Heterodimerization and cross-desensitization between the μ-opioid receptor and the chemokine CCR5 receptor

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Abstract

Cross-desensitization between μ-opioid receptor agonists and CC chemokines was shown to occur in immune cells and in the central nervous system. However, these cells do not permit examination of potential mechanisms at cellular levels due to low levels and mixed populations of receptors. In this study, we investigated possible interactions and biochemical mechanisms of cross-desensitization between the μ-opioid and chemokine CCR5 receptors coexpressed in Chinese hamster ovary (CHO) cells. Hemagglutinin (HA)-tagged μ-opioid receptor coimmunoprecipitated with FLAG (Asp–Tyr–Lys–Asp–Asp–Asp–Asp–Lys)-tagged chemokine receptor CCR5 in cells expressing the two receptors, but not in a mixture of cells transfected with one of the two receptors, indicating that the two receptors form heterodimers. Treatment with the μ-opioid receptor agonist DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin), the chemokine RANTES (Regulated on Activation, Normal T cell-Expressed and -Secreted) (CCL5), or both, did not affect the level of coimmunoprecipitation. DAMGO and RANTES (CCL5) induced chemotaxis in CHO cells coexpressing both receptors, and preincubation with either DAMGO or RANTES (CCL5) profoundly inhibited chemotaxis caused by the other. DAMGO pretreatment enhanced phosphorylation of the chemokine CCR5 receptor and reduced RANTES (CCL5)-promoted [35S]GTPγS binding. Conversely, RANTES (CCL5) preincubation slightly increased phosphorylation of the μ-opioid receptor and significantly reduced DAMGO-induced [35S]GTPγS binding. These results indicate that activation of either receptor affected G protein coupling of the other, likely due to enhanced phosphorylation of the receptor. Heterodimerization between the two receptors may contribute to the observed cross-desensitization.

Introduction

Opiates and opioids interact with specific receptors to produce unique physiological and pharmacological effects, most notably modulation of pain perception and regulation of mood. The presence of at least three types of opioid receptors—μ, δ, and κ—was established in the 1970s and 1980s by pharmacological, binding, and anatomical distribution analyses. μ, δ, and κ opioid receptors have been cloned (for reviews, see Kieffer, 1995, Knapp et al., 1995). Activation of opioid receptors, coupled via pertussis toxin (PTX)-sensitive G proteins, induces a number of cellular effects, including inhibition of adenylyl cyclase, increase in K+ conductance and decrease in Ca2+ conductance, and stimulation of the p42/p44 mitogen-activated protein (MAP) kinase pathways (for a review, see Law et al., 2000).

Exposure to opiates and opioids has been reported to inhibit cellular immune responses and induce chemotactic responses in immune cells (for reviews, see Peterson et al., 1998, McCarthy et al., 2001). These effects on the immune system may contribute to impaired immune function (Des Jarlais et al., 1988) and a higher rate of infection by the human immunodeficiency virus (HIV) in chronic heroin users (Holmberg, 1996). These effects are, at least in part, mediated by opioid receptors as the mRNAs of μ, δ, and κ opioid receptors have been shown to be present in immune cells (for a review, see McCarthy et al., 2001).

Chemokines and chemokine receptors play crucial roles in the functions of the immune system, including leukocyte development, and function and clearance of infectious organisms. Chemokine receptors CCR5 and CXCR4 act as major coreceptors, along with CD4, for the entry of HIV into cells (Cairns and D'Souza, 1998). In addition, recent studies have shown that chemokine and chemokine receptors are present in the central nervous system and play important roles in brain development, regulation of neurotransmission, and pathophysiological states in which inflammation persists (Bajetto et al., 2002). To date, 19 chemokine receptors have been identified, and many chemokines bind to multiple receptors and most chemokine receptors recognize several chemokines (for a review, see Proudfoot, 2002). Activation of chemokine receptors, coupled via G proteins (Gi/o, Gq, and G16) (Arai and Charo, 1996), results in cellular effects such as inhibition of adenylyl cyclase (Zhao et al., 1998); stimulation of phospholipases A, C, and D; activation of p42/p44 MAP kinases; stimulation of phosphatidylinositol-3-kinase; and enhancement of nonreceptor tyrosine kinase activities (for reviews, see Bokoch, 1995, Maghazachi, 1999).

Opioid treatment of immune cells desensitized the chemotactic response induced by several chemokines; in turn, pretreatment with some chemokines reduced the chemotaxis induced by some opioids Grimm et al., 1998a, Grimm et al., 1998b, Choi et al., 1999, Rogers et al., 2000, Miyagi et al., 2000. Heterologous desensitization of CCR5 may reduce susceptibility to HIV infection Cairns and D'Souza, 1998, Shen et al., 2000, Szabo et al., 2003. In addition, administration of some chemokines into the periaqueductal gray of the rat brain reduced the antinociceptive effects of μ-opioid receptor agonists (Szabo et al., 2002). Cross-talk between the opioid and chemokine systems may serve as a modulatory mechanism to fine-tune cellular function (for a review, see McCarthy et al., 2001).

Like many other G protein-coupled receptors (GPCRs), both the μ-opioid and chemokine CCR5 receptors are desensitized following prolonged agonist exposure Zhang et al., 1996, Aramori et al., 1997. Two major types of desensitization have been characterized: homologous and heterologous. In homologous desensitization, only the activated receptor is desensitized, while in heterologous desensitization, activation of a receptor causes reduced responsiveness of another receptor. Homologous desensitization of GPCRs shares similar mechanisms; however, the mechanisms underlying heterologous desensitization of GPCRs are less uniform. Multiple processes may be involved in heterologous desensitization, including changes at the levels of receptors, G proteins, and second messenger pathways Ali et al., 1999, Willars et al., 1999.

GPCRs have been shown to form dimers with the same receptor or a different receptor (for reviews, see Milligan, 2001, Devi, 2001, Angers et al., 2002). μ-Opioid receptors undergo heterodimerization with several GPCRs including the δ-opioid and β2-adrenergic receptors. The ligand binding, signaling properties, and cellular function of a number of GPCRs have been reported to be modified as a result of receptor dimerization (for reviews, see Milligan, 2001, Devi, 2001, Angers et al., 2002).

In the present study, we investigated whether the μ-opioid and the chemokine CCR5 receptors formed heterodimers, and examined the early cellular signaling events following agonist binding, which may contribute to receptor cross-desensitization. We were not able to conduct such studies in immune cells and the central nervous system where cross-desensitization between opioids and chemokines has been reported, since these cells contain low levels of a heterogeneous population of opioid and chemokine receptors. The low levels of receptors do not permit biochemical characterization and the presence of several different chemokine and/or opioid receptors does not lend themselves to the unequivocal characterization of actions at a specific receptor type. We thus used a cell model, Chinese hamster ovary (CHO) cells stably coexpressing the CCR5 and μ-opioid receptors, for the study.

Section snippets

Materials

[35S]GTPγS (∼1250 Ci/mmol), [3H]diprenorphine (∼58 Ci/mmol), and [32P]orthophosphate (∼8500 Ci/mmol) were purchased from Perkin Elmer Life Science (Boston, MA) and [125I]MIP-1â was from Amersham Pharmacia Biotech (Piscataway, NJ). Naloxone was a gift from DuPont/Merck (Willmington, DE). Rabbit anti-FLAG polyclonal antibody (F-7425), Dulbecco's modified Eagle's medium, GDP, and GTPγS were obtained from Sigma (Louis, MO). The following reagents were purchased from the indicated companies:

Heterodimerization of the μ-opioid receptor and the chemokine receptor CCR5

CHO-HA-μ cells expressed ∼0.5 pmol/mg protein of HA-tagged μ-opioid receptor as determined by [3H]diprenorphine binding. FLAG-CCR5 cDNA was transfected into CHO or CHO-HA-μ cells to approximately 1 pmol/106 cells. CHO-HA-μ/FLAG-CCR5 cells, or a mixture of CHO-HA-μ and CHO cells transfected with the FLAG-CCR5 receptor was solubilized and incubated with anti-FLAG antibody to immunoprecipitate FLAG-CCR5, and immunoprecipitated materials were resolved by SDS-PAGE followed by immunoblotting with

Discussion

We have shown that the μ-opioid and chemokine CCR5 receptors coexpressed in CHO cells form heterodimers and cross-desensitize each other. Cross-desensitization is, at least in part, due to enhanced receptor phosphorylation and reduced receptor/G-protein. Heterodimerization of the two receptors may contribute to cross-desensitization.

Acknowledgments

This work was supported by NIH grants DA04745, DA06650, DA11263, and DA13429.

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