Abstract
Using Xenopus laevis oocytes coexpressing mammalian μ-opioid receptors (MORs), β-adrenergic receptor kinase 2 (β-ARK2) [also called G protein-coupled receptor kinase (GRK3)], and β-arrestin 2 (β-arr 2), we compared the rates of β-ARK2 (GRK3)- and β-arr 2-mediated homologous receptor desensitization produced by treatment with opioid agonists of different efficacies. The response to MOR activation was measured using two-electrode voltage clamp as an increase in the conductance of the coexpressed G protein-coupled inwardly rectifying potassium (heteromultimer of KIR3.1 and KIR3.4) channels. Treatment with opioids of high efficacy, either [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin, fentanyl, or sufentanyl, produced a GRK3- and β-arr 2-dependent reduction in response in <20 min, whereas treatment with the partial agonist morphine produced receptor desensitization at a significantly slower rate. Because GRK3 requires activation and membrane targeting by free G protein βγ subunits released after agonist-mediated activation of G proteins, a low efficacy agonist such as morphine may produce weak receptor desensitization as a consequence of poor GRK3 activation. To address this hypothesis, we substituted GRK5, a GRK that does not require activation by G protein βγ. In oocytes expressing GRK5 instead of GRK3, both [d-Ala2,N-MePhe4,Gly-ol5]enkephalin and fentanyl, but not morphine, produced desensitization of MOR-activated potassium conductance. Thus, μ-opioid agonists produced significant receptor desensitization, mediated by either GRK3 or GRK5, at a rate dependent on agonist efficacy.
The processes underlying the clinically observed tolerance to opioid drugs are complex and may involve learning mechanisms, compensatory changes in neuronal circuits, and desensitization of signal transduction mechanisms (Nestler and Aghajanian, 1997; Ramsay and Woods, 1997). Studies both in vivo and in vitro have documented that the response to opioids desensitize after prolonged exposure to opioids (Cox, 1978). One of the important molecular events that has been shown to be involved in the desensitization of the response to opioids is opioid receptor desensitization. The molecular basis for opioid receptor desensitization was shown to be a reduction in opioid agonist efficacy (Chavkin and Goldstein, 1982, 1984; Porreca and Burks, 1983) that manifests as a reduction in the rate of G protein activation by the agonist-bound receptor complex. For example, continuous infusion of guinea pigs or rats with morphine results in an uncoupling of μ-opioid receptors from associated G proteins as measured biochemically (Werling et al., 1989; Tao et al., 1993), cytochemically (Sim et al., 1996), or electrophysiologically (Christie et al., 1987). The uncoupling of MOR from G proteins is likely to result from the agonist-dependent phosphorylation of MOR mediated by a GRK (Kovooret al. 1997). Because clinically useful opioids differ in their intrinsic efficacies, the rates of tolerance development for different opioid agonists might be expected to differ (Stevens and Yaksh ,1989a). However, the relationship between agonist efficacy and opioid tolerance is controversial. Stevens and Yaksh (1989a, 1989b) reported that tolerance to the analgesic effects of opioids measured by the hot-plate response of rats was greater for agonists with lower efficacy when continuously infused at equieffective doses. Duttaroy and Yoburn (1995) confirmed that the amount of analgesic tolerance after continuous infusion with opioids was inversely proportional to agonist efficacy but that intrinsic efficacy had no effect on the magnitude of tolerance produced by intermittent administration of opioid agonist to mice.
The complexity of the pharmacological parameters controlling thein vivo situation confounds the analysis of the relationship between efficacy and tolerance because differences in receptor occupancy, drug distribution, metabolism, and receptor selectivity affect the measured response and subsequent changes after prolonged drug administration. Stevens and Yaksh (1989a, 1989b) explained their results by suggesting that agonists with higher efficacy had a larger fraction of spare receptors and, hence, tolerance development to the more efficacious agonists was slower. However, this assumes that agonists with different efficacies produce receptor desensitization at the same rate when they bind the same fraction of receptors. Thus, to better elucidate the molecular mechanisms underlying the relationship between efficacy and receptor desensitization, we used Xenopus laevis oocytes coexpressing the MOR and the G protein-gated inwardly rectifying potassium channel complex (KIR3.1 and KIR3.4) to reconstitute a typical neuronal opioid response (North et al., 1987). To reconstitute a homologous receptor desensitization mechanism, we also coexpressed GRKs and β-arr 2. Using this gene expression system, we previously showed that coexpression of β-ARK2 (or GRK3) and β-arr 2 synergistically produced an agonist-dependent homologous desensitization of the β2-adrenergic receptor, the δ-opioid receptor, and the MOR activation of the KIR3 response (Kovoor et al., 1997). Recently, Yu and colleagues (1997) reported that opioids with high receptor efficacies were better able to stimulate MOR phosphorylation than were opioids with low efficacies. Similarly, partial agonists were previously reported to be less effective at promoting β-adrenergic receptor phosphorylation by β-ARK (i.e., GRK2) than full agonists (Benovic et al., 1988). In the current study, we asked whether μ-opioid agonists with different efficacies produce GRK/β-arr 2-mediated desensitization at different rates.
Materials and Methods
Chemicals.
DAMGO was obtained from Peninsula Laboratories (San Carlos, CA). Fentanyl, buprenorphine, and naloxone were from Research Biochemicals International (Natick, MA). Sufentanyl was a gift from Janssen Pharmaceuticals (Brussels, Belgium). All other chemicals were from Sigma Chemical (St Louis, MO).
Complementary DNA clones and cRNA synthesis.
Except for GRK5, the cDNA clones used and cRNA synthesis methods have been described previously (Kovoor et al., 1997). The rat MOR clone was obtained from Dr. Lei Yu (GenBank accession No, L13069). cDNA for the KIR3.1 (GIRK1) channel was obtained from Dr. Henry Lester (GenBank accession No. U01071). The KIR3.4 (GIRK4) clone provided by Dr. John Adelman (GenBank accession No. X83584) and the β-arr 2 cDNA (clone provided by Dr. Robert Lefkowitz, GenBank accession No. M91590) were first amplified by the use of Amplitaq DNA Polymerase (Perkin-Elmer Cetus, Norwalk, CT) in a standard polymerase chain reaction using oligonucleotides designed to add a T7 promoter region and a 45-base poly(A)+ tail. The rat GRK3 cDNA (GenBank accession No. M87855) was provided by Dr. Shaun Coughlin in the pFROGY vector. The human GRK5 clone (GenBank accession No. L15388) was obtained from Dr. Jeffrey Benovic (Kunapuli et al., 1993). Plasmid templates for all constructs were linearized before cRNA synthesis, and mMESSAGE MACHINE kits (Ambion, Austin, TX) were used to generate capped cRNA from the cDNA templates, which contained either T7, T3, or SP6 promoters to direct synthesis of sense transcripts.
Oocyte culture and injection.
Stage IV oocytes were prepared as described previously (Kovoor et al., 1995). cRNA was injected (50 nl/oocyte) using a Drummond automatic microinjector, and then oocytes were incubated at 18° for 3–7 days in normal oocyte saline buffer (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mmCaCl2, and 5 mm HEPES, pH 7.5) solution supplemented with sodium pyruvate (2.5 mm) and gentamycin (50 μg/ml).
Electrophysiology.
Oocytes were clamped at −80 mV with two electrodes filled with 3 m KCl having resistances of 0.5–1.5 MΩ using a Geneclamp 500 amplifier and pCLAMP 6 software (Axon Instruments, Foster City, CA). Data were digitally recorded (Digidata 1200, Axon Instruments, and Intel 386PC) and filtered. Membrane current traces also were recorded using a chart recorder. To facilitate the inward potassium current flow through the KIR3 channels, normal oocyte saline buffer was modified to increase KCl concentration to 16 mm as stated in Results. All drug responses were evaluated in this high K+-containing solution. In the experiments in which sufentanyl desensitization was evaluated, the high K+ solution in which drug responses were measured also contained 0.1% bovine serum albumin (Sigma). The concentration of NaCl was correspondingly decreased to maintain isoosmolarity. Opioid activation of KIR3 conductance was measured as described previously (Kovoor et al., 1997).
Statistical analysis.
The Student’s t test (with two-tailed p values) was used for comparison of the independent mean values. Dose-response curves were fitted to a simple Emax model using the nonlinear regression analysis PCNONLIN v4.2 (SCI Software) to determine the EC50 values and 95% confidence intervals.
Results
Dose-response relationship of μ-opioid agonists in X. laevis oocytes expressing MOR and KIR3.
As reported previously (Kovoor et al., 1995, 1997), activation of MOR by DAMGO, a selective MOR agonist, elicited a G protein-activated inwardly rectifying potassium conductance in X. laevis oocytes injected with cRNA for MOR and the KIR3 channel (Fig.1). MOR-activated KIR3 channel response was measured as the increase in inward current produced in oocytes clamped at −80 mV. The opioid-induced increase in potassium conductance was blocked by naloxone and required oocyte injection with both MOR and KIR3.1 cRNA. As reported, coexpression of KIR3.1 and KIR3.4 resulted in the formation of heteromultimeric channels, enhanced the agonist-activated response, reduced the amount of channel cRNA required, and greatly attenuated the amount of heterologous desensitization produced on prolonged agonist treatment (Krapivinskyet al., 1995; Kovoor et al., 1997). In this study, we generated cumulative concentration-response curves for activation of KIR3 by the MOR ligands DAMGO, fentanyl, sufentanyl, buprenorphine, and morphine. To normalize for daily differences in receptor expression, dose-response data are presented as a percentage of the average maximal stimulation produced by DAMGO as measured in the same oocyte batch and under the same conditions.
Sufentanyl was the most potent opioid tested followed with fentanyl and DAMGO (Fig. 1, Table 1). As expected, morphine was found to be a less efficacious agonist than DAMGO, sufentanyl, or fentanyl; the maximal response to morphine was smaller than that to the other agonists tested. In oocytes injected with a low dose of cRNA for MOR (0.08 ng), morphine was unable to elicit any measurable KIR3 currents (Fig. 1) but instead was a competitive antagonist of the DAMGO-activated response. Schild analysis of the antagonist potency of morphine under these conditions gave a Kd value of 98 nm (data not shown). For morphine to produce a MOR-mediated KIR3 response, receptor expression was increased by increasing the amount of MOR cRNA injected/oocyte to 0.5 ng (Fig. 1). At this dose of MOR cRNA, the EC50 value of morphine was 90 nm (Table 1), a value consistent with theKd value determined by Schild analysis. A second, weak opioid receptor agonist, buprenorphine, also was tested under these conditions in an attempt to extend the analysis, but 10 μm buprenorphine did not produce detectable MOR activation at the MOR cRNA amounts injected (0.08–0.5 ng).
In oocytes injected with 0.08 ng of MOR cRNA, the maximal responses produced by sufentanyl, DAMGO, or fentanyl were not significantly different. For oocytes injected with 6.3-fold higher (0.5 ng) MOR cRNA, the maximal stimulation produced by DAMGO was increased (Fig. 1,inset). Similarly, increasing the amount of MOR injected to 1 ng also increased the maximal receptor-stimulated currents (data not shown). These results suggest that in oocytes injected with either 0.08 or 0.5 ng of MOR cRNA, there were no spare MORs for DAMGO, fentanyl, or sufentanyl because the maximal receptor-stimulated currents were limited by receptor expression. Thus, although sufentanyl was more potent than either DAMGO or fentanyl, all three drugs were equally efficacious in this expression system.
Effect of DAMGO, fentanyl, and morphine treatment on GRK3- and β-arr 2-mediated homologous desensitization of MOR.
In oocytes expressing only MOR and the KIR3 channel subunits KIR3.1 and KIR3.4, no significant homologous desensitization of the receptor-elicited KIR3 current was observed (Fig.2A). However, as demonstrated previously (Kovoor et al., 1997), coexpression of both GRK3 (i.e., β-ARK 2) and β-arr 2 led to a significant homologous desensitization of the DAMGO-activated KIR3 current (Fig. 2A). Using oocytes coexpressing GRK3 and β-arr 2 with the receptor and channel, we compared the DAMGO-induced desensitization with the desensitization induced by either 1 μm fentanyl or 1 μm morphine, each at the receptor-saturating concentration of agonist. Responses elicited by DAMGO or fentanyl desensitized by ∼50% during 8 min of continuous agonist exposure. In the same group of oocytes, the morphine-activated responses showed no desensitization over the same period. To test whether the lack of acute desensitization by morphine was a result of its lower activity, we tested a concentration of DAMGO that evoked a response similar to the maximal morphine response and found that it also produced little receptor desensitization (data not shown). In the absence of either GRK3 or β-arr 2, neither fentanyl nor DAMGO treatment produced acute homologous receptor desensitization (Fig. 2C and data not shown).
Effect of GRK5 substitution for GRK3 on MOR desensitization.
We also tested whether another member of the GRK family, GRK5, was able to modulate MOR activity (Fig. 3). Brief treatment (8 min) with either DAMGO, fentanyl, or morphine (1 μm) did not produce significant desensitization in MOR-activated KIR3 responses in oocytes that had been coinjected with cRNA for GRK5 and β-arr 2. However, prolonged DAMGO or fentanyl treatment did produce significant desensitization in oocytes coexpressing both GRK5 and β-arr 2. In this experiment, oocytes were exposed for 12–14 hr to either DAMGO, fentanyl, or morphine in normal oocyte saline buffer and then the drug was removed by perfusion with saline buffer for 5 min. The amount of receptor desensitization produced by each agonist was assessed by measuring the response to a challenge with 1 μm DAMGO; this is important because the degree of desensitization may otherwise vary depending on the efficacy of the test agonist.
In oocytes expressing GRK5 and β-arr 2, pretreatment with DAMGO or fentanyl significantly diminished the amplitude of the inward current elicited by subsequent DAMGO application compared with oocytes not injected with GRK5 and β-arr 2 (Fig. 3, A and B). In contrast, pretreatment with morphine did not significantly diminish the inward current elicited by DAMGO application. The data demonstrate that GRK5 also can mediate agonist-dependent MOR desensitization after treatment with DAMGO or fentanyl and can substitute for GRK3 in this system. However, the kinetics of GRK5-mediated desensitization were slower than that of GRK3 under these expression conditions. The results obtained showed further that morphine pretreatment was significantly less effective than DAMGO or fentanyl pretreatment at producing GRK5-mediated receptor desensitization. Because GRK5 does not require Gβγ activation, the difference between morphine- and DAMGO- or fentanyl-mediated desensitization did not result from the weaker production of Gβγ by the partial agonist morphine.
Although acute treatment of oocytes with morphine did not produce opioid receptor desensitization (Fig. 2), we next tested whether a longer treatment with morphine was effective. After the protocol described in Fig. 3, but with GRK3 instead of GRK5, oocytes were treated for 12–14 hr with either 1 μm DAMGO or 1 μm morphine (Fig. 4). As expected, oocytes coexpressing GRK3 and β-arr 2 showed complete desensitization after 12–14-hr treatment with 1 μmDAMGO. Significantly less desensitization was produced by DAMGO in oocytes not expressing GRK3 and β-arr 2; however, in this group of oocytes, the responses observed were significantly smaller than evident in control oocytes not pretreated with DAMGO. Interestingly, prolonged treatment with morphine also produced significant desensitization in oocytes coexpressing GRK3 and β-arr 2, and significantly less desensitization was produced by morphine in oocytes not expressing GRK3 and β-arr 2. These results suggest that morphine can produce GRK/β-arr 2-dependent receptor desensitization in this expression system, but it does so at a significantly slower rate than does the more efficacious agonists, DAMGO and fentanyl.
Finally, we also compared the GRK3- and β-arr 2-mediated desensitization produced by treatment with DAMGO with that produced by treatment with the more potent but equally efficacious MOR ligand sufentanyl. After 20 min of agonist pretreatment, the amounts of homologous, GRK/β-arr 2-dependent desensitization produced by DAMGO and sufentanyl were not significantly different (Fig. 4). These results suggest that the rate of homologous desensitization of the MOR depends on agonist efficacy.
Discussion
Here, we report that in addition to regulation by GRK3, MOR also may be uncoupled in an agonist-dependent manner by a GRK with different properties, GRK5. We also show that the highly efficacious μ-opioid agonists DAMGO, sufentanyl, and fentanyl produce a GRK- and β-arr-mediated uncoupling of the MOR. In our investigation, efficacy directly correlated with GRK3- and β-arr 2-mediated receptor uncoupling. The highly efficacious ligands DAMGO, fentanyl, and sufentanyl caused a greater amount of receptor desensitization than morphine at saturating concentrations of agonist.
The faster rate of GRK-mediated desensitization caused by treatment with either DAMGO, fentanyl, or sufentanyl compared with that produced by morphine suggests that the more efficacious agonists induce a conformation of the receptor that is a better substrate for the kinase. Because Gβγ subunits are required for recruitment of GRK3 to the plasma membrane and activation of the kinase (Pitcher et al., 1995, Muller et al., 1997), the more efficacious ligands would be expected to activate the GRK3 more effectively than does morphine. However, we demonstrated here that GRK5, which differs from GRK3 in that it is not bound or activated by Gβγ, also produces a faster rate of MOR desensitization after DAMGO or fentanyl treatment. The more efficacious agonists thus allow more effective desensitization of the receptor independent of Gβγ release. The slower rate of GRK5-mediated desensitization evident in our study is consistent with the results of Kunapuli et al. (1994), who showed that purified GRK5 had a lower Vmax than GRK2 at other G protein-coupled receptor substrates.
Sternini et al. (1996) and Keith et al. (1996)reported that in the HEK 293 cell line and in neurons in vivo, high efficacy agonists such as etorphine produced MOR internalization. However, morphine, which effectively activated the receptor, did not produce MOR internalization in HEK 293 cells orin vivo. Recent experiments suggest that in addition to uncoupling the β2-adrenergic receptor from G proteins, phosphorylation of the β2-adrenergic receptor by receptor kinases followed by β-arr binding also is important for receptor internalization (Goodman et al., 1998). β-Arr serves as an adapter protein that links the agonist-bound receptor to the cellular internalization machinery. Using the X. laevis oocyte expression system, we have shown a synergistic action of the GRKs and β-arr on MOR desensitization. If phosphorylation of the MOR by receptor kinases followed by β-arr binding to MOR also is important for MOR internalization, then our demonstration that morphine only very weakly stimulates GRK-mediated arrestin binding to MOR may provide a mechanistic explanation for the observations of Keith and colleagues. Under conditions in which the levels of receptor expression are high or when the amount of receptor activation is greatly amplified by the effector system used to monitor receptor activity, poorly efficacious agonists such as morphine may be expected to produce maximal responses. However, for poorly efficacious ligands, the fraction of bound receptors that are in the active state at any time would be small, and this would reduce the probability for GRK phosphorylation and subsequent β-arr binding. In support of that hypothesis, we observed that the concentration of DAMGO that produced a response similar to that evoked by saturating doses of morphine also produced little receptor desensitization. Our results predict that the minimal dose of etorphine or DAMGO producing the same response as the dose of morphine tested also would fail to produce receptor internalization.
There are two discrepancies between the conclusions presented in the current study and the recent report by Yu et al. (1997). They found that sufentanyl was more efficacious than DAMGO in activating MORs expressed in oocytes and concluded that agonist-dependent receptor phosphorylation occurred in oocytes without adding GRK-cRNA. Under the conditions in which no cRNA for GRK or β-arr is injected (Yu et al., 1997), we previously established that the response desensitization that occurs is heterologous and occurs via adaptive changes downstream of the receptor (e.g., without receptor phosphorylation) (Kovoor et al., 1995). In oocytes coexpressing MOR and the 5-hydroxytryptamine1A receptor, stimulation of either receptor resulted in heterologous desensitization of the subsequent response to the other and injection of the nonhydrolyzable GTP analogue guanosine-5′-O-(3-thio)triphosphate did not affect the rate of desensitization (Kovoor et al., 1995). Furthermore, the heterologous desensitization of receptor/KIR3 coupling was greatly attenuated by the coexpression of the KIR3 channel subunits KIR3.1 and KIR3.4 (Kovooret al., 1997). After testing a series of agonists with differing efficacies at MOR in the X. laevis oocyte expression system, they determined that the amount of desensitization of the agonist-induced responses was related directly to the efficacy of the agonist and concluded that the more efficacious agonists produced a greater receptor desensitization. However, Yu et al. (1997) did not show that receptor phosphorylation resulted in homologous receptor desensitization because their results simply indicate that heterologous desensitization (e.g., changes occurring downstream of the receptor, presumably at the channel) also is dependent on agonist efficacy.
Furthermore, under the conditions used by Yu et al., the high levels of MOR cRNA and low levels of channel expression (caused by the limiting expression of endogenous KIR3.5) are likely to have resulted in spare opioid receptors for DAMGO, sufentanyl, and fentanyl. In X. laevis oocytes injected with cRNA for only the KIR3.1 subunit (as done by Yuet al.), the expression of functional channels is dependent on the formation of heteromultimers between the exogenously expressed KIR3.1 subunit and the endogenously expressed KIR3.4 homologue KIR3.5 (Krapivinsky et al., 1995, Hedin et al., 1996). In oocytes injected only with cRNA for the KIR3.1, it has been shown that channel, and not receptor, is limiting (Hedin et al., 1996). This conclusion is supported by the lower EC50 values obtained (Yu et al., 1997). Under conditions in which there are spare opioid receptors, it thus is surprising that sufentanyl produced greater responses than DAMGO (Yu et al., 1997). We failed to replicate that finding.
Previously, Blake et al. (1997) stated that treatment of MORs expressing HEK 293 cells with buprenorphine abolished the ability of other μ opioids to inhibit adenylyl cyclase, and they interpreted these results to argue that buprenorphine produced a profound receptor desensitization. This conclusion is in direct contrast to our results, which indicate that less efficacious agonists produce less receptor desensitization. To reconcile this discrepancy, we tested 10 μm buprenorphine and found that it did not activate KIR3 channels at any of the levels of functional MOR. Buprenorphine is an agent that binds the MOR with very high affinity but very low efficacy, and we found that 1 μmbuprenorphine acted as a pseudoirreversible antagonist consistent with previous findings (Thomas and Hoffman, 1993). Thus, the reduction in response previously reported may have resulted from receptor antagonism rather than desensitization.
There is an apparent contradiction in the relationship between ligand efficacy and receptor desensitization observed in this study and the opposite relationship between opiate tolerance and ligand efficacy observed in vivo (Stevens and Yaksh, 1989a, 1989b; Duttaroy and Yoburn, 1995). Highly efficacious agonists produced less tolerancein vivo while producing greater GRK-mediated receptor phosphorylation and receptor desensitization in vitro. The discrepancy can be reconciled if we assume that a very large fraction of spare receptors mediates the analgesic response to the highly efficacious opioids but that morphine has relatively few, if any, spare receptors in vivo. That assumption is supported by previous studies demonstrating a large fraction of spare opioid receptorsin vivo (Chavkin and Goldstein, 1981; Perry et al., 1982). Furthermore, a drug with low efficacy such as morphine may act as a full agonist in tissues that express an excess of MOR and act as a partial agonist in tissues with lower receptor expression. After the desensitization or inactivation of an identical number of receptors, a less efficacious drug will show a greater shift in the dose-response curve than a more efficacious drug with an extremely large receptor reserve based on mass action kinetic principles. Thein vivo tolerance studies were confounded because the amount of tolerance produced by prolonged exposure to an agonist was evaluated by examining the shift of the dose response curve to the same agonist. Under these conditions, a reduction in receptor reserve would be expected to produce a greater shift in the dose response of a partial agonist than a full agonist. A clearer measure of the degree of change at the MOR after prolonged treatment with various toleragens would have been obtained by evaluating the shifts in the dose-response curves for a single, receptor-selective test agonist.
In studies using established cell lines, heterologous gene expression systems, or real neurons, efforts to define the relationship between agonist efficacy and tolerance or desensitization have been hindered by the confounding presence of spare opioid receptors. In the current study, we deliberately used a system in which there were no spare receptor receptors for any of the opioids tested (increases in MOR cRNA injection gave proportional increases in agonist-activated maximal response). Thus, further work is required to define the molecular changes in opioid signaling after prolonged in vivo exposure to drug. Nevertheless, the results obtained using in vitromodel systems predict that highly efficacious opioid agonists produce a higher rate of receptor phosphorylation in animals treated with drug for extended times.
Acknowledgments
We thank Dr. Gregory Terman for helpful discussion. We thank Dr. Robert Lefkowitz (Howard Hughes Medical Institute, Durham, NC) for the β-arr 2 clone and for permission to use the GRK3 (β-ARK 2) clone, Dr. Shaun Coughlin (Dept. of Pathology, University of Claifornia, San Francisco, CA) for the rat GRK3 clone, Dr. John Adelman (Vollum Institute, Portland, OR) for KIR3.4 cDNA (GIRK 4), Dr. Lei Yu (Dept. of Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH) for the rat MOR cDNA, Dr. Jeffry Benovic (Dept. of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA) for the GRK5 clone, and Dr. Henry Lester (Division of Biology, California Institute of Technology, Pasadena, CA) for the rat KIR3.1 (GIRK 1) and β2-adrenergic receptor clones.
Footnotes
-
Send reprint requests to: Dr. Charles Chavkin, Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280. E-mail: cchavkin{at}u.washington.edu
-
This work was supported by United States Public Health Service Grant DA04123 from the National Institute on Drug Abuse. A.K. and J.P.C. contributed equally to this work.
- Abbreviations:
- MOR
- μ-opioid receptor
- DAMGO
- [d-Ala2,N-MePhe4,Gly-ol5]enkephalin
- β-ARK
- β-adrenergic receptor kinase
- β-arr
- β-arrestin
- GRK
- G protein-coupled receptor kinase
- HEK
- human embryonic kidney
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- Received May 27, 1998.
- Accepted June 17, 1998.
- The American Society for Pharmacology and Experimental Therapeutics