Introduction

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Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL₁) receptor (Meunier et al., 1995; Reinscheid et al., 1995). The ORL₁ receptor has high sequence homology to different opioid receptor subtypes but does not exhibit high-affinity binding for opioid ligands. The mRNA of ORL₁ and the peptide precursor of N/OFQ are widely distributed in the central nervous system (Mollereau et al., 1994; Nothacker et al., 1996). The biological functions associated with the ORL₁ receptor appear to be somewhat opposite to those of the opioid receptors. ORL₁ receptors expressed in the central nervous system mediate pain regulation, and intraventricular injection of N/OFQ into the mouse brain resulted in enhanced painful reactivity (Meunier et al., 1995).

The ORL₁ receptor produces its biological effects through different members of the G protein superfamily. Like opioid receptors, the ORL₁ receptor is known to inhibit adenylyl cyclases (Mollereau et al., 1994) and mediate ion channel activities (Connor et al., 1996) by coupling to the pertussis toxin (PTX)-sensitive G_i and G_o proteins, respectively. We have demonstrated that the ORL₁ receptor is also capable of coupling to PTX-insensitive G_z, G_14, G₁₆, and, to a lesser extent, G₁₂ to regulate the activities of different second messenger systems (Chan et al., 1998; Yung et al., 1999). The ability of the ORL₁ receptor to interact with multiple G proteins may in part explain the diverse physiological effects of N/OFQ, ranging from modulation of nociceptive function to locomotor activity (Reinscheid et al., 1995) and stress adaptation (Koster et al., 1999).

In less than a decade, we have come to the realization that many G protein-coupled receptors regulate cell proliferation and differentiation through the mitogen-activated protein kinases (MAPKs). MAPKs are serine/threonine protein kinases capable of phosphorylating several transcription factors (Price et al., 1996), and hence, regulate subsequent transcriptional events. There are at least three subtypes of MAPK, the extracellular signal-regulated kinases (ERKs) are mainly stimulated by growth factors (Boulton et al., 1991), whereas c-Jun NH₂-terminal kinases (JNKs) and p38 MAPK are more responsive to cellular stress (Han et al., 1994; Kyriakis et al., 1994). Recent studies suggest that the signaling of ORL₁ receptors involves the activation of ERK and p38 MAPK (Lou et al., 1998; Zhang et al., 1999) to modulate the activities of the downstream transcription factors Elk-1 and Sap1a (Bevan et al., 1998). However, the capability of the ORL₁ receptor to activate JNK remains unknown.

JNK modulates the activities of several transcription factors. Depending on the JNK isoforms involved, c-Jun, ATF-2, and Elk-1 can be actively phosphorylated upon JNK activation (Gupta et al., 1996), whereas the phosphorylation of NFAT4 by JNK prevents its translocation to the nucleus (Chow et al., 1997). Therefore, activation of JNK may regulate gene transcription in both directions to up-regulate the expression of certain genes and to down-regulate others. There is evidence to suggest that ORL₁ receptor-mediated MAPK stimulation and the subsequent modulation of transcriptional events may have some physiological significance. Chronic activation of the ORL₁ receptor leads to supersensitization of adenylyl cyclase (Chan and Wong, 1999), and this adaptive response may involve gene transcription as in the case of immediate-early genes transcription associated with supersensitization of dopamine receptor functions (LaHoste et al., 1993). In this report, we used COS-7 cells transiently expressing the ORL₁ receptor, as well as neuroblastom × glioma hybrid NG108-15 cells, which endogenously express the receptor, to investigate the characteristics of N/OFQ-induced JNK activation. Our results clearly demonstrated the capability of ORL₁ receptor to stimulate JNK activity in terms of active phosphorylation of c-Jun and ATF-2.

	Materials and Methods

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Reagents. The cDNAs encoding ORL₁ receptor and JNK-HA were kindly provided by Dr. Gang Pei (Shanghai Institute of Cell Biology, Shanghai, China) and Dr. Tatyana A. Voyno-Yasenetskaya (University of Illinois, Chicago, IL), respectively. The cDNAs of the dominant-negative mutants of Ras (RasS17N) and Rac (RacT17N) were generous gifts from Dr. Eric J. Stanbridge (University of California, Irvine, CA). [-³²P]ATP was purchased from DuPont NEN (Boston, MA). Anti-phospho-JNK, anti-JNK, anti-phospho-c-Jun, and anti-phospho-ATF-2 antibodies were obtained from New England BioLabs (Beverly, MA). PTX and 12CA5 (anti-HA) antibody were purchased from List Biological Laboratories (Campbell, CA) and Roche Molecular Biochemicals (Indianapolis, IN), respectively. N/OFQ was obtained from Research Biochemicals International (Natick, MA). Cell culture reagents, including LipofectAMINE PLUS were obtained from Life Technologies (Gaithersburg, MD) and all other chemicals were purchased from Sigma (St. Louis, MO).

Cell Culture and Transfection. COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 50 units/ml penicillin and 50 µg/ml streptomycin, and grown at 37°C in an environment of 5% CO₂. In the case of NG108-15 cells, fetal calf serum was reduced to 5%. Transfection was performed on 3 × 10⁶ COS-7 cells in a 10-cm plate by means of LipofectAMINE PLUS reagents following the supplier's instructions.

In Vitro JNK Assay. COS-7 cells were transferred to six-well plates at 3 × 10⁵ cells/well 12 h after transfection, and then kept in the growth medium for 36 h. The cells were then serum starved for 18 h in the presence or absence of PTX before drug treatment. In case of the wortmannin sensitivity assay, an additional treatment of wortmannin (100 nM, 15 min) was applied to the starved cells. The assay used was basically similar to that previously described (Berestetskaya et al., 1998). Transfected COS-7 cells in six-well plates were treated with the assay medium (Dulbecco's modified Eagle's medium with 20 mM HEPES) in the presence or absence of N/OFQ for 30 min at 37°C. Reactions were terminated by washing the cells with ice-cold phosphate-buffered saline, followed by addition of 500 µl 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 µM Na₃VO₄, 100 µM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 0.7 µg/ml pepstatin) and then gently shaken on ice for 30 min. A supernatant was collected for each sample by centrifugation at 16,000g for 5 min. Fifty microliters of each supernatant was used for the detection of JNK-HA expression, and the remaining was incubated for 1 h at 4°C with anti-HA antibody (2 µg/sample), followed by incubation with 30 µl of 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 µM Na₃VO₄]. Washed immunoprecipitates were resuspended in 40 µl of kinase assay buffer containing 5 µg of GST-c-Jun per reaction, and the kinase reactions were initiated by the addition of 10 µl of ATP buffer (50 µM ATP containing 2 µCi of [-³²P]ATP per sample). After a 30-min incubation at 30°C with occasional shaking, the reactions were terminated by 10 µl of 6× sample buffer, and the samples were resolved by 12% SDS-polyacrylamide gel electrophoresis. The radioactivity incorporated into GST-c-Jun was detected by autoradiogram, and the signal intensity was quantified by PhosphorImager (Molecular Dynamics 445 SI).

Western Blot. NG108-15 cells were seeded on six-well plates at a density of 1.5 × 10⁵ cells/well and were kept in the growth medium overnight. The cells were then serum starved for 18 h either in the presence or absence of PTX, followed with a wortmannin treatment (100 nM, 15 min) if necessary. The cells were stimulated with N/OFQ (100 nM) for 30 min and then lysed in 500 µl of lysis buffer. Supernatants were collected by centrifugation at 16,000g for 5 min. Eighty microliters of each supernatant was resolved by 12% SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The presence of actively phosphorylated JNK, c-Jun, and ATF-2 were detected by phospho-specific antibodies as mentioned under Reagents, followed with horseradish peroxidase-conjugated secondary antibody. The blot was 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).

	Results

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ORL₁ Receptor Stimulates JNK Activity in COS-7 Cells. It has been established that the ORL₁ receptor inhibits adenylyl cyclase by activating the G_i proteins (Mollereau et al., 1994). Because G_i-coupled receptors can activate JNK (Coso et al., 1996), we examined whether the functional coupling between ORL₁ receptor and G_i proteins has any effect on JNK activity. Agonist treatment on COS-7 cells transfected with JNK-HA alone had no observable changes in the kinase activity upon application of 100 nM N/OFQ (Fig. 1A). However, COS-7 cells cotransfected with JNK-HA and ORL₁ receptor exhibited increased JNK activity upon stimulation with N/OFQ (Fig. 1A). These results indicate that the COS-7 cells probably do not express the ORL₁ receptor, or the receptor is only present at an extremely low level and does not affect our functional assay. The agonist-induced kinase activity was characterized by a dose dependence on the concentration of N/OFQ, reaching a maximum activity at 100 nM (Fig. 1B). When the transfected cells were stimulated with 100 nM N/OFQ for different durations, the JNK activity increased gradually and became saturated around 15 to 30 min of drug treatment (Fig. 1C). To examine whether the N/OFQ-induced stimulation of JNK required G_i proteins, we pretreated the transfectants with PTX before stimulation with N/OFQ. As shown in Fig. 1A, PTX treatment significantly reduced the N/OFQ-induced activation of JNK. This result indicated that the ORL₁ receptor-mediated stimulation of JNK was primarily via endogenous PTX-sensitive G_i proteins. However, because the increased kinase activity could not be completely abolished by PTX (Fig. 1A), we could not exclude the possibility that the ORL₁ receptor may also stimulate the kinase activity through other pathways, for example, via endogenous PTX-insensitive G proteins.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Stimulation of JNK by the ORL1 receptor in transfected COS-7 cells. COS7-cells were transfected with the cDNAs of JNK-HA alone, or cotransfected with the ORL1 receptor. JNK assay was performed as described under Materials and Methods. The JNK activity was determined at 30 min (A and B) in the absence (basal) or presence of 100 nM N/OFQ with or without PTX pretreatment (100 ng/ml, 18 h), or stimulated for the indicated periods (C). Values shown represent the mean ± S.E. from three separate experiments. The phosphorylation of GST-c-Jun and the expression of JNK-HA in the cell lysates are shown on the right-hand side. *, N/OFQ significantly stimulated the JNK activities in the transfected cells (Bonferroni t test, P < .05). **, PTX treatment significantly inhibited the N/OFQ-induced JNK stimulation (Bonferroni t test, P < .05). In B, the JNK activity was significantly higher than the basal values when the N/OFQ concentration was >1 nM (Bonferroni t test, P < .05).

ORL₁ Receptor-Mediated JNK Activity Is Dependent on the Low-Molecular-Weight GTPases and Is Insensitive to Wortmannin Pretreatment. Low-molecular-weight GTPases have been shown to play an important role in the activation of MAPK activities (Thomas et al., 1992; Minden et al., 1995). The involvement of small GTPases such as Ras and Rac is often demonstrated with the use of dominant-negative mutants. By transfecting the dominant negative mutants of Ras (RasS17N) and Rac (RacT17N), we found that the ORL₁ receptor-mediated JNK activity was significantly inhibited (Fig. 2A). Coexpression of the two dominant-negative mutants did not produce additive inhibition of the kinase activity (Fig. 2A). On the other hand, the role of phosphatidylinositol-3 kinase (PI3K) in modulating JNK activity has been studied by several groups, and both up-regulation (Lopez-Ilasaca et al., 1998) and down-regulation (Kwon et al., 2000) of JNK activity have been proposed. By pretreating the transfected cells with the specific PI3K inhibitor wortmannin, we observed no significant effect on the induced JNK activation (Fig. 2B). These results suggested that the low-molecular-weight GTPases, but not PI3K, served as signaling intermediates in the ORL₁ receptor-mediated JNK activation.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   ORL1 receptor-mediated JNK activity is dependent on the low-molecular-weight GTPases and is insensitive to wortmannin pretreatment. COS-7 cells were transfected with the cDNAs of JNK-HA and ORL1 receptor in the absence (B and the control in A) or presence of RasS17N and/or RacT17N (A). JNK activity is shown at 30 min in the absence (basal) or presence of 100 nM N/OFQ with or without wortmannin pretreatment (100 nM, 15 min) as indicated. Values shown represent the mean ± S.E. from three independent experiments. The phosphorylation of GST-c-Jun and the expression of JNK-HA in the cell lysates are shown below the graphs. *, N/OFQ-induced JNK activation was significantly inhibited in the presence of RasS17N and RacT17N (Bonferroni t test, P < .05).

N/OFQ-Induced Activation of JNK via PTX-Insensitive G Proteins. Because the ORL₁ receptor-mediated JNK activity was not completely abolished by PTX (Fig. 1A), PTX-insensitive G proteins endogenously expressed in COS-7 cells may link ORL₁ receptors to the stimulation of JNK. The activated mutants of PTX-insensitive G₁₂, G₁₃, G_q/11, and G₁₆ are capable of stimulating the JNK activity (Heasley et al., 1996; Voyno-Yasenetskaya et al., 1996). Our previous reports demonstrated that the ORL₁ receptor is incapable of activating G_q, but it is functionally coupled to G_z, G₁₄, G₁₆, and to a lesser extent to G₁₂ (Chan et al., 1998; Yung et al., 1999). Whether these functional couplings are linked to activation of JNK remains unknown. We coexpressed the ORL₁ receptor, JNK-HA, and the -subunit of different PTX-insensitive G proteins in COS-7 cells, and examined the ORL₁ receptor-mediated JNK activation in the presence of PTX. Functional coupling of the ORL₁ receptor to the coexpressed PTX-insensitive G protein should enhance the PTX-resistant JNK activity. No enhancement of N/OFQ-induced JNK activity was associated with cells coexpressing either PTX-insensitive G_s or G₁₃ compared with the control cells (Fig. 3). In contrast, COS-7 cells transfected with G₁₂ was associated with a larger increase of JNK activity upon N/OFQ treatment, and this enhancement was even greater when PTX-insensitive G_z, G₁₄, or G₁₆ was coexpressed instead (Fig. 3). These results indicated that the ORL₁ receptor is capable of activating JNK through the PTX-insensitive G_z, G₁₂, G₁₄, and G₁₆. However, it should be noted that the expression of G₁₆ is restricted to hematopoietic cells (Amatruda et al., 1991), and COS-7 cells do not express endogenous G_z (our unpublished data). Hence, the residual N/OFQ-induced JNK activity after PTX pretreatment was probably mediated through G₁₂ and G₁₄, which are ubiquitously expressed in different tissues (Strathmann and Simon, 1991) and found in kidney cells (Nakamura et al., 1991), respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Functional coupling of ORL1 receptor with PTX-insensitive G proteins leads to JNK activation. COS-7 cells were transfected with the cDNAs of JNK-HA, ORL1 receptor, in the absence (control) or presence of a G-subunit (Gs, Gz, G12, G13, G14, or G16). Transfected cells were pretreated with PTX (100 ng/ml, 18h). JNK activity is shown at 30 min after the addition of 100 nM N/OFQ. Values shown represent the mean ± S.E. from three separate experiments. The expression of JNK-HA and the phosphorylation of GST-c-Jun are also illustrated. *, coexpression of the G-subunit (Gz, G12, G14, and G16) significantly enhanced the N/OFQ-induced kinase activity compared with the control (Bonferroni t test, P < .05).

Stimulation of Endogenous ORL₁ Receptor in NG108-15 Cells Resulted in Enhanced JNK Activity. To verify that the ORL₁ receptor-mediated JNK activation can occur in vivo, we applied N/OFQ treatment on NG108-15 cells that endogenously express ORL₁ receptors, followed by subsequent detection of actively phosphorylated JNK by specific antibodies. Two immunoreactive bands, presumably representing JNK1 and JNK2, were detected by the JNK-specific antibodies. Treatment of N/OFQ induced the activation of JNK in a PTX-sensitive manner (Fig. 4A). Densitometric quantification revealed a doubling of the JNK phosphorylation (JNK1 and JNK2) upon agonist treatment. Similar to the results obtained in the kinase assay of transfected COS-7 cells (Fig. 1A), the ORL₁-mediated JNK activation in NG108-15 cells was also associated with a PTX-insensitive component (Fig. 4A). The N/OFQ-induced kinase activity is insensitive to wortmannin (Fig. 4B). Interestingly, the basal JNK activity was enhanced upon wortmannin pretreatment (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Stimulation of endogenous ORL1 receptor in NG108-15 cells resulted in enhanced JNK activity in a PTX-sensitive but wortmannin-insensitive manner. NG108-15 cells were serum starved for 18 h in the absence or presence of PTX (100 ng/ml) before stimulation with 100 nM N/OFQ for 30 min (A), or accompanied by an additional wortmannin pretreatment (100 nM, 15 min) before the application of N/OFQ (B). Phosphorylated JNKs were detected by phospho-specific antibodies against JNKs and quantified by densitometry. Values shown represent the mean ± S.E. from three separate experiments. *, N/OFQ induced a significant increase of JNK phosphorylation in the control, PTX-treated (A) and wortmannin-treated (B) cells (Bonferroni paired t test, P < .05). **, PTX significantly inhibited N/OFQ-induced JNK phosphorylation (A), whereas wortmannin treatment (B) enhanced the basal JNK phosphorylation (Bonferroni t test, P < .05).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   ORL1 receptor mediates JNK activation and the phosphorylation of c-Jun and ATF-2 in a time-dependent manner. Serum-starved NG108-15 cells were stimulated by 100 nM N/OFQ for different durations. Phosphorylated JNKs (A), and c-Jun and ATF-2 (B) were detected by phospho-specific antibodies, followed with subsequent quantification by densitometry. Values shown represent the mean ± S.E. from three separate experiments.

	Discussion

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The ORL₁ receptor bears high resemblance to the opioid receptors in terms of its signal transduction properties. Given that the opioid receptors regulate neural development and synaptic plasticity by modulating neuronal survival and translational control, it is important to establish whether the ORL₁ receptor can regulate similar events. The present study demonstrated that the functional coupling of ORL₁ receptor with PTX-sensitive G_i proteins was associated with an increased activity of JNK. Because free G-subunits are more effective regulators than G_i subunits for the stimulation of JNK activity (Coso et al., 1996; Yamauchi et al., 2000), the observed JNK activation might be mediated mainly through G-subunits released during the activation of G_i proteins by receptor stimulation. Indeed, the -subunits of G_o do not possess the ability to activate JNK (Yamauchi et al., 2000). Interestingly, PTX-insensitive G proteins such as G_z, G₁₂, G₁₄, and G₁₆ can also link the ORL₁ receptor to the activation of JNK in heterologous expression systems. Further analysis revealed that the N/OFQ-induced JNK activity was dependent on Ras and Rac but not on PI3K. N/OFQ-induced JNK signaling as well as the phosphorylation of c-Jun and ATF-2 were observed in NG108-15 cells that endogenously express the ORL₁ receptor.

The mechanism by which G protein-coupled receptors regulate JNK activity is rather complicated. It has been suggested that stimulation of JNK by G_i-coupled receptors is G dependent, and the G-induced JNK activation involves PI3K for signal transduction and is therefore wortmannin sensitive (Lopez-Ilasaca et al., 1998). In contrast, wortmannin-insensitive JNK activation mediated by G has also been described (Yamauchi et al., 1999). The G-mediated pathway probably involves nonreceptor tyrosine kinases, which act on specific guanine nucleotide exchange factors to activate low-molecular-weight GTPases such as Ras and Rac (Kiyono et al., 1999). A different perspective on the functional role of PI3K signaling is illustrated by the observations that Akt inhibits Rac-GTP binding, whereas wortmannin stimulates JNK activity (Kwon et al., 2000). The wortmannin insensitivity of our experimental results did not support the activating role of PI3K signaling on the ORL₁ receptor-mediated JNK activation. Elevation of basal JNK activity in wortmannin-treated NG108-15 cells, in fact, suggested the presence of a PI3K inhibitory pathway in these cells. The characteristics of communication between PI3K and G protein-coupled receptor-mediated JNK signaling pathway may differ from one receptor to another, and cell-type specific.

It is generally believed that selective activation of the JNK signaling cascade is associated with the Rho subfamily of GTPases, particularly with Rac and Cdc42 (Minden et al., 1995). MEKK1 is a ubiquitously expressed MAPK kinase kinase that binds Rac1 in a GTP-dependent manner, and both Rac1 and MEKK1 can activate the JNK pathway (Fanger et al., 1997). The substantial decrease of N/OFQ-induced JNK activation in the presence of a dominant-negative Rac indicated the Rac dependence of the ORL₁ receptor-mediated JNK activation. However, we also demonstrated that Ras might also contribute to the N/OFQ-induced JNK activation. For some G protein-coupled receptors the activation of ERK is mediated via G-subunits and is Ras dependent (Crespo et al., 1994). Direct interaction between Ras and MEKK1 may result in the latter being stimulated (Russell et al., 1995). It has recently been shown that EPS8, E3B1, and SOS-1 form a tri-complex with Rac-specific guanine nucleotide exchange factor activity, and probably transmit intracellular signals from Ras to Rac (Scita et al., 1999). Our results showed that coexpression of dominant-negative mutants of both Ras and Rac did not produce additive inhibitory effect on the N/OFQ-induced JNK activity. Thus, the ORL₁ receptor might transmit its activating signaling from Ras to Rac, and then through MEKK1 to stimulate JNK.

Inhibitory effects of ORL₁ receptor on adenylyl cyclases through PTX-sensitive G_i proteins are well established (Mollereau et al., 1994). However, previous reports have shown that ORL₁ receptor may also act through PTX-insensitive pathways to regulate the activity of adenylyl cyclase as well as phospholipase C (Chan et al., 1998; Yung et al., 1999). Because the N/OFQ-induced JNK activation could not be completely inhibited by PTX in both transfected COS-7 cells and NG108-15 cells, PTX-insensitive G proteins may actually participate in the ORL₁ receptor-mediated JNK activation. Many PTX-insensitive G proteins are characterized by differential tissue distribution, for example, G_z is mainly expressed in neuronal cells; G₁₄ is found in spleen, pancreatic islets (Zigman et al., 1994), kidney, and early myeloid cells (Nakamura et al., 1991); and G₁₆ is predominantly expressed in hematopoietic cells (Amatruda et al., 1991). However, G₁₂ is a ubiquitously expressed G protein, and a strong activator for the Rho-dependent biological activities, including differentiation and apoptosis (Berestetskaya et al., 1998). The finding of ORL₁ receptor expression in neuronal cells and lymphocytic cells implied that coexpression of the ORL₁ receptor with these PTX-insensitive G proteins is likely. Our results clearly demonstrated a PTX-insensitive component of the ORL₁ receptor-mediated JNK activation in NG108-15 cells that endogenously express G_z. Further studies are needed to explore the physiological relevance of the functional coupling between the ORL₁ receptor and PTX-insensitive G proteins.

As a subgroup of MAPK, JNK phosphorylates and activates the activator protein-1 transcription factor component; c-Jun, therefore induces activator protein-1 transcriptional activity (Van Dam et al., 1993). Other transcription factors such as ATF-2 and Elk-1 can also be activated by different isoforms of the JNK family (Gupta et al., 1996). Stimulation of ORL₁ receptor has been shown to activate both ERK and p38 MAPK (Zhang et al., 1999), and the activating effects of these two subtypes of MAPK on different transcriptional factors have been extensively studied (Price et al., 1996). The ability of the ORL₁ receptor to activate different subtypes of MAPK indicated the importance of N/OFQ signaling at the nuclear level. In fact, growth, differentiation, and even apoptotic events of neuronal cells are highly dependent on the activities of MAPK, and the effects of N/OFQ on neuronal differentiation have been proposed (Saito et al., 1997). We have recently demonstrated that chronic activation of ORL₁ receptor induces supersensitization of adenylyl cyclases (Chan and Wong, 1999). On the other hand, supersensitivity of dopamine receptor functions is tightly associated with the transcription of immediate-early genes (LaHoste et al., 1993). Hence, the capability of ORL₁ receptor to stimulate both MAPK and transcription factors may imply that the resulting transcriptional events of immediate-early genes (e.g., c-Jun and ATF-2) could be critically important for N/OFQ signaling. In this report, we demonstrated the stimulatory effect of ORL₁ receptor on JNK via PTX-sensitive and PTX-insensitive G proteins, and suggested the involvement of Ras and Rac as important intermediates in the signaling. The participation of c-Jun and ATF-2 in the ORL₁ receptor-mediated pathway was also proposed. Further studies on the co-operativity of MAPK subtypes in the ORL₁ receptor-mediated neuronal cell activity will provide us with a refined picture for the N/OFQ signaling, and reveal the inter-relationship between the N/OFQ-induced MAPK activities and the resulting physiological consequences.