Elsevier

Neuropharmacology

Volume 39, Issue 3, March 2000, Pages 353-363
Neuropharmacology

A cluster of Ser/Thr residues at the C-terminus of μ-opioid receptor is required for G protein-coupled receptor kinase 2-mediated desensitization

https://doi.org/10.1016/S0028-3908(99)00174-4Get rights and content

Abstract

To investigate the functional role of G protein-coupled receptor kinases (GRK) in homologous desensitization of the μ-opioid receptor, human embryonic kidney (HEK) 293 cells, which express a significant level of GRK2, were stably transfected with the cDNA encoding the rat μ-opioid receptor. Wild-type μ-opioid receptors developed homologous desensitization after 30 min pretreatment with DAMGO ([d–Ala2,N–methyl–Phe4,Gly–ol5]enkephalin), a specific μ-opioid receptor agonist. The ability of μ-opioid receptors to develop homologous desensitization was greatly impaired following the transfection of a cDNA fragment encoding the GRK2(495–689) polypeptide, which is believed to block Gβy-mediated transduction events including the membrane translocation and activation of GRK2. The μ(CΔ45) receptor, a deletion mutant that lacks 45 C-terminal amino acids, failed to exhibit homologous desensitization after 30 min pretreatment of DAMGO. The μ(CΔ41) receptor, which differs from the μ(CΔ45) receptor by having four more Ser/Thr residues (Thr354Ser355Ser356Th357), developed GRK2-mediated desensitization. These results suggest that homologous desensitization of rat μ-opioid receptors results from the activation of GRK2 and that a cluster of Ser/Thr residues (Thr354Ser355Ser356Thr357) at the intracellular carboxyl tail plays an important role in GRK2-mediated μ-opioid receptor desensitization.

Introduction

Endogenous opioid peptides and opiate drugs are believed to produce various biological effects including analgesia by the activation of μ-, δ- and κ-opioid receptors (Loh and Smith, 1990, Reisine and Bell, 1993). A recent study demonstrated that morphine failed to induce analgesic and reward effects in transgenic mice lacking the μ-opioid-receptor gene, suggesting that pharmacological effects induced by morphine-like drugs mainly result from the activation of μ-opioid receptors in vivo (Matthes et al., 1996). Continuous administration of morphine-like drugs leads to the development of tolerance and dependence (Nestler, 1993, Nestler, 1996). In the face of sustained or repeated exposure to agonists, μ-opioid receptors are also rapidly inactivated by a process referred to as desensitization. Therefore, the homologous desensitization of μ-opioid receptors in vivo is likely to be involved in the development of morphine-induced tolerance (Nestler, 1996). A better understanding of the molecular mechanism underlying μ-opioid receptor desensitization should be useful in designing therapeutic agents that prevent morphine-induced tolerance.

Multiple lines of evidence suggest that the desensitization of G protein-linked receptors results from receptor phosphorylation by G protein-coupled receptor kinase (GRK) (Sterne-Marr and Benovic, 1995, Lefkowitz, 1998). According to this proposal, agonist binding to G protein-coupled receptors causes the dissociation of G proteins which consist of α (Gα) and βγ subunits (Gβγ). In addition to the subsequent Gα- or Gβγ-mediated activation of effector systems, Gβγ subunits also bind to GRK and translocate GRK from the cytoplasm to the cell membrane. After being phosphorylated by GRK, the receptors bind to inhibitory proteins, β-arrestins, and are uncoupled from G proteiens resulting in homologous desensitization. Up to now, six subtypes of GRKs have been cloned and characterized (Haga et al., 1994, Lefkowitz, 1998). Among these GRKs, GRK2 is widely distributed in the body and is likely to mediate receptor desensitization. Consistent with this hypothesis, GRK2 has been shown to phosphorylate α-adrenergic, β-adrenergic, muscarinic, type 1A angiotensin II and thrombin receptors (Haga et al., 1994, Ishii et al., 1994, Freedman et al., 1995, Diviani et al., 1996 Jewell-Motz and Liggett, 1996, Oppermann et al., 1996). Overexpression of a dominant negative mutant GRK2 blocked agonist-induced desensitization exhibited by A2-adenosine, α1B-adrenergic, β-adrenergic, and type IA angiotensin II receptors (Kong et al., 1994, Diviani et al., 1996, Oppermann et al., 1996, Mundell et al., 1997). Further studies also suggested that GRK2 mediates receptor desensitization by phosphorylating serine and threonine residues located in the third or C-terminal intracellular loop (Haga et al., 1994, Ishii et al., 1994, Lefkowitz, 1998).

Molecular cloning studies have indicated that the μ-opioid receptor is a member of the G protein-coupled receptor family (Chen et al., 1993, Thompson et al., 1993, Standifer and Paternak, 1997). Recently, it has been shown that when μ-opioid receptors expressed in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells, short term desensitization of the receptors was accompanied by phosphorylation of the receptor (Zhang et al., 1996, Yu et al., 1997, Zhang et al., 1998). Furthermore, overexpression of GRK2 promotes the agonist-induced phosphorylation of the μ-opioid receptor (Zhang et al., 1998). These findings raise the possibility that GRK2-mediated phosphorylation leads to the homologous desensitization of μ-opioid receptors, Gβγ is believed to mediate the membrane translocation and activation of GRK2 by interacting with the C-terminal domain of GRK2 (Koch et al., 1993). A recent study demonstrated that the carboxyl tail of GRK2 containing Gβγ-binding domain functions as a specific Gβγ antagonist when expressed in intact cells and blocks various Gβγ-mediated transduction events (Koch et al., 1994). Therefore, in the present study, the cDNA of the rat μ-opioid receptor was stably transfected into HEK 293 cells, which express a significant level of endogenous GRK2 and β-arrestin (Freedman et al., 1995). Subsequently, the functional role played by GRK2 in μ-opioid receptor desensitization was investigated by transfecting the cDNA fragment encoding the C-terminal Gβγ binding domain of GRK2 into HEK 293 cells stably expressing μ-opioid receptors. Similar to other G protein-coupled receptors, GRK2 is likely to induce μ-opioid receptor desensitization by phosphorylating serine and threonine residues in the intracellular carboxyl tail. This hypothesis was also tested using various C-terminal deletion mutants of the μ-opioid receptor. The present study proposes that short term desensitization of the rat μ-opioid receptor results from the activation of GRK2 and that a cluster of Ser/Thr residues (Thr354Ser355Ser356Thr357) at the C-terminus plays an important role in GRK2-mediated μ-opioid receptor desensitization.

Section snippets

Molecular cloning of the rat μ-opioid receptor

A full-length cDNA clone encoding the rat μ-opioid receptor was obtained by performing PCR amplification using cDNA prepared from the brainstem mRNA as the template. PCR was carried out in a programmable thermal controller (Minicycler, NJ Research Inc., Watertown, MA) with the following oligonucleotide primers: (a) a forward primer: 5′ATGGACAGCAGCACCGGCCCA3′ corresponding to nucleotides 1–21 of the rat μ receptor (Chen et al., 1993, Thompson et al., 1993). (b) a reverse primer:

Results

Saturable and specific binding sites for [3H]DAMGO, a selective μ-opioid receptor agonist, were detected in HEK 293 cells stably transfected with the cDNA encoding the rat μ-opioid receptor. Scatchard analysis of [3H]DAMGO binding revealed the expression of a single population of high-affinity binding sites (Table 1; Bmax=548±40 fmol/mg protein; KD=3.1±0.2 nM). No specific [3H]DAMGO binding was observed in non-transfected HEK 293 cells (data not shown). DAMGO inhibited forskolin (20

Discussion

Multiple lines of evidence indicate that GRK-mediated phosphorylation of G protein-coupled receptors in their active or stimulated conformations results in homologous desensitization (Haga et al., 1994, Sterne-Marr and Benovic, 1995, Lefkowitz, 1998). It has been shown that μ-opioid receptors undergo rapid agonist-induced phosphorylation, most likely by GRK, which correlates with the development of short term desensitization (Zhang et al., 1996, Yu et al., 1997, Zhang et al., 1998). By

Acknowledgements

The author thanks M.J. Yen and W.T. Chang for their technical assistance. This work was supported by the National Science Council (NSC87-2314-B182-085 and NSC88-2314-B182-069) and Chang Gung Research Foundation (CMRP 555).

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