Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

An action of opioids is to inhibit the modulation of synaptic transmission in both the central and peripheral nervous systems. Opioids induce their biological actions through association with three classes of receptors: delta, kappa and mu. Each of these receptors has recently been cloned and is a member of the seven-transmembrane spanning superfamily of G protein-coupled receptors (Chen et al., 1993; Evans et al., 1992; Kieffer et al., 1992; Yasuda et al., 1993). The cloned opioid receptors are ~65% identical in amino acid sequence and are most closely related to somatostatin receptors (Reisine and Bell, 1993). In fact, the mouse delta opioid receptor was isolated from a cDNA library using probes selective for the transmembrane spanning regions of the previously cloned somatostatin receptors (Yasuda et al., 1993).

At the cellular level, opioid receptors modulate various effector systems. All three subtypes of opioid receptors inhibit adenylyl cyclase (Attali et al., 1989; Chen et al., 1993; Frey and Kebabian, 1984; Sharma et al., 1975; Yasuda et al., 1993) and Ca⁺⁺ conductance (Gross and MacDonald, 1987; Hescheler et al., 1987; Schroeder et al., 1991) while stimulating K⁺ conductance (Chen and Yu, 1994; North, 1993; Wimpey and Chavkin, 1991), and Na⁺/H⁺ exchange (Isom et al., 1987). Opioid receptors are coupled to these effector systems by G proteins. G proteins are heterotrimeric complexes consisting of alpha, beta and gamma subunits. Many subtypes of alpha, beta and gamma subunits have been cloned, and the myriad of possible combinations may provide the basis for the divergent cellular actions of neurotransmitters and hormones (Simon et al., 1991).

Most of the cellular effects of delta opioid receptors are reported to be blocked by prior treatment with PTX. PTX ADP-ribosylates G_i/G_o (G_i1, G_i2, G_i3, G_o1 and G_o2) subtypes and effectively disrupts their activation by receptors. This result implies that any or all of the G_i/G_o subtypes might be mediating cellular actions of delta opioid receptors. The coupling of G subunits with delta opioid receptors has been examined by many methods with varying results. In a reconstituted system, delta opioid receptors were first shown to be linked by G_o to the inhibition of Ca⁺⁺ conductance (Hescheler et al., 1987). More recently, in NG108-15 cells, an approach using PTX-insensitive mutants indicates that G_o1 couples the delta opioid receptor to the inhibition of Ca⁺⁺ conductance (Taussig et al., 1992). In contrast, other studies in NG108-15 cells that measure cholera toxin-induced ADP-ribosylation conclude that G_o2 associates with delta opioid receptors in these cells (Roerig et al., 1992). To study delta receptor/G_i interactions, antisera have been used to uncouple the signaling pathway between delta opioid receptors and adenylyl cyclase. This approach in NG108-15 cells indicates that G_i2-directed antiserum disrupts delta agonist-stimulated GTPase activity and inhibition of adenylyl cyclase activity (McKenzie and Milligan, 1990). The same technique in SH-SY5Y cells reveals that both G_i and G_o directed antisera uncouple delta opioid receptor signaling to adenylyl cyclase (Carter and Medzhradsky, 1993). Furthermore, labeling techniques including ³²P-azidoanilide show many different combinations of G_i/G_o subtypes coupling with delta opioid receptors (Laugwitz et al., 1993; Offermans et al., 1991; Roerig et al., 1992). These varying results may be due to the fact that different cell lines and techniques are used in the measurement of delta opioid receptor/G associations.

For somatostatin receptors, which are most closely related to opioid receptors, association with G proteins has been investigated by an immunoprecipitation approach (Law et al., 1991, 1993; Law and Reisine, 1992). These studies indicate that rat brain and AtT-20 cell somatostatin receptors associate with G_i1, G_i3 and G_o and the cloned somatostatin receptor SSTR2A associates with G_i3 and G_o. This correlates with the functional analyses, which have shown that G_i1 couples somatostatin receptors to adenylyl cyclase (Tallent and Reisine, 1992), G_o2 couples the receptor to Ca⁺⁺ channels (Kleuss et al., 1991; Taussig et al., 1992) and G_i3 couples the receptor to K⁺ channels (Yatani et al., 1987). Therefore, the coupling of distinct G protein subunits with somatostatin receptors may determine which signaling pathways can be activated.

Investigations into the coupling of delta opioid receptors with G proteins have yielded many contradictory results. With the cloning of the delta opioid receptor, the ability to study its association with various G protein subunits in isolation from other opioid receptors may allow insight into the signaling process from delta opioid receptors to effector systems. To determine which G protein subunits associate with the cloned delta opioid receptor, a similar approach was used as previously described for somatostatin receptor/G protein coupling (Law et al., 1991, 1993; Law and Reisine, 1992). Our results show that delta opioid receptors associate with G_i1, G_i3, G_o, G₁ and G₂ subunits. On binding of agonist to the receptor, the G protein association changes so that a greater proportion of the receptor associates with G_i than with G_o, much less G_i1 is coupled to the receptor and the receptor forms a new association with G_i2 and remains coupled to G_i3, G₁ and G₂. Furthermore, a component of the high-affinity agonist binding to the delta opioid receptor was found to be PTX insensitive but GTPS sensitive. It was then discovered that the delta opioid receptor associates with G_z and G_q in both the absence and presence of agonist. Our findings for the first time reveal alterations in delta receptor/G protein coupling that occur as a result of agonist binding and, most importantly, show interactions of the receptor with PTX-insensitive G proteins. These dynamic changes in delta receptor/G protein association may be the underlying basis of activation for the delta opioid receptor signal transduction pathway.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Stable cell line. The mouse delta opioid receptor has been stably expressed in CHO-DG44 variant cell line (Yasuda et al., 1993). The pharmacological characteristics of the cloned delta opioid receptor have been studied in detail (Raynor et al., 1994) and are similar to the delta receptor endogenously expressed in NG108 cells, a cell line from which the receptor was cloned. The density of delta receptors in CHO cells is 1251 fmol/mg of protein (Raynor et al., 1994).

Western blotting and antisera. Conditions for gel electrophoresis and the transfer of proteins to nitrocellulose membranes to analyze G proteins have been previously described (Carlson et al., 1989). The primary antibodies bound to proteins on the nitrocellulose membranes were detected with the Protoblot alkaline phosphatase kit (Promega, Madison, WI). All antisera were used at a dilution of 1:250 for Western blotting.

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Solubilization of delta opioid receptors. In a typical experiment, ~10 million CHO cells stably expressing the delta opioid receptor grown to confluency would yield sufficient solubilized receptor for one immunoprecipitation sample (e.g., either the nonimmune serum or one G protein-directed antiserum immunoprecipitation). The delta receptor/G protein complexes from CHO cells were solubilized by removing media, washing the plated cells with 50 mM Tris · HCl (pH 7.8) and then scraping the cells from the flasks. The cells were centrifuged at 24,000 × g for 7 min at 4°C, and the supernatant was discarded. To the pelleted cells, 2 ml of buffer was added containing 50 mM Tris · HCl (pH 7.8), 1 mM EGTA, 5 mM MgCl₂, 10 µg of leupeptin, 2 µg of pepstatin and 200 µg of bacitracin (buffer A). For these studies, buffer A also contained 4 µl of 50 mM phenylmethylsulfonyl fluoride in ethanol. The sample was homogenized with a Brinkmann Polytron and centrifuged at 120 × g for 10 min at 4°C. The resulting supernatant was removed and centrifuged at 45,000 × g for 15 min at 4°C. The supernatant was subsequently discarded, and 1 ml of buffer A was added to the membrane pellet, which was then homogenized as before. Next, the cell membranes were incubated with the delta receptor-selective agonist 1 µM DPDPE or buffer A for 30 min at 30°C and then chilled for 10 min at 4°C. Then, 250 µl of solubilization buffer (50 mM CHAPS and 50% glycerol) was added to the samples, and the samples were placed on ice with constant stirring for 30 min. The sample was then centrifuged at 100,000 × g for 60 min at 4°C. The supernatant was diluted 1:3 in buffer A with 7.5% glycerol and 0.5 µg/ml aprotinin and concentrated to a final volume of 1 ml. The 1-ml sample was loaded onto a Sephadex G-50 column (0.7 × 15 cm, BioRad, Melville, NY) and run in the following buffer: 50 mM Tris (pH 7.8), 1 mM EGTA, 5 mM MgCl₂, 5% glycerol and 2 mM CHAPS. Eluted fractions were assayed for specific binding to the opioid receptor agonist ¹²⁵I--endorphin. Fractions containing such activity were pooled, concentrated with Centricon 30 (Amicon) ultrafiltration devices and placed in the immunoprecipitation assay. The recovery of delta receptors after solubilization was ~10% compared with the amount of receptor present in the starting membrane preparation.

Immunoprecipitation of delta opioid receptor/G protein complexes. Solubilized delta opioid receptors were incubated with G protein-specific antisera (table 1) at a final dilution of 1:20. The amount of antisera used was similar to that used in previous studies of somatostatin and alpha-2 adrenergic receptors (Law et al., 1991, 1993; Law and Reisine, 1992; Okuma and Reisine, 1992) and was optimal for the immunoprecipitation of delta opioid receptor/G protein complexes. Higher concentrations of antisera had no further effect. A 1:12 dilution of 50% (w/v) protein A-Sepharose beads (CL-4B, Sigma Chemical, St. Louis, MO), washed three times and diluted in buffer A, was also added. The samples were then placed on a rotator at 4°C and incubated overnight. Following this, the samples were centrifuged at 10,000 rpm for 4 minutes in an Ependorf microcentrifuge. The supernatant was removed, and the presence of solubilized delta opioid receptors was detected in this portion of the sample using the ¹²⁵I--endorphin binding assay. The immunoprecipitate was washed in buffer A and centrifuged. The supernatant was then discarded, the immunoprecipitate was resuspended in buffer A and the presence of delta opioid receptors detected using the ¹²⁵I--endorphin binding assay.

¹²⁵I--Endorphin binding assay. The presence of solubilized delta opioid receptors was detected with the high-affinity agonist ¹²⁵I--endorphin (specific activity, 2200 Ci/mmol; Amersham, Arlington Heights, IL). Solubilized delta opioid receptors were incubated with 25 pM ¹²⁵I--endorphin in a total volume of 0.3 ml of buffer A. Nonspecific binding of ¹²⁵I--endorphin was determined as the amount of binding remaining in the presence of 1 µM -endorphin or DSLET and accounted for <20% of the total ¹²⁵I--endorphin binding. The binding reaction was carried out at 25°C for 60 min. Under these conditions, the binding reaction reached equilibrium. The binding reaction was terminated by the addition of 9 ml (three consecutive additions of 3 ml) of 50 mM Tris · HCl (pH 7.8) at 4°C. The samples were vacuum filtered over Whatman (GF/F) glass-fiber filters that had been presoaked in 0.5% polyethylenimine at 4°C. The filters were dried, and radioactivity was measured in a gamma counter (80% efficiency). A similar procedure was used to detect immunoprecipitated delta opioid receptors. In these studies, after immunoprecipitation of delta opioid receptor/G protein complexes with antisera directed against different G protein subunits, the immunoprecipitate was resuspended in an appropriate volume with buffer A, and the presence of delta opioid receptors was detected using the ¹²⁵I--endorphin binding assay.

PTX treatment. CHO cells stably expressing the delta opioid receptor were treated overnight with media containing 100 ng/ml PTX (List Biologicals, Campbell, CA). Membranes were then prepared and the membrane binding assay was performed as previously described (Raynor et al., 1994). The CHO cell membranes were incubated with 25 pM ¹²⁵I--endorphin in a total volume of 0.3 ml of buffer A. Nonspecific binding of ¹²⁵I--endorphin was determined as the amount of binding remaining in the presence of 1 µM -endorphin or DSLET and accounted for <20% of the total ¹²⁵I--endorphin binding. The binding reaction was carried out at 25°C for 60 min, when equilibrium was accomplished. The binding assay was terminated by rapid vacuum filtration, and the filters were washed with 12 ml of a Tris · HCl buffer (pH 7.8) and counted in a gamma counter (80% efficiency).

Data analysis programs. Inhibition curves were analyzed using the National Institutes of Health computer-based PROPHET program as previously described (Raynor et al., 1994). Immunoprecipitation data were analyzed by the statistical program Number Cruncher Statistical Systems, Version 501 (Kaysville, UT).

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

The cloned mouse delta opioid receptor expressed in CHO (DG44 variant) cells has pharmacological properties similar to those of the delta-2 opioid receptor endogenously expressed in brain and NG108 cells (Raynor et al., 1994). After solubilization of the receptor from CHO cells with the nonionic detergent CHAPS, the receptor was labeled with ¹²⁵I--endorphin. Specific binding was inhibited by the opioid agonists -endorphin and the delta receptor-selective agonist DPDPE as well as the antagonists diprenorphine and naloxone (fig. 1). The specific binding was not affected by dextrorphan.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Pharmacological characterization of the solubilized delta opioid receptor. The specific binding of 125I--endorphin to the solubilized delta opioid receptor was inhibited by varying concentrations of -endorphin (), DPDPE (), naloxone (), diprenorphine () or dextrorphan (). Data are mean results of three different experiments.

The solubilized delta opioid receptor sample was examined by Western blot analysis to determine its G protein complement. All of the G_i and G subtypes as well as G_o, G_q and G_z were present (fig. 2). Thus, any or all of these subunits could potentially couple the delta opioid receptor to various cellular effector systems.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   G proteins present in CHO (DG44 variant) cells expressing the cloned delta opioid receptor. Western blotting was preformed on CHO cells stably expressing the cloned delta opioid receptor to detect the G subunits Gi (antiserum 8730), Go (antiserum 9072), Gq (antiserum 946) and Gz (antiserum 2919) (A); the Gi subtypes Gi1 (antiserum 3646), Gi2 (antiserum 1521) and Gi3 (antiserum 1518) (B); and the G subunits G1 (antiserum 8132), G2 (antiserum 8129) and G1/2 (antiserum 8136 detects both G1 and G2).

Delta receptor/G interactions. Peptide directed antisera against G_o and G_i were used to investigate delta receptor/G associations. Antiserum 8730, which is directed against the carboxyl terminus of G_i and selectively detects G_i1, G_i2 and G_i3, does not immunoprecipitate or uncouple delta receptor/G_i complexes (fig. 3, A and B). However, 8730 is very effective in immunoprecipitating G_i (Law and Reisine, 1992). This indicates that either G_i is not associated with the delta opioid receptor or the 8730 epitope on G_i is inaccessible. In contrast, antiserum 9072, which is directed against the carboxyl terminus of G_o, uncouples delta receptor/G_o complexes (fig. 3, A and B). This is demonstrated by a loss of binding sites in the supernatant but no immunoprecipitation of these sites. A similar result was seen previously as antiserum 9072 uncoupled the cloned somatostatin receptor SSTR2 from G_o (Law et al., 1993). The differences seen with the two carboxyl-terminally directed antisera, the uncoupling by antiserum 9072 and the inability of antiserum 8730 to have an effect may indicate that the delta receptor contact sites are different for G_o vs. G_i. Antiserum 2918, which is directed against an internal sequence of G_o, immunoprecipitates delta receptor/G_o complexes (fig. 3, A and B). This is indicated by the appearance of specific ¹²⁵I--endorphin binding sites in the 2918 immunoprecipitate and the corresponding loss of delta receptor binding sites in the supernatant. The data obtained with the G_o-directed antisera show that the cloned delta opioid receptor associates with G_o.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   The association of Gi and Go with the cloned delta opioid receptor. Solubilized delta opioid receptor/G protein complexes were immunoprecipitated with various G protein antisera. Delta opioid receptors in the immunoprecipitate or supernatant were detected with 125I--endorphin, and the resulting specific binding was compared with nonimmune serum. Data are the mean ± S.E.M. specific cpm bound in seven different experiments. A, Solubilized delta opioid receptor/G protein complexes were immunoprecipitated with Gi (8730) and Go (9072 and 2918)-directed antiserum or nonimmune serum (NI). Open bars, specific binding of 125I--endorphin in the immunoprecipitates. B, Hatched bars, delta receptor/G protein complexes detected by 125I--endorphin in the supernatants from A. C, Solubilized delta opioid receptor/G protein complexes were immunoprecipitated with antisera against Gi1 (3646), Gi2 (1521) or Gi3 (1518) and nonimmune serum (NI). Open bars, specific binding of 125I--endorphin in the immunoprecipitates. D, Hatched bars, delta receptor/G protein complexes detected by 125I--endorphin in the supernatants from C.

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Effect of agonist treatment on delta receptor/G coupling. The effect of agonist on delta receptor/G interactions was examined by incubating the receptor before solubilization with the delta-specific agonist DPDPE for 30 min. It should be noted that the levels of ¹²⁵I--endorphin binding to delta receptors in the nonimmune controls varied little between the untreated and agonist-treated samples. Thus, similar levels of delta receptor binding were added in the immunoprecipitation procedure. Furthermore, residual free agonist was removed through column chromatography and was not present in the sample during the binding assay because fractions eluted from the gel filtration column were detected by their ability to bind the agonist ¹²⁵I--endorphin, which could not have labeled the solubilized receptor if the receptor were still bound to DPDPE. In the presence of agonist, antiserum 8730 is able to coimmunoprecipitate the delta opioid receptor (fig. 4, A and B), which is in contrast to its inability to immunoprecipitate the agonist free receptor (fig. 3, A and B). Antisera 2918 and 9072 are able to coimmunoprecipitate and uncouple, respectively, the agonist-bound delta receptor from G_o (fig. 4, A and B), indicating that G_o remains coupled to the delta receptor after the binding of DPDPE. However, the relative levels of delta receptor able to associate with G_o and G_i changed after agonist binding to the receptor so that a similar proportion of the receptor associated with G_i and G_o (compare figs. 3 and 4). Agonist binding to the delta receptor may induce conformational changes in the delta receptor and/or G_i so that the carboxyl terminus of G_i is no longer occluded and is freely accessible to antiserum 8730, which can then coimmunoprecipitate delta receptor/G_i complexes and may also facilitate receptor/G_i interactions.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Interaction of the agonist DPDPE with the delta receptor changes delta receptor/Gi associations. Delta opioid receptor/G protein complexes treated with the delta receptor agonist DPDPE and then solubilized were immunoprecipitated with various G protein antisera. Delta opioid receptors in the immunoprecipitate or supernatant were detected with 125I--endorphin, and the resulting specific binding was compared with nonimmune serum. Data are the mean ± S.E.M. specific cpm bound in at least four different experiments. A, Delta receptor/G protein complexes were immunoprecipitated with antisera against Gi (8730), Go (9072 or 2918) and the nonimmune serum (NI). Open bars, specific binding in the immunoprecipitate. B, Hatched bars, specific binding in the supernatants from A. C, Delta receptor/G protein complexes were immunoprecipitated with antisera against Gi1 (3646), Gi2 (1521) or Gi3 (1518) and nonimmune serum (NI). D, Hatched bars, specific binding in the supernatants from C.

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Delta receptor/G interactions. In addition to G_i1, G_i3 and G_o, G₁ and G₂ form stable complexes with the delta receptor. Antisera 8132, 8129 and 8136, which are directed against G₁, G₂ and G_common, respectively, coimmunoprecipitate the delta receptor (fig. 5, A and B). Immunoprecipitation of delta receptor/G₁ complexes by antiserum 8132 could be blocked by the peptide to which the antiserum was generated with no effect seen by the peptide alone. However, analysis of the supernatant binding results show that antiserum 8132 potentiates binding above the nonimmune control level (fig. 5, A and B). The potentiation by antiserum 8132 was blocked to the level of the nonimmune control when the antiserum was incubated with 8132 peptide. This probably represents a full blockade because the 8132 peptide completely blocks the ability of antiserum 8132 to immunoprecipitate delta receptor/G complexes. The potentiation by 8132 may be due to the ability of the antiserum to alter G conformation, thereby promoting the delta receptor high-affinity state. The 8132 peptide itself could significantly decrease delta receptor binding in the supernatant (fig. 5, A and B). It is possible that this peptide contains the receptor/G recognition domain and competes with other G subunits for these sites, thus causing a decrease in specific binding.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   The association of G subunits with solubilized delta opioid receptors. Delta opioid receptor/G protein complexes treated with or without the delta receptor agonist DPDPE, solubilized and then immunoprecipitated with various G protein antisera. Delta opioid receptors in the immunoprecipitate or supernatant were detected with 125I--endorphin, and the resulting specific binding was compared with nonimmune serum controls. Data are the mean ± S.E.M. specific cpm bound in five different experiments. A, In the absence of agonist, delta receptor/G protein complexes were immunoprecipitated with antisera against G1 (8132), G2 (8129) and an antiserum against both G subunits (8136) or nonimmune serum (NI). Open bars, specific binding in the immunoprecipitate. B, Hatched bars, specific binding in the supernatants from A. C, In the presence of the delta opioid agonist DPDPE, delta receptor/G protein complexes were immunoprecipitated with the antisera listed in A. Open bars, specific binding in the immunoprecipitate. D, Hatched bars, specific binding in the supernatants from C. 8132p is CEGNVRSRELAGHTGY.

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Delta receptor association with PTX-insensitive G proteins. The possibility that PTX-insensitive G proteins associate with delta opioid receptors was investigated with PTX and the nonhydrolyzable GTP analog GTPS. As figure 6 indicates, there is a portion of specific high-affinity agonist binding to delta opioid receptors that is unaffected by PTX treatment but is sensitive to GTPS. Higher concentrations of PTX did not further reduce ¹²⁵I--endorphin binding, and a similar concentration of PTX abolished agonist binding to the cloned somatostatin receptor SSTR2. PTX reduced ¹²⁵I--endorphin binding to approximately half that reduced by GTPS. These results suggest that delta opioid receptors may associate with PTX-insensitive G proteins. To examine this possibility, the immunoprecipitation approach was used to determine whether two of the PTX-insensitive G proteins, G_z and G_q, couple with delta opioid receptors. In the absence of agonist, antiserum 2919 directed against G_z immunoprecipitates delta receptor/G protein complexes, whereas antiserum 946 directed against G_q uncouples these complexes (fig. 7, A and B). Therefore, both of the PTX-insensitive G proteins tested appear to associate with delta opioid receptors. In the presence of the opioid agonist DPDPE, the interaction between the delta receptor and G_z remains unchanged, whereas the association of G_q is altered so that antiserum 946 is able to immunoprecipitate instead of uncouple the delta receptor/G protein complexes (fig. 7, C and D). This is the first report that delta opioid receptors physically associate with PTX-insensitive G proteins and that there are differences in these associations depending on the presence of agonist.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   The effect of GTPS and PTX pretreatment on the specific binding of 125I--endorphin to the cloned delta opioid receptor. CHO cells (DG44 variant) expressing either the cloned delta opioid receptor (open bars) or the cloned somatostatin receptor SSTR2 (hatched bars) (S) were either pretreated overnight with PTX or with medium alone. Delta receptors were detected by the binding of the agonist 125I--endorphin. SSTR2 was detected with the somatostatin agonist 125I-MK678 (specific binding defined with 1 µM SRIF). Binding to SSTR2 was used as a control to establish the effectiveness of the PTX treatment that abolished specific 125I MK678 binding. Specific binding of the radioligands to membranes from control cells or cells pretreated with PTX was measured in either the presence or absence of 10 µM GTPS. Data are the mean ± S.E.M. of three different experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   The association of Gz and Gq with the cloned delta opioid receptor. Delta opioid receptor/G protein complexes treated with or without the delta receptor agonist DPDPE and then solubilized were immunoprecipitated with various G protein antisera. Delta opioid receptors in the immunoprecipitate or supernatant were detected with 125I--endorphin, and the resulting specific binding was compared with nonimmune serum. Data are the mean ± S.E.M. specific cpm bound in four different experiments. A, In the absence of agonist, delta receptor/G protein complexes were immunoprecipitated with antisera against Gz (2919), Gq (946) or nonimmune serum (NI). Open bars, specific binding in the immunoprecipitate. B, Hatched bars, specific binding in the supernatants from A. C, In the presence of the delta opioid agonist DPDPE, delta receptor/G protein complexes were immunoprecipitated with the antisera listed in A. Open bars, specific binding in the immunoprecipitate. D, Hatched bars, specific binding in the supernatants from C.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Delta opioid receptors mediate the physiological actions of endogenous opioid neurotransmitters by coupling to various effector systems, such as adenylyl cyclase (Chen et al., 1993; Evans et al., 1992; Kieffer et al., 1992; Sharma et al., 1975; Yasuda et al., 1993), Ca⁺⁺ and K⁺ channels (Gross and MacDonald, 1987; Hescheler et al., 1987; North, 1993; Wimpey and Chavkin, 1991) and the Na⁺/H⁺ exchanger (Isom et al., 1987). G proteins link delta opioid receptors to these effector systems, and G proteins may determine which effector systems are activated.

In this study, the physical interactions between delta opioid receptors and G proteins were investigated. Both G_i and G_o associate with delta receptors, and changes in receptor/G protein association occur after agonist binding to the receptor. These changes may be involved in the activation of the delta receptor signal transduction pathway. An important new finding is that PTX-insensitive G proteins couple to the delta receptor. These interactions may be involved in mediating the effects of delta agonists on phospholipase C activation and changes in intracellular phosphoinositol turnover. In fact, Tsu et al. (1995) recently reported that G_z can couple the cloned delta receptor to adenylyl cyclase and phospholipase C.

In our studies, we used a CHO cell line transfected to express the cloned mouse delta receptor. The levels of receptor in these cells are higher than those found naturally in the body. However, it is unlikely that the high levels of receptor resulted in a distorted G protein association. Law et al. (1994a) reported that the cloned delta receptor expressed in CHO cells at 1.4 (comparable to our levels of 1.25 pmol), 0.7 and 0.27 pmol/mg of protein gave similar levels of DPDPE-induced inhibition of cAMP accumulation (83-61%), indicating that varying the densities of delta receptor did not significantly affect the interaction of the receptor with the G proteins involved in coupling the receptor to adenylyl cyclase. Furthermore, the agonist affinities for the cloned delta receptor in our CHO cells is comparable to those reported by Evans et al. (1992), Kieffer et al. (1992), Yasuda et al. (1993), Tsu et al. (1995) and Blake et al. (1995), who performed their studies in different cells and at different receptor densities. Because agonist affinities are dependent on G protein association, the similar agonist affinities in our cells and those of others suggest that the receptor/G protein associations we identified are not unusual because of the high receptor densities.

A carboxyl-terminally directed antiserum against G_i, 8730, did not immunoprecipitate delta receptor/G_i complexes in the absence of agonist. The inability of antiserum 8730 to immunoprecipitate receptor/G_i complexes in the absence of agonist was not due to a lack of G_i association with the receptor because antisera directed against internal regions of G_i1 and G_i3 were able to immunoprecipitate or uncouple the delta receptor/G_i complex. This suggests that the inability of antiserum 8730 to immunoprecipitate or uncouple the agonist free receptor/G_i complex is likely to be due to the lack of epitope accessibility. This is in contrast to the G_o antiserum 9072, which is directed against the same epitope as 8730 that is a domain of G_a that differs in amino acid sequence between G_i and G_o and uncoupled the agonist free delta receptor from G_o. These findings imply that differences exist in the coupling of the delta receptor with G_i and G_o.

The binding of agonist to the delta receptor permitted antiserum 8730 to coimmunoprecipitate the delta receptor. Agonist binding to the delta receptor may therefore increase the accessibility of the 8730 epitope in G_i. This alteration could be due to conformational changes in the carboxyl terminus of G_i, which result from agonist association with the delta opioid receptor. The carboxyl terminus of G_i subunits is thought to be important for association with receptors, and recent results with somatostatin receptors have shown the carboxyl terminus is essential for mediating somatostatin inhibition of adenylyl cyclase activity (Law et al., 1994b).

The physical association of G_o with the delta opioid receptor is consistent with results of Roerig et al. (1992), who showed in NG108 cells that delta agonists increase the ADP-ribosylation of G_o. Our results are also consistent with functional studies showing that this alpha subunit selectively couples delta receptors to Ca⁺⁺ channels (Hescheler et al., 1987; Taussig et al., 1992). G_o was the predominant alpha subunit associated with the agonist-free delta receptor. Agonist binding to the receptor reduced the proportion of delta receptor coupled to G_o compared with G_i. This shift in association of the receptor with G_o to G_i may further reflect the dynamic changes that result from receptor activation.

In the absence of agonist, G_i1 was found to associate with the delta receptor. However, in the presence of agonist, much less association of G_i1 with the delta receptor was detected. These findings suggest two possibilities: either agonist binding to the receptor may promote the dissociation of G_i1 from the receptor, or the epitope for the G_i1-directed antiserum becomes inaccessible on agonist binding. The former possibility is more plausible given that the G_i1- and G_i2-directed antisera were generated against the same region of G, which differs in sequence, and that the G_i2-directed antiserum can immunoprecipitate receptor/G protein complexes in the presence of agonist. Thus, both G_i1 and G_i2 associate with delta opioid receptors. However, agonist binding to the receptors may cause G_i1 to dissociate and G_i2 to associate with the receptor. Both G_i1 and G_i2 have been proposed to couple delta receptors to adenylyl cyclase. The possible dissociation of G_i1 from the receptor after agonist binding might be expected to increase accessibility of G_i1 to effector systems. The coupling of G_i2 with the receptor might link the receptor to membrane-associated forms of adenylyl cyclase. Our findings are consistent with the results of previous studies showing a role for both G_i1 and G_i2 in delta receptor signaling and provide the first evidence for dynamic changes in delta receptor/G_i interaction induced by agonists.

Preliminary studies (Blake et al., 1995) have shown that the cloned delta receptor expressed in HEK 293 cells couples to adenylyl cyclase and mediates agonist inhibition of cAMP formation. These cells have been shown previously to express G_i1 and G_i3 immunoreactivity but not G_i2 or G_o immunoreactivity (Law et al., 1993), suggesting that G_i2 is not required for delta receptors to couple to adenylyl cyclase and that either G_i1 and/or G_i3 can couple the receptor to this enzyme. These studies do not exclude a role for G_i2 in coupling delta receptors to adenylyl cyclase. Behavioral studies (Sanchez-Blazquez and Garcon, 1995) have suggested that G_i2 is critical for mediating the analgesic effects of delta-selective agonists because a "knockdown" of G_i2 in mice using antisense blocked delta agonist-induced analgesia.

G_i3 was the only G_i subtype consistantly associated with both the agonist-free and -bound receptor. Agonist binding did increase the apparent stability of the delta receptor/G_i3 complexes because they were only coimmunoprecipitated in the presence of agonist. The ability of G_i3 to associate with the receptor and the lack of coupling of G_i1 with the agonist-bound delta receptor are of interest because these proteins are 94% identical in amino acid sequence, indicating that only a few residues are responsible for the major differences in physical association with the delta receptor. In contrast, the cloned somatostatin receptor SSTR3 was coupled to adenylyl cyclase by G_i1 but not G_i2 or G_i3 (Law et al., 1994b). Therefore, receptor-specific coupling to various G subunits may be due to the recognition by receptor contacts of divergent regions of these subunits.

Our results showing that delta receptors associate with G_i2 and G_i3 are consistent with those reported by Roerig et al. (1992) in NG108 cells, which showed that agonists can increase ADP-ribosylation of G_i2 and G_i3. Furthermore, McKenzie and Milligan (1990) reported that delta receptors in NG108 cells interact with G_i2 to transduce agonist inhibition of adenylyl cyclase activity. Unique to our findings is the association of the delta receptor with G_i1, a G protein that was not found to be expressed in NG108 cells by McKenzie and Milligan (1990). Because brain does express G_i1, our studies reveal for the first time the potential importance of this G protein in delta receptor signaling.

G subunits are important for the modulation of particular effector systems such as adenylyl cyclase (Tang and Gilman, 1991; Taussig et al., 1993). Thus, the determination of specific G coupling with receptors may yield clues regarding which of the increasing number of G-mediated effector functions might be activated by a particular receptor. Both G₁ and G₂ subunits formed stable complexes with the delta receptor. This multiplicity of G subunit association with the delta receptor differs from the findings with the somatostatin and alpha-2 adrenergic receptors, which primarily associate with G₁ (Law et al., 1991; Okuma and Reisine, 1992). The additional coupling of G₂ with the delta receptor may allow it to couple with a different set of effector systems not activated by somatostatin or alpha-2 adrenergic receptors. The potentiation of specific high-affinity delta receptor binding with the addition of the G₁-directed antiserum may indicate that this antiserum modulates various delta receptor/G protein contacts in the absence of agonist. However, on agonist binding, a more stable complex may be formed between the receptor and G_/ subunits that does not allow G₁-directed antiserum to modify the complex. Further study of this region involving site-directed mutagenesis would be useful because there is only one amino acid change in the G₁ vs. G₂ epitope domain.

The study of opioid signal transduction pathways has primarily focused on PTX-sensitive signaling mechanisms. However, we provide data indicating that delta opioid receptors physically associate with PTX-insensitive G proteins. Delta receptors in both the presence and absence of agonist associate with two of the PTX-insensitive G proteins, G_z and G_q. These two proteins in turn may lead to the activation of signaling systems such as phospholipase C. In fact, recent studies have shown that the cloned delta opioid receptor expressed in Xenopus oocytes can mediate agonist activation of phospholipase C (Miyamae et al., 1993). Furthermore, the delta receptor-selective agonist DPDPE has been reported to increase intracellular Ca⁺⁺ levels in NG108 cells, presumably via a phospholipase C mechanism (Connor et al., 1994). The cloned delta receptor coexpressed with G_z in 293 cells was reported to couple to adenylyl cyclase in a PTX-insensitive manner (Tsu et al., 1995). Furthermore, G_z reconstituted delta receptor coupling to phospholipase C in Ltk cells (Tsu et al., 1995). Therefore, G_z and possibility G_q may serve critical roles in delta receptor signaling.

We have shown that there are dynamic changes in G protein association with the delta opioid receptor after agonist binding and that delta receptors associate with PTX-insensitive as well as -sensitive G proteins. Thus, the specific association of particular G proteins with the delta opioid receptor may provide the diversity and direction for the various opioid-attributed signaling events.