therefore, Gbc

0

1

2

3

0 10 20 30 40 50 60

*

(a)

Time (min.)0 5 15 30 45 60

JNK

acti

vity

(fol

dst

imul

atio

n)

JNK-HA

GSTc-Jun

P0 1 5 15 30 45

0

1

2

3

0 10 20 30 40

**

TotalJNK

p54P

p46P

B -10 -9 -8 -7 -6Log[DAMGO] (M)

0

1

2

3

-10 -9 -8 -7 -6

* *

(b)

JNK

acti

vity

(fol

dst

imul

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JNK-HA

GSTc-Jun

PB -10 -9 -8 -7 -6

0

1

2

3

-10 -9 -8 -7 -6

* *

TotalJNK

p54P

p46P

COS-7 SH-SY5Y

COS-7 SH-SY5Y

Fig. 1 l-Opioid receptor-induced JNK activation in COS-7 cells and

SH-SY5Y cells. COS-7 cells were transiently cotransfected with the

cDNAs of JNK-HA and l-opioid receptor (0.5 lg for each). The

transfectants were serum-starved for 18 h, before application of

DAMGO. The lysate of COS-7 cells were subjected to in vitro kinase

assay as described under ‘Materials and methods’. SH-SY5Y cells

were serum-starved for 4 h prior to stimulation with DAMGO, and

activated JNK in the cell lysates was detected by anti-phospho-JNK

antiserum. (a) Time course of JNK activation by 100 nM DAMGO. (b)

Dose–response of JNK activation following DAMGO treatment of

COS-7 cells for 15 min or SH-SY5Y cells for 5 min. Representative

autoradiograms illustrate phosphorylated GST-c-Jun, total JNK-HA,

phosphorylated and total JNK. Band intensities were quantified by

densitometry. Data are presented as fold increase in JNK activity over

non-stimulated cells (Con), and represents the mean ± SEM from four

or five separate experiments. *DAMGO significantly induced JNK

activation (Bonferroni t-test, p < 0.05).



appeared to play an essential role in regulating l-opioidstimulated-JNK activity. As determined by immunodetection

with anti-Gat antiserum, coexpression of Gat did not alter theamount of JNK (shown in the anti-HA immunoblot).

Small GTPases Rac and Cdc42 contribute to JNK

activation by l-opioids

Small GTPases are believed to be a link between G protein-

coupled receptors (GPCRs) or receptor tyrosine kinases and

MAPKs. They may operate upstream of Raf in ERK

activation or MEKK in JNK activation. Belcheva et al.

(1998) have demonstrated that the three classical opioid

receptors require Ras GTPases to activate ERK via Raf and

MEK1/2. Like the ERK pathway, several small GTPases act

as upstream activators of JNK and they are members of the

Rho family, Rac and Cdc42 (Coso et al. 1995). In agreement

with this observation, we have previously found that the

pathway from the d-opioid receptor to JNK primarily

requires Rac or Cdc42 (Kam et al. 2003). To determine

which GTPases participate in signaling from the l-opioidreceptor toward JNK, dominant-negative mutants, including

RasS17N, RacT17N, Cdc42T17N or RhoT19N, were co-

transfected together with l-opioid receptor and HA-JNK into

the COS-7 cells. These inhibitory mutants are stable in a

GDP conformation, and so competitively prevent endog-

enous GTPases from accessing their specific guanine

nucleotide exchange factors. Like the d-opioid receptor, the

ability of the l-opioid receptor to stimulate JNK was

essentially abolished by RacT17N and Cdc42T17N, whereas

RasS17N was ineffective in inhibiting the JNK activation

(Fig. 3a). RhoT19N produced a slight but statistically

insignificant inhibition on DAMGO-induced JNK activity

(Fig. 3a). These results indicate that Rac and Cdc42 GTPases

link the l-opioid receptor to the regulation of JNK. In orderto further validate the important roles of Rac and Cdc42, we

(a)

(b)

Fig. 2 JNK stimulation by l-opioid receptor is attenuated in the

presence of PTX or Gbc-scavenging proteins. (a) The l-opioid

receptor and JNK-HA were coexpressed in COS-7 cells as in legend to

Fig. 1. Transfectants, with or without PTX pretreatment (100 ng/mL,

18 h), were incubated in the absence (basal) or presence of DAMGO

(100 nM) for 15 min. Similarly, serum-starved SHSY-5Y cells were

pretreated with or without PTX (100 ng/mL, 18 h) prior to application of

DAMGO (100 nM) in the absence or presence of CTOP (10 lM) for

5 min. (b) COS-7 cells were cotransfected with cDNAs encoding JNK-

HA and l-opioid receptor, together with vector or Gat (0.5 lg for each).

Serum-starved cells were exposed to 100 nM DAMGO for 15 min.

Values shown represent the mean ± SEM from five or six separate

experiments. *DAMGO significantly stimulated JNK activity (Bonfer-

roni t-test, p < 0.05).

(a)

(b)

Fig. 3 Role of small GTPases Ras, Rac, Cdc42 and Rho in l-opioid-

dependent JNK activation. (a) COS-7 cells were cotransfected with the

cDNAs of l-opioid receptor and JNK-HA, together with dominant

negative RasS17N, RacT17N, N17Cdc42 or N17Rho (0.5 lg each).

Transfectants were exposed to 100 nM DAMGO for 15 min. Results

are the mean ± SEM from eight independent experiments. *DAMGO

significantly stimulated JNK activity (Bonferroni t-test, p < 0.05). (b)

COS-7 cells expressing l-opioid receptors were stimulated by 100 nM

DAMGO for the indicated time, activation of Cdc42 and Rac was

determined by GST-PAK-PBD pull down assay and western blotting

as described in ‘Materials and Methods’. Total cell lysates were also

immunoblotted with Rac and Cdc42 antibodies to determine protein

expressions.



next investigated the activation state of Rac and Cdc42

following stimulation of the l-opioid receptor. This was

achieved by using GST-PAK-PBD pull-down assay. Because

the p21-binding domain (PBD) of the PAK protein binds

only to the activated GTP-bound Rac and Cdc42, the amount

of such activated GTPases in the assay was measured by anti-

Rac/Cdc42 immunoblotting. As shown in Fig. 3(b), stimu-

lation of the l-opioid receptor led to a time-dependent

activation of Rac and Cdc42, with a peak at around

15–30 min incubation of DAMGO. After 60 min, activations

of both GTPases decreased to basal levels.

The guanine nucleotide exchange factor (GEF)

Son-of-sevenless (Sos) mediates l-opioid-stimulated

JNK activity

Activations of small GTPases occur through stimulation of

specific GEF by promoting their exchange of GDP for GTP.

It was therefore required to identify which Rac-GEFs and/or

Cdc42-GEFs provide the link between l-opioid receptor andJNK activation. Sos proteins belong to Dbl family GEF and

are widely expressed. They specifically activate Rac but not

Cdc42 (Han et al. 1998; Nimnual et al. 1998). Hawes et al.

(1998) have demonstrated that l-opioid receptor requires Sosto regulate the activation of ERK, implying that the

stimulated receptor may transmit signal to Sos. Additionally,

expression of the Dbl homology domain of Sos (DH-Sos) has

been shown to induce a robust JNK stimulation in COS-1

cells (Nimnual et al. 1998). Sos therefore was considered as

the candidate to mediate the signaling from l-opioid receptortoward JNK. The inhibition on the activity of Sos is achieved

by expressing their proline-rich domains (Sos-Pro) at the

C-terminus, as Sos-Pro compete with the endogenous Sos for

binding to the SH3 domain of Grb2 (van Biesen et al. 1995).

As shown in Fig. 4, expression of Sos-Pro blocked the JNK

activation in response to DAMGO, indicating that Sos is able

to provide a relay from the l-opioid receptor to Rac and

subsequently JNK.

Like Sos protein, Vav2 is also a ubiquitously expressed

member of Dbl family, but displays overlapping GEF activity

for Rac1, Cdc42 and RhoA (Abe et al. 2000). To determine

the participation of Vav2 in the l-opioid-dependent JNKactivation, a dominant negative mutant of Vav2 was coex-

pressed in COS-7 cells together with the receptor and JNK-

HA. However, as the dominant negative mutant of Vav2

robustly increased the basal activity of JNK (data not

shown), it is difficult to observe the effect of DAMGO on the

JNK activity in the presence of the mutant. Owing to this

technical limitation, involvement of Vav2 in the l-opioid-signaling could not be determined.

l-Opioid receptor, but not j-opioid receptor requires

PI3Kc to stimulate JNK activity

As illustrated in Fig. 4, Sos proteins provide the link between

the l-opioid receptor and JNK activation. Numerous studies

revealed that Sos catalyze exchange for Rac in response to

lipid products of phosphatidylinositol 3-kinase (PI3K) such

as PI-3,4,5-P3 via binding to their Pleckstrin Homology (PH)

domains (PH-Sos; Han et al. 1998; Nimnual et al. 1998). In

addition, overexpression of PI3Kc significantly stimulates

JNK in COS-7 cells (Lopez-Ilasaca et al. 1998). We then

asked whether the l-opioid receptor can activate PI3K,

which in turn evoked the Rac and Cdc42-dependent JNK

activations via specific GEFs such as Sos. Agonist stimula-

tion of the l-opioid receptor in COS-7 cells was demonstra-ted to increase the phosphorylation of Akt at Ser-473, a

downstream target for the activated PI3K (Fig. 5a, immuno-

blot of antiphospho-Akt in left panel). Thus, it implied the

stimulatory effect of l-opioids on PI3K. To assess the

requirement of PI3K for l-opioid receptor signaling toward

Rac, Cdc42 and JNK, a specific inhibitor of PI3K wortman-

nin was utilized. The inhibitor significantly abrogated JNK

phosphorylation by the l-opioid receptor in both transfectedCOS-7 cells and SH-SY5Y cells (Fig. 5a). Wortmannin

(100 nM, 15 min) also attenuated the l-opioid receptor-

induced Rac and Cdc42 stimulation in COS-7 cells as

determined with the GST-PAK-PBD pull down assay (data

not shown). Surprisingly, these results were inconsistent with

the previous observation that no inhibitory effect by wort-

mannin was observed when JNK was stimulated by d-opioidreceptor (Kam et al. 2003). Hence, the effect of wortmannin

on the j-opioid signaling was also examined. COS-7 cells

were transiently expressed j-opioid receptor and HA-JNK.

As shown in Fig. 5(b) (left panel), JNK activity was

significantly increased upon stimulation with j-opioidU-50,488H, irrespective of whether COS-7 cells were

preincubated with wortmannin or not. In order to confirm

the insensitivity of wortmannin in j-opioid-stimulated JNK

Fig. 4 Suppression of l-opioid-stimulated JNK activation by SOS-

Pro. The l-opioid receptor and JNK-HA were coexpressed in COS-7

cells, together with vector or SOS-Pro (0.5 lg each). JNK activity was

determined at 15 min in the absence (basal) or presence of DAMGO

(100 nM). Results are the mean ± SEM from five independent

experiments. *DAMGO significantly induced JNK activation (Bonfer-

roni t-test, p < 0.05).



pathway, human monocytic THP-1 cells endogenously

expressing the j-opioid receptor were utilized. Administra-

tion of U-50,488H in quiescent THP-1 cells increased the

phosphorylation of JNK (Fig. 5b; right panel). In agreement

with studies in COS-7 cells, wortmannin still had no

inhibitory effect on the JNK activation in the j-opioid-treated THP-1 cells. Hence, the crucial role for PI3K was

only observed in the Rac and Cdc42-dependent JNK

activation by l-opioid receptor, but not j- and d-opioidreceptors. Unlike COS-7 cells, elevations of basal JNK

activity were observed in wortmannin-treated SH-SY5Y and

THP-1 cells. The reason may be the presence of a PI3K

inhibitory pathway in the naive cells. The immunoblots

against Akt and phospho-Akt in Fig. 5 revealed that

pretreating COS-7 cells with wortmannin substantially

reduced the Akt activity, while total amount of Akt was

not altered, confirming the inhibitory effect of wortmannin

on PI3K.

Wortmannin inhibits all isoforms of PI3K and the

c-isoform has been well documented to be dependent on

Gbc subunits (Stephens et al. 1994). Although PI3Kc is

mainly expressed in hematopoietic cells, its limited expres-

sion is still detected in COS-7 cells (Murga et al. 1998).

Moreover, COS-7 cells expressing p101 (a regulatory subunit

specific for c isoform) alone can elicit a level of Akt

activation similar to that induced by PI3Kc overexpression,

implicating that p101 functionally interacts with endogenous

PI3Kc isoform in COS-7 cells to stimulate Akt (Murga et al.

1998). Therefore, the function of PI3Kc in the JNK

activation by l-opioid was determined by using a kinase-

deficient mutant (PI3KcK832R). Coexpression of

PI3KcK832R suppressed JNK activation in response to

DAMGO, but not U-50,488H (Fig. 6a). Collectively, these

findings reveal a marked difference in signaling between the

three opioid receptors, whereby only l-opioid receptor

regulates JNK activity through PI3Kc-dependent pathway.Expression of PI3KcK832R clearly reduced the activity of

Akt in comparison with the wild type of PI3Kc (data not

shown), confirming the negative effect of the kinase-deficient

mutant on endogenous PI3K signaling.

Akt phosphorylation by l-opioid receptor is not essential

for JNK activation

Although activation of l- and j-opioid receptors increased

the Akt phosphorylation in the absence of wortmannin

(Fig. 5), only the l-opioid receptor required PI3K to

stimulate JNK. To test whether the observed phosphorylation

of Akt correlated with l-opioid receptor-mediated JNK

activation, a kinase deficient Akt containing a Lys179 to Met

mutation was utilized (Xu and Wu 2000). Overexpression of

this kinase deficient Akt mutant in mouse C2C12 has been

shown to suppress insulin-like growth factor-1-mediated

myogenin expression (Xu and Wu 2000). Furthermore, Akt-

dependent phosphorylations of glycogen synthase kinase 3

and tuberin induced by platelet-derived growth factor (30 ng/

mL, 5 min) were attenuated in COS-7 cells transiently

overexpressing this Akt mutant (data not shown). However,

the Akt mutant failed to inhibit the PI3K-dependent JNK

activation induced by l-opioid receptor (Fig. 6b, left panel),therefore excluding the role for Akt in the pathway. Similar

to the insensitivity to wortmannin and PI3KcK832R,expression of the kinase deficient Akt still had no demon-

strable effect on the j-opioid receptor-stimulated JNK

(Fig. 6b, right panel). Taken together, these studies indicate

that signal transductions from l- and j-opioid receptors to

JNK and Akt activation might occur independently.

Fig. 5 Wortmannin inhibits JNK activation by l-opioid receptor, but

not j-opioid receptor. (a) COS-7 cells were cotransfected with the

cDNAs of l-opioid receptor and JNK-HA (0.5 lg each). Transfectants

were serum-starved for 18 h and pretreated with or without wort-

mannin (100 nM, 15 min). The JNK activity was stimulated with 100 nM

DAMGO (15 min). Fifty microliters of lysate of COS-7 cells were

probed for phosphorylated and total Akt. SH-SY5Y cells were similarly

pretreated with wortmannin and then stimulated with DAMGO

(100 nM, 5 min). (b) COS-7 cells coexpressing j-opioid receptor and

JNK-HA were preincubated with or without wortmannin (100 nM,

15 min) prior to application of 100 nM U-50,488H (U-50; 15 min). THP-

1 cells were similarly preincubated with wortmannin after 18 h star-

vation, and then stimulated with 100 nM U-50 for 5 min. Values shown

represent the mean ± SEM from five to six separate experiments.

*Opioid agonists significantly induced JNK activation (Bonferroni t-test,

p < 0.05). **Wortmanin significantly increased the basal JNK activity

as compared to untreated cells (Bonferroni t-test, p < 0.05).



l-Opioid receptor stimulates JNK through Src family

tyrosine kinases but not transactivation of EGF receptor

Li and Smithgall (1998) have demonstrated that v-src

transformed Rat-2 cells induce JNK activation. Furthermore,

Src kinase activity can be stimulated by transient over-

expression of Gbc (Luttrell et al. 1996) or a2-adrenergic andmuscarinic m1 receptors (Chen et al. 1994). Therefore, these

studies suggest that Src family tyrosine kinases seem to

participate in the mitogenic signaling by G protein-coupled

receptors, in addition to PI3Kc. A pyrazolopyrimidine PP2 is

a potent and selective inhibitor of Src family tyrosine kinase.

COS-7 and SH-SY5Y cells were preincubated with PP2

(10 lM) for 30 min before stimulation by DAMGO. The

JNK stimulation in response to DAMGO was completely

suppressed following pretreatment with PP2 (Fig. 7a,b, left

panels). We next examined the sole contribution of the

ubiquitous Src kinase in the pathway. Upon coexpression of

dominant negative Src kinase with the l-opioid receptor in

COS-7 cells, a complete inhibition of JNK was observed

JNK

act

ivit

y (f

old

stim

ulat

ion)

JNK-HA

GSTc-Jun

PWT DN

0

1

2

3*

WT DN0

1

2

3

* *

Basal DAMGO U50(a)

Vector AktDN

JNK

act

ivit

y (f

old

stim

ulat

ion)

0

1

2

3*

*

0

1

2

3

**

Vector AktDN

JNK-HA

GSTc-Jun

P

Basal DAMGO U50(b)

PI3Kγ

Fig. 6 Effect of dominant negative PI3Kc and Akt on l- and j-opioid

receptor-mediated stimulation of JNK. (a) cDNAs encoding l- (left

panel) or j- (right panel) opioid receptor and JNK-HA were cotrans-

fected into COS-7 cells, together with wild-type (WT) or dominant

negative (DN) PI3Kc (0.5 lg each). (b) COS-7 cells were cotrans-

fected with the cDNAs encoding l- (left panel) or j- (right) opioid

receptor and JNK-HA in the presence of vector or dominant negative

Akt (Akt DN). Transfected cells were stimulated for 15 min with 100 nM

DAMGO or U-50,488H. Results are the mean ± SEM from four sep-

arate experiments. *Opioid agonists significantly induced JNK activa-

tion (Bonferroni t-test, p < 0.05).

Fig. 7 Differential involvement of Src family tyrosine kinase and EGF

receptor in the activation of JNK by opioid receptors. (a) The l-opioid

receptor and JNK-HA were coexpressed in COS-7 cells in the

absence or presence of vector or dominant negative Src (Src DN;

0.5 lg each). In case of inhibitor pretreatments, transfectants were

preincubated with either PP2 (10 lM, 3 h) or AG1478 (500 nM, 30 min)

as indicated. JNK activity was determined at 15 min in the absence

(basal) or presence of DAMGO (100 nM). (b) SH-SY5Y cells were

pretreated with either PP2 or AG1478 (as in a) before application of

100 nM DAMGO for 5 min. (c) Serum starved SH-SY5Y cells were

stimulated with 100 nM DAMGO for the indicated durations. Activated

and total Src were determined by western blotting using antiphospho-

Src-Y416 and antic-Src antisera, respectively. *DAMGO significantly

stimulated JNK activity (Bonferroni t-test, p < 0.05).



following incubation of DAMGO (Fig. 7a, right panel).

Furthermore, DAMGO treatment of SH-SY5Y cells led to a

time-dependent increase in phosphorylation of c-Src at Tyr-

416, which represented the activated c-Src (Fig. 7c). The

maximal stimulation was obtained at 5–15 min. These results

suggest that Src kinases are essential mediators for l-opioidreceptor to activate JNK.

Recently, Belcheva et al. (2001) have demonstrated

increased EGF receptor phosphorylation upon DAMGO

treatment in HEK293 cells stably expressing l-opioidreceptors. Moreover, l-opioid receptor-mediated ERK acti-

vation has also been shown to require calmodulin-dependent

transactivation of EGF receptor, where a metalloprotease is

involved in promoting the shedding of membrane-bound

EGF. The contribution of EGF receptor transactivation to

activation of JNK by l-opioid receptor remains unclear. As

shown in Fig. 7a,b, inhibition of endogenous EGF receptor

tyrosine kinase in transfected COS-7 and SH-SY5Y cells by

500 nM of the specific tyrphostine AG1478 (30 min) did not

abolish l-opioid-elicited JNK activation. Therefore, transac-

tivation of EGF receptor is not required in l-opioid receptorssignaling toward JNK. The Src-dependent, but EGF receptor-

independent JNK activation by l-opioid receptor displays thesame signaling properties as that of the d-opioid receptor

(Kam et al. 2003).

Discussion

The present study together with our previous findings (Kam

et al. 2003) suggested that molecular mechanisms behind

opioid-stimulated JNK were similar among three opioid-

receptors, except for the differential participation of PI3K.

The l-opioid receptor-activated JNK was transduced by Gbcreleased upon Gi/o stimulation, and required Src, Rac and

Cdc42, as shown previously for the d-opioid receptor (Kam

et al. 2003). Furthermore, l- and d-opioid receptors did not

entail EGF transactivation to stimulate JNK. One of Rac-

GEFs, Sos, appeared to transmit signals from l-opioidreceptor to JNK. Remarkably, a significant difference

between l-, d- and j-opioid receptors signaling was

observed, whereby PI3K was involved in l-, but not d-and j-opioid receptors-induced JNK activation. Presumably,

an alternative pathway from l-opioid receptor to JNK might

exist.

The l-opioid receptor-activated JNK was completely

inhibited by PTX (Fig. 2a), suggesting Gi/o as the primary

mediator for functional coupling of the receptor to JNK.

Notably, opioid receptors also utilize PTX-insensitive G

proteins, such as Gz to regulate downstream effectors under

certain circumstances. When SH-SY5Y cells are differenti-

ated by retinoic acid, the expression of Gz increases (Ammer

and Schulz 1994) and ORL1 receptor-inhibited cAMP

accumulation becomes PTX-resistant (Chan et al. 1998). It

may be interpreted as functional coupling between ORL1

receptor and Gz upon differentiation of the cells. It remains to

be determined if the l-opioid receptor utilizes Gz to stimulate

JNK in differentiated SH-SY5Y cells. Activation of the

l-opioid receptor releases free Gbc and Gai-GTP, which thenmediates downstream signals. In endothelial cells, JNK is

regulated by mechanisms involving Gbc, Ras and tyrosine

kinases, but not by Gai2 (Jo et al. 1997). Predictably,

sequestration of Gbc by transducin blocked l-opioid recep-

tor-induced JNK activation, implicating the involvement of

Gbc (Fig. 2b). It is further supported by a study where Gbcoverexpression in COS-7 cells potently stimulates JNK,

whereas activated Gai2 fails to do that (Coso et al. 1996).

Yet, a different involvement of Gai in JNK activation is

illustrated by Yamauchi et al. (2000), wherein activated Gai2can stimulate JNK through MKK4- and MKK7-independent

pathways in HEK 293 cells. This disparate JNK regulation

by Gai might be attributed to cell-type specificity.

JNK is potently stimulated by constitutively activated Rac

and Cdc42, but is inhibited by their dominant-negative

mutants, so they appear as upstream mediators of the JNK

cascade (Coso et al. 1995). Similarly, dominant-negative Rac

and Cdc42, but not Ras and Rho, significantly abrogated the

l-opioid receptor-activated JNK (Fig. 3a). In the PAK pull-

down assay, participation of Rac and Cdc42 were further

highlighted by the induction of GDP/GTP exchange after

l-opioid incubation (Fig. 3b). This finding is compatible withour previous study revealing Rac- and Cdc42-dependent JNK

regulation by d-opioid receptor (Kam et al. 2003). Then,

specific GEF in the pathway was identified here. Dominant-

interfering Sos attenuated the l-opioid receptor-stimulated

JNK (Fig. 4), implying that Sos may link the receptor to Rac

and subsequently JNK. DH-Sos can activate JNK via Rac, but

does not affect ERK activity (Nimnual et al. 1998). PH-Sos,

on the other hand, negatively regulates the activity of its DH

domain by structural contact (Nimnual et al. 1998). Although

it is unclear how Sos couples the l-opioid receptor to Rac-

dependent JNK activation, several reports provide a glimpse

of the pathway. An interaction between the PH-Sos and Gb1c2(Sawai et al. 1999) provides a possible means for Gbc to

regulate Sos activity. Moreover, the inhibition of PH domain

on Sos activity toward Rac is disrupted by interaction with

PI3K-generated PI-3,4,5-P3 and PI-3,4-P3 (Nimnual et al.

1998). Because Akt activity was increased after incubation of

DAMGO (Fig. 5) and a report documented Akt activation

requires PI-3,4,5-P3 and PI-3,4-P3 (Andjelkovic et al. 1997),

we speculate that activated l-opioid receptor may trigger

PI3K stimulation and so produce such lipid products.

Collectively, a putative pathway from l-opioid receptors to

JNK is proposed. PI3K stimulations by l-opioid receptors

increase the amount of intracellular PI-3,4,5-P3, which

subsequently bind to the PH-Sos. Consequently, exposed

DH-Sos catalyzes the GDP/GTP exchange specifically on

Rac and initiates the JNK cascade. However, at this point, the

identitiy of Cdc42-GEF for the pathway remains ambiguous.



The crucial role for PI3K is only observed in JNK

activation by l-opioid receptor, but not j- and d-opioidreceptors (Figs 5 and 6a; Kam et al. 2003). PI3K thus

differentially mediates their JNK regulations, but the reason

for this discrepancy is unclear. Coexpression of kinase-

deficient Akt had no effect on l- and j-opioid receptor-

induced JNK activations (Fig. 6b). This exclusion of Akt

narrows down the potential targets of PI3K that might

contribute to the disparate opioid signaling. Indeed, signifi-

cant differences in many signalings regulated by l-, d- andj-opioid receptor have been observed. The l- but not

j-opioid receptor utilizes overexpressed Gaz to trigger acuteERK activation in a PTX-insensitive manner (Belcheva et al.

2000). When COS-7 cells express the l- or j-opioid receptortogether with Gaz or Ga12, EGF-stimulated ERK is sup-

pressed by chronic l-agonist, but not j-agonist, after PTXtreatment (Belcheva et al. 2000). Additionally, DAMGO has

a synergistic effect with NGF on the survival of chick dorsal

root ganglion (DRG) neurons, but DPLPE (d-agonist) andU-50 488 failed to do that (Sakaguchi et al. 1999).

An interesting role of PI3K is implicated in neuronal

protection from apoptosis. Cannabinoids require PI3K to

protect oligodendrocyte progenitors from apoptosis in the

absence of trophic support (Molina-Holgado et al. 2002).

Similarly, recent studies have reported the association of

PI3K signaling with l-opioid-promoted neuronal survival.

Morphine dose-dependently decreases peroxynitrite-induced

cell death of rat neonatal astrocytes via Gi and PI3K (Kim

et al. 2001). Furthermore, PI3K participates in DAMGO-

induced anti-apoptotic effect on differentiated SH-SY5Y

cells and cortical neurons following serum withdrawal

(Iglesias et al. 2003). Based on these reports and our results

demonstrating the involvement of PI3K in l-opioid receptor-stimulated JNK in SH-SY5Y cells (Figs 5 and 6A), we

postulate that the ability of l-opioid receptor-activated JNK

through PI3K will be ultimately involved in the neuronal

survival, especially since JNK has been shown to possess

neuronal protection capacity. In hindbrain and forebrain of

jnk1–/–jnk2–/– mice at E10.5, apoptosis is augmented (Sab-

apathy et al. 1999). Hence, future efforts should be directed

to confirm the physiological significance of this signaling

cascade. Moreover, the differential involvement of PI3K in

JNK pathway regulated by three opioid receptors possibly

indicates their functional specificity in certain biological

responses or in specific cell types. For example, l-opioidreceptor exclusively has a survival-promoting effect on chick

DRG neurons in the presence of NGF, whereas d- and j-opioid receptors have no such effect (Sakaguchi et al. 1999).

Our experiments support Src participations in l-opioidreceptors-activated JNK, with no role for EGF receptor

(Fig. 7a,b). The importance of Src is further confirmed by

Src activations after DAMGO administration (Fig. 7c).

Present understanding of the molecular mechanism by which

GPCRs stimulate Src is limited. A recent study by Ma et al.

(2000) has successfully demonstrated a direct linkage

between Gai1 and Src, whereas Gbc failed to directly

stimulate the kinase activity. Nevertheless, this observation in

a reconstituted system may not totally reflect the situation

in a cellular environment. Luttrell et al. (1996) have

µ-Opioid Receptor

JNKJNK

RacCdc42

PI3KPI3K

SosGEF?GEF?

Src

. GTP

MKK4/7

MEKK

AC

Gα

γ

β

i

Fig. 8 A schematic diagram illustrating the putative pathway from the

l-opioid receptor to JNK activation. The JNK activation induced by the

l-opioid receptor is initiated by free Gbc subunit released from Gi/o

protein, and subsequently leads to activations of Src family tyrosine

kinase and PI3K. The activated PI3K is able to generate lipid products,

that in turn increase the activities of Sos and/or other GEFs. The

activated Sos and other GEFs catalyze the exchange of GDP for GTP

on Rac and Cdc42 and thus stimulate the JNK cascade.



demonstrated that overexpressed Gbc in COS-7 cells elicits

c-Src activation. Intracellular signal-adapters might be required

for Gbc-stimulated Src activity. JNK activation by l-opioidreceptor is primarily mediated by Gbc due to a complete

suppression of the response after transducin coexpression

(Fig. 2b). Either direct activation of Src by Gai is insufficientto activate JNK or a Gbc-dependent signal is concomitantlyrequired for l-opioid receptor-induced JNK activation.

It is generally believed that PI3K and Src lie upstream of

Rho family GTPases (Han et al. 1998; Nagao et al. 1999).

The next issue is the relative position of PI3K and Src within

the l-opioid-activated JNK pathway. We employed an

experimental model in which JNK activation by SrcWT

was studied in contransfection system with PI3KcK832R, asSrcWT can functionally act as the active mutant to stimulate

MAPK (Della Rocca et al. 1997). A potent JNK stimulation

by SrcWT was partially suppressed by the coexpression of

PI3KcK832R (approximate 40% reduction; data not shown).

Thus, Src is presumably located upstream of PI3K in the

pathway. The incomplete reduction on the JNK activation

may be due to the presence of PI3K-independent pathway

from Src toward JNK. In agreement with a study, PI3K

stimulation by factors-VIIa was sensitive to Src inhibitor PP1

(Versteeg et al. 2000), indicating that Src is perhaps located

upstream of PI3K.

The present study provides a putative mechanism by

which l-opioid receptor activated JNK (Fig. 8). l-Opioidreceptor stimulated JNK in a PTX-sensitive and Gbc-dependent manner. The signal transduction undergoes

through Src family tyrosine kinase, Sos, Rac and Cdc42,

but not EGF receptor. However, one intermediate step

exclusively occurs in the l-opioid signaling, whereby l-,but not j- and d-opioid receptor requires PI3K to mediate

JNK activity. Hence, this is a distinct difference in the

signaling pathway between three opioid receptors.

Acknowledgements

We are extremely grateful to the following individuals for kindly

providing the cDNAs: Dr L. Yu for rat l-opioid receptor,

T. VoynoYasenetskaya for the JNK-HA, Dr Matthias P. Wymann

for wild-type and dominant negative mutant of PI3Kc, Dr S. Lin forwild-type and dominant negative mutant of Src, Dr E. J. Stanbridge

for RasS17N and RacT17N, Dr M. Symons for Cdc42T17 N

and RhoT17N, Dr Udo Schmitz for PAK1-PBD as well as

Dr R. J. Lefkowitz for Sos-Pro. This work was supported in part

by grants from the University Grants Committee of Hong Kong

(AoE/B-15/01), the Research Grants Council of Hong Kong

(HKUST 6115/00 M and 2/99C) and the Hong Kong Jockey Club.

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Phosphatidylinositol-3 kinase is distinctively required for �-, but not ?-opioid receptor-induced activation of c-Jun N-terminal kinase