Introduction

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The opioid receptor-like (ORL₁) receptor is a guanine nucleotide-binding regulatory protein (G protein)-coupled receptor whose complementary DNA sequence bears substantial homology to those of the opioid receptors (Mollereau et al., 1994). Despite its resemblance to opioid receptors, the ORL₁ receptor does not bind any of the known opioid ligands with high affinity and appears to regulate distinct physiological functions. The endogenous ligand for the ORL₁ receptor has been identified as a novel heptadecapeptide termed nociceptin (Meunier et al., 1995), or orphanin FQ (Reinscheid et al., 1995; henceforth referred to as nociceptin/OFQ). Nociceptin/OFQ has been shown to regulate nociception (Meunier et al., 1995) and may also be involved in numerous physiological processes as diverse as blood pressure (Champion et al., 1997) and feeding (Pomonis et al., 1996). The diverse function of the ORL₁ receptor must invariably be mediated via the heterotrimeric () signal-transducing G proteins. Like opioid receptors, the ORL₁ receptor uses the pertussis toxin (PTX)-sensitive G_i/G_o proteins to inhibit adenylyl cyclase (Mollereau et al., 1994) and to regulate the activity of ion channels (Connor et al., 1996; Mattes et al., 1996). The ORL₁ receptor can thus be considered as a multifunctional "G_i-coupled" receptor.

It has become increasingly apparent that multifunctional receptors possess the capacity to interact with G proteins belonging to more than one subfamily. Many G_i-coupled receptors can signal through G proteins other than the G_i/G_o proteins. The µ-opioid receptor, a close relative of the ORL₁ receptor, can interact with G proteins from both the G_i and G_q subfamily (Chan et al., 1995; Offermanns and Simon, 1995; Lee et al., 1998). Among the seven known G proteins that can interact with the µ-opioid receptor, at least two are PTX insensitive (G_z and G₁₆). Given that the ORL₁ receptor is closely related to the opioid receptor family, it may likewise activate PTX-insensitive G proteins to regulate disparate effector pathways. To gain further insights on the signaling capacity of the ORL₁ receptor, we screened for potential coupling of the receptor to a panel of PTX-insensitive G proteins.

A number of approaches have been developed to monitor receptor-G protein interactions. In heterologous expression systems, chimeric G subunits can be adopted to convert receptor-generated signals into detectable outputs. Coupling of type 3 somatostatin receptor to G₁₄ and G₁₆ (two members of the G_q subfamily of G proteins) was predicted and proven with the use of chimeric G_s/₁₄ and G_s/₁₆ constructs (Komatsuzaki et al., 1997). Another approach relies on the detection of the complex, which is concomitantly released upon receptor-induced activation of heterotrimeric G proteins. Numerous reports have demonstrated that G_i-coupled receptors can stimulate type 2 adenylyl cyclase (AC2) via the subunits released from G_i (Federman et al., 1992; Chan et al., 1995; Tsu et al., 1995a,b; Yung et al., 1995). Activation of AC2 by the subunits requires either the presence of GTP-bound G_s (Federman et al., 1992) or phosphorylation by protein kinase C (Tsu and Wong, 1996). Because receptor-induced activation of any G protein will unavoidably lead to the release of subunits, -mediated stimulation of AC2 is an ideal index for receptor-G protein interactions. Productive coupling between the ORL₁ receptor and its associated G proteins should therefore give rise to -mediated stimulation of AC2. In this report, we present evidence that the cloned human ORL₁ receptor may in fact interact with the PTX-insensitive G_z, G₁₂, G₁₄, and G₁₆. Our studies further show that G₁₄, like G₁₆, can be activated by the human ORL₁ receptor to stimulate phospholipase C (PLC).

	Materials and Methods

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Materials. The cDNA encoding the  subunit of G_L1 (the bovine homolog of G₁₄; henceforth referred to as G₁₄ for generality) was generously provided by Dr. T. Nukada (Tokyo Institute of Psychiatry). Other cDNAs were obtained as previously described (Wong et al., 1991, 1992). PTX was purchased from List Biological Laboratories (Campbell, CA). Human embryonic kidney 293 (ATTC CRL-1573) and COS-7 (ATCC CRL-1651) cells were obtained from the American Type Culture Collection (Rockville, MD). [³H]Adenine and [³H]myo-inositol were purchased from Amersham International (Buckinghamshire, UK) and DuPont NEN (Boston, MA), respectively. Nociceptin/OFQ was purchased from Research Biochemicals Inc. (Natick, MA). Plasmid purification columns were obtained from Qiagen (Hilden, Germany). Antisera against G_q/11 (3A-180) and G₁₄ (3A-195) were purchased from Gramsch Laboratories (Schwabhausen, Germany). Anti-G₁₆ polyclonal antibodies were from Calbiochem (San Diego, CA). Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) and all other chemicals were purchased from Sigma (St. Louis, MO).

Cell Culture and Transfection. Human embryonic kidney (HEK) 293 cells were maintained and transfected as reported previously (Wong et al., 1991). Briefly, cells were cultured in Eagle's minimum essential medium (MEM) containing 10% (v/v) fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin in 5% CO₂ at 37°C. Cells were seeded on 12-well plates at approximately 1 × 10⁵ cells/well. One day later, cells were transfected with medium containing the desired cDNAs along with 400 µg/ml DEAE-dextran and 0.1 mM chloroquine for up to 2 h at 37°C. The cells were then shocked with 10% (v/v) dimethyl sulfoxide in phospate-buffered saline (PBS) and returned to growth medium. COS-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. COS-7 cells were transfected as with HEK 293 cells except that the DEAE-dextran concentration was reduced to 250 µg/ml and the transfection time increased to 4 h.

cAMP Accumulation and Inositol Phosphates (IPs) Formation. The transfected HEK 293 cells were labeled 1 day later with [³H]adenine (1 µCi/ml) in MEM containing 1% (v/v) FCS. Where indicated, 100 ng/ml PTX was added simultaneously. After 16 to 20 h the cells were assayed for cAMP levels in response to various drugs as described previously (Wong et al., 1991). cAMP accumulations were determined in the presence of 1 mM 1-methyl-3-isobutylxanthine at 37°C for 30 min. Results are expressed as the ratios of [³H]cAMP to total [³H]ATP, [³H]ADP, and [³H]cAMP pools. Absolute values for cAMP accumulation varied between experiments, but variability within a given experiment was less than 10% in general.

Preparation of Plasma Membranes and Immunodetection of G Subunits. COS-7 cells were grown on 150-mm dishes to 70 to 80% confluence and transfected as described for 12-well plates with proper adjustments to the volumes and amounts of the reagents used. Transfected cells were harvested 48 h later in PBS (Ca⁺⁺ and Mg⁺⁺ free) containing 10 mM EDTA. Cells were resuspended in lysis buffer (50 mM Tris-HCl containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine-HCl, 1 mM EGTA, 5 mM MgCl₂, and 1 mM dithiothreitol, pH 7.4) and lysed by one cycle of freezing and thawing followed by 10 passages through a 27-gauge needle. After removal of nuclei by centrifugation, membranes were collected, washed, and resuspended in lysis buffer. Protein concentrations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). For each sample, 50 µg of membrane proteins was separated on a 12.5% polyacrylamide-SDS gel and electrophorectically transferred to polyvinylidene difluoride (PVDF) membranes. Localization of protein markers on the PVDF membrane was by Ponceau S staining. Antigen-antibody complexes were visualized by chemiluminescence using the enhanced chemiluminescence kit from Amersham International.

	Results

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Identification of G₁₄ as a Signal Transducer for ORL₁ Receptor. By adopting the AC2 system to monitor receptor-G protein interactions, we have successfully examined the G protein-coupling capacity of the formyl peptide (Tsu et al., 1995a), melatonin 1c (Yung et al., 1995), and µ-opioid (Chan et al., 1995) receptors. Although this approach is indirect, it has proven to be a convenient and reliable functional assay. We began by examining the ability of the ORL₁ receptor to stimulate AC2 via the endogenous G proteins in HEK 293 cells. HEK 293 cells were cotransfected with cDNAs encoding the ORL₁ receptor, AC2, and G_s-Q227L (a constitutively active mutant of G_s). Inclusion of G_s-Q227L provided the system with the precondition for AC2 to become responsive to subunits (Federman et al., 1992). Activation of the ORL₁ receptor by 100 nM nociceptin/OFQ stimulated the cAMP accumulation by 80 to 120% over basal values (Fig. 1). The nociceptin/OFQ-induced stimulatory response was apparently mediated by G_i-like proteins as the stimulation was blocked by PTX treatment (Fig. 1). When the G_s-Q227L cDNA was omitted from the transfection cocktail, AC2 became unresponsive to nociceptin/OFQ (data not shown). This result implied that the ORL₁ receptor could not activate G_s.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Activation of AC2 by the ORL1 receptor. HEK 293 cells were transiently cotransfected (DEAE-dextran method) with cDNAs encoding the ORL1 receptor (0.25 µg/ml), AC2 (0.25 µg/ml), and Gs-Q227L (0.05 µg/ml). Transfected cells were labeled with [3H]adenine (1 µCi/ml) in the absence or presence of PTX (100 ng/ml) and then assayed for cAMP accumulation in response to 100 nM nociceptin/OFQ. Data are mean ± S.E. (n = 3). *Nociceptin/OFQ significantly increased cAMP accumulation over basal; Bonferroni's t test, P < .05.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Coupling of the ORL1 receptor to PTX-insensitive G proteins. HEK 293 cells were transfected and labeled as described in the legend to Fig. 1 except with the inclusion of an additional cDNA (0.25 µg/ml): Gz, Gq, G11, G12, G13, G14, or G16. Cotransfected cells were labeled, treated with PTX, and assayed for cAMP accumulation in the presence of 100 nM nociceptin/OFQ. Data are mean ± S.E. (n = 4). Results are expressed as percent stimulation of cAMP formation in the presence of nociceptin/OFQ, compared with that measured in the absence of nociceptin/OFQ. The basal values expressed as the ratio (×103) of cAMP to total adenine nucleotides ranged from 3.13 ± 0.35 to 6.28 ± 0.71. *Nociceptin/OFQ significantly stimulated cAMP accumulation even after PTX treatment; Bonferroni's t test, P < .05.

ORL₁ Receptor Stimulates PLC via G₁₄. To confirm the putative coupling of G₁₄ to the ORL₁ receptor, we examined the ability of nociceptin/OFQ to stimulate PLC through G₁₄. As COS-7 cells generally support a more robust stimulation of PLC than HEK 293 cells, we used COS-7 cells for subsequent transfections. COS-7 cells were transiently transfected with the ORL₁ receptor cDNA at 0.25 µg/ml in the absence or presence of varying amounts of G₁₄ cDNA (Fig. 3A). In the absence of G₁₄, activation of the ORL₁ receptor by nociceptin/OFQ (100 nM) did not stimulate the activity of PLC. By increasing the amounts of G₁₄ cDNA used in the transfections, nociceptin/OFQ induced IP formation in a cDNA dose-dependent manner. Maximal stimulation produced a 3-fold increase in IP levels and was achieved with a G₁₄ cDNA concentration of approximately 0.25 µg/ml (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   G14-dependent stimulation of PLC by nociceptin/OFQ. A, COS-7 cells were cotransfected with the ORL1 receptor cDNA (0.25 µg/ml) and varying concentrations of cDNA encoding G14 (0.001-1 µg/ml). Transfected cells were labeled with [3H]myo-inositol for 16 to 24 h and assayed for IP production in the absence (basal) or presence of 100 nM nociceptin/OFQ. Maximal response corresponded to 0.25 µg/ml of G14 cDNA. 100 nM nociceptin/OFQ significantly stimulated the production of IP at G14 cDNA concentrations of 0.003 µg/ml; t test, P < .05. B, COS-7 cells were cotransfected with G14 and the ORL1 receptor cDNAs (0.25 µg/ml per construct). Transfected cells were assayed for IP production in the absence or presence of varying concentrations of nociceptin/OFQ (0.3 nM to 100 nM). The estimated EC50 was 5 nM and a maximal response was obtained at approximately 30 nM nociceptin/OFQ. The IP formation was significantly increased by nociceptin/OFQ at concentrations 3 nM; t test, P < .05. Data are mean ± S.E. (n = 3).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Immunodetection of G subunits. COS-7 cells were transiently cotransfected with cDNAs encoding the ORL1 receptor in the absence or presence of G14. Plasma membranes were prepared 48 h posttransfection. Fifty micrograms of membrane proteins was separated on a 12.5% polyacrylamide-SDS gel and electrophorectically transferred to PVDF membranes. Protein markers were localized by Ponceau S staining. The expressions of Gq/11, G14, and G16 were assessed by antisera 3A-180, 3A-195, and anti-G16, respectively. Two independent experiments with different batches of membrane proteins yielded similar results.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Agonist selectivity of ORL1-mediated stimulation of PLC. COS-7 cells were cotransfected as described in the legend to Fig. 3B and subsequently assayed for IP formation after exposure to various ligands including nociceptin/OFQ, U50,488, [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAGO), or [D-Pen2,5]enkephalin (DPDPE; each at 100 nM). Where indicated, 10 µM naloxone was added together with nociceptin/OFQ. Basal indicates IP production in the absence of any ligand. Data represent mean ± S.E. (n = 3). *Nociceptin/OFQ significantly increased the IP production over the basal response; Bonferroni's t test, P < .05.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   G14-mediated stimulation of PLC is resistant to PTX. COS-7 cells were cotransfected as described in the legend to Fig. 3B and subsequently labeled with [3H]myo-inositol in the absence or presence of PTX. IP formation was determined with or without 100 nM nociceptin/OFQ. Data represent mean ± S.E. (n = 3). *Nociceptin/OFQ significantly increased the IP production over the basal response; Bonferroni's t test, P < .05.

	Discussion

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As a recently discovered close relative of the opioid receptors, the ORL₁ receptor holds promise for new insights on the regulation and perception of noxious stimuli. It is important to understand the cellular actions of the ORL₁ receptor. The present study was primarily designed to address the G protein-coupling capability of the ORL₁ receptor, without the need to clearly define the downstream effector pathways. Our choice of the -stimulated AC2 readout system has previously been validated with other receptor classes (Chan et al., 1995; Tsu et al., 1995a,b; Yung et al., 1995). In the presence of PTX, activation of the ORL₁ receptor by nociceptin/OFQ led to stimulation of AC2 when the  subunit of the PTX-insensitive G_z, G₁₂, G₁₄, or G₁₆ was coexpressed with the receptor. None of these PTX-insensitive G proteins has ever been shown to interact with the ORL₁ receptor, although its coupling to G_z and G₁₆ can be predicted from similar studies on opioid receptors (Chan et al., 1995; Tsu et al., 1995b; Lee et al., 1998).

An increasing number of G_i-coupled receptors have now been shown to interact with a variety of PTX-insensitive G proteins. Examples include the coupling between alpha₂ adrenoceptor and G_q, C5a receptor and G₁₆, dopamine D₂ receptor and G_z (Wong et al., 1992), and type 3 somatostatin receptor and G₁₄ (Komatsuzaki et al., 1997). The present study shows that the ORL₁ receptor may possess the ability to use more than one PTX-insensitive G proteins for signal propagation. Because the  subunit of these G proteins lacks the cysteine residue required for ADP ribosylation by PTX, their associated signaling pathways are insensitive to PTX treatment. Although the known pathways regulated by nociceptin/OFQ are all PTX sensitive, detailed functional analysis of the ORL₁ receptor in different tissues may unveil its linkage to PTX-insensitive pathways. For example, the nociceptin/OFQ-induced inhibition of adenylyl cyclase in retinoic acid-differentiated SH-SY5Y neuroblastoma cells is only partially sensitive to PTX, suggesting possible coupling to the PTX-insensitive G_z (L. Yung and Y.H. Wong unpublished data). There is ample evidence of diverse coupling to G proteins in other receptor classes. At the most diversified end, the human thyrotropin receptor is capable of stimulating all four G protein subfamilies (Laugwitz et al., 1996). Such findings indicate that multiple signaling pathways are used by many G protein-coupled receptors with obscure functional implications.

Numerous G protein-coupled receptors exhibit high specificity for coupling to G subunits within the same G protein subfamily. The beta₂ adrenergic receptor does not regulate PLC via G_q and G₁₄ but it can do so through G₁₆ (Wu et al., 1995a). Likewise, type 3 somatostatin receptor selectively activates G_i2, G₁₄, and G₁₆ but not other members of the G_i or G_q subfamily (Komatsuzaki et al., 1997). The ability of the ORL₁ receptor to differentially recognize G₁₄ and G₁₆ within the G_q subfamily is therefore not unique. In this regard, the ORL₁ receptor resembles the type 3 somatostatin receptor by not being able to interact with G_q and G₁₁. Interestingly, the somatostatin receptors and the opioid receptors share substantial homologies in their amino acid sequences (Mollereau et al., 1994). It is not clear whether the ORL₁ receptor can discriminate against individual members of the G_i subfamily. Its close relative, the µ-opioid receptor, has the capacity to interact with all members of the G_i subfamily with the exception of the transducins (Chan et al., 1995). The functional implication of G₁₄ coupling to the ORL₁ receptor has yet to be discovered. G₁₄ is detected in pancreatic islets (Zigman et al., 1994), taste tissue (McLaughlin et al., 1994), spleen, lung, kidney, uterus, testis, and bone marrow stromal cells, early myeloid cells, and progenitor B cells (Nakamura et al., 1991; Wilkie et al., 1991). Although the ORL₁ receptor is found abundantly in the central nervous system (Mollereau et al., 1994), it is also expressed peripherally in the intestine, vas deferens, liver, lymphocytes, lung, and spleen (Halford et al., 1995; Wick et al., 1995). Interestingly, the peripheral tissues and cells that express both G₁₄ and the ORL₁ receptor are components of the immune system. Nociceptin/OFQ may possess some immune functions that require coupling to G₁₄. Preliminary attempts to identify a suitable immune cell line as a model system to study the coupling of G₁₄ to the ORL₁ receptor were unfruitful (A.S.L. Chan and Y.H. Wong, unpublished data).

Among the various members of the G_q subfamily, G₁₄ has received the least attention. Apart from the type 3 somatostatin receptor (Komatsuzaki et al., 1997), several other receptors have been shown to interact with G₁₄. Transient overexpression of G₁₄ in rat aortic smooth muscle cells that express parathyroid hormone-related protein receptor allows parathyroid hormone-related protein to increase intracellular calcium and IP formation (Maeda et al., 1996). The G_s-coupled histamine H₂ receptor has also been found to interact with G₁₄ and other members of the G_q family (Bernhard et al., 1996). However, one of the more provocative roles of G₁₄ is its possible involvement in mediating inhibition of phosphoinositide metabolism (Nakamura et al., 1994). In frog oocytes, the expressed metabotropic glutamate receptor subtype 1 (mGluR1) can either stimulate or inhibit PLC, depending on whether it is coupled to G₁₁ or G₁₄, respectively (Nakamura et al., 1994). The mechanism by which G₁₄ inhibits phosphoinositide metabolism has not been established. Interestingly, both in terms of coupling to the muscarinic m1 receptor and activation of PLC in reconstituted lipid vesicles, G₁₄ is equivalent to G_q and G₁₁ (Nakamura et al., 1995). Contrary to the reconstitution studies, the muscarinic m1 receptor does not appear to use G₁₄ for calcium mobilization in the rat basophilic leukemia cell line RBL-2H3 (Dippel et al., 1996). This may suggest a more complex picture of the specificity of coupling between receptors and G proteins. Different factors present in different cell types may be involved in determining the outcome of coupling. If the ORL₁ receptor can indeed use G₁₄ to stimulate PLC, nociceptin/OFQ should ultimately lead to the activation of protein kinase C. Nociceptin/OFQ-induced activation of protein kinase C has recently been reported, and such a response is believed to be mediated via a PLC pathway (Lou et al., 1997).

Our dose-response study shows that the EC₅₀ value for G₁₄-mediated stimulation of PLC by nociceptin/OFQ was about 5 nM, which is higher than its reported EC₅₀ values for inhibition of forskolin-stimulated cAMP accumulation (Reinscheid et al., 1995). The significance of this difference is presently unclear. Mechanistically, differences in the EC₅₀ values imply that activation of ORL₁ receptor will lead to inhibition of adenylyl cyclase first, before stimulation of PLC occurs. The EC₅₀ values for G₁₄-mediated PLC activation by other receptors are comparable with our results. Agonist-induced activation of the MCP-1Rb and alpha_1B adrenergic receptors produced half-maximal stimulation of PLC at 7 nM and approximately 90 nM, respectively (Wu et al., 1995b; Kuang et al., 1996), in cells coexpressing G₁₄. Our results show that nociceptin/OFQ is relatively potent in stimulating PLC activity via G₁₄. The ORL₁ receptor may be an excellent candidate for studying the molecular details of interactions between receptors and G₁₄.

The potential interplay between the ORL₁ receptor and G₁₂ deserves further comment. G₁₂ has been shown to stimulate the Jun kinase/stress-activated protein kinase pathway (Vara Prasad et al., 1995). Additionally, G₁₂ has been demonstrated to stimulate Rho-dependent stress fiber formation, inhibit the ubiquitously expressed Na⁺/H⁺ exchanger, and activate Ras. These studies strongly implicate G₁₂ in the control of cell growth and differentiation. Indeed, expression of constitutively activated G₁₂ in NIH-3T3 (Xu et al., 1993) and Rat-1 (Voyno-Yasenetskaya et al., 1994) fibroblasts leads to neoplastic transformation. The present study suggests that the ORL₁ receptor can activate G₁₂ and, if so, nociceptin/OFQ may regulate cellular proliferation in cells coexpressing the ORL₁ receptor and G₁₂. This hypothesis is being actively pursued in our laboratories, especially when nociceptin/OFQ has recently been shown to stimulate the mitogen-activated protein kinase in Chinese hamster ovary cells (Lou et al., 1998).

In summary, we have shown that the ORL₁ receptor possesses the ability to interact with multiple G proteins belonging to three distinct subfamilies (the G_i, G_q, and G₁₂ subfamilies). Our data show that G₁₄ can interact with the ORL₁ receptor leading to activation of PLC. The G₁₄-mediated stimulation of PLC is agonist dose dependent and PTX insensitive. Given the restricted distribution of G₁₄, its coupling to the ORL₁ receptor should be explored further, especially when considering the possible role of nociceptin/OFQ in the modulation of immune responses.