Bi-directional heterologous desensitization between the major HIV-1 co-receptor CXCR4 and the κ-opioid receptor

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Abstract

We previously characterized multiple interactions between chemokine and opioid G protein-coupled receptors (GPCR), and we found both μ and δ-opioid receptors cross-desensitize CCR1, CCR2, CCR5, but not CXCR4. Here we report that the κ-opioid receptor (KOR) is able to cross-desensitize CXCR4, and this phenomenon is bi-directional. Chemotactic responses by KOR activation are diminished with prior activation of CXCR4. Additionally, calcium mobilization assays show these cross-desensitization processes occur within seconds of receptor activation, and target receptor internalization is not responsible for desensitization between these receptors. These results have implications for several essential processes including neuronal and lymphocyte development, inflammatory responses, and pain/sensitivity.

Introduction

Opioids have roles in the central and peripheral nervous systems for the control of pain sensations. They also function as regulators of cellular and humoral immune responses (Carr et al., 1996, McCarthy et al., 2001) and have been shown to regulate expression of chemokines as well as chemokine receptors (Steele et al., 2003). The opioid receptors and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) super-family. Three primary types of opioid receptors have been cloned and are designated δ, κ, and μ (Chen et al., 1993, Evans et al., 1992, Kieffer et al., 1992, Li et al., 1993, Yasuda et al., 1993). Chemokines and chemokine receptors are involved in leukocyte recruitment to sites of inflammation, and are also involved in neuronal development (Stumm and Hollt, 2007, Stumm et al., 2003). Classification of the chemokine receptors, designated C, CC, CXC, or CX3C, is based on the location of amino-terminal cysteine residue(s) (Murphy et al., 2000).

CXCR4 has been under intense study as a participant in a number of disease-related processes angiogenesis and human immunodeficiency virus (HIV) infection by virtue of its role as a major HIV-1 co-receptor (Gerard and Rollins, 2001, Hesselgesser et al., 1997, Moore et al., 1997, Proudfoot et al., 1999). CXCR4 is widely expressed in many tissues and cells, including the brain (Baggiolini et al., 1997, Feil and Augustin, 1998, Hesselgesser et al., 1997, Ohtani et al., 1998, Tanabe et al., 1997, Volin et al., 1998). At the same time, the opioid receptors have been found on neuronal cells of the brain, as well on certain leukocytes (Belkowski et al., 1995, Bidlack et al., 1992, Carr et al., 1989, Chuang et al., 1994, Sedqi et al., 1995).

The desensitization of one GPCR following agonist binding by another unrelated receptor is termed heterologous desensitization. This is accomplished via an intracellular signaling process involving Gαi proteins with the participation of a secondary messenger-dependent kinase (protein kinase A or protein kinase C) (Ali et al., 1999, Zhang et al., 2003). As a part of this process, target receptor signaling is inhibited, and in some cases, the target receptor becomes internalized (Ali et al., 1999, Lefkowitz, 1998, Olbrich et al., 1999). Whether the receptor is internalized or not, the final result is loss of receptor function. Analysis of the heterologous desensitization between several GPCRs suggests a hierarchy exists with respect to the susceptibility to cross-desensitization (Zhang et al., 2003). Moreover, it appears that receptors that are resistant to cross-desensitization are typically strong desensitizers of other GPCRs and receptors that are easily desensitized are typically weak desensitizers of other GPCRs.

Our laboratories, and others, have shown that several cross-desensitization interactions occur between opioid and chemokine receptors, based on studies carried out both in vitro and in vivo (Chen et al., 2007, Grimm et al., 1998, Rogers et al., 2000, Szabo et al., 2002, Zhang et al., 2003). For example, it is apparent that both μ- and δ-opioid receptors (MOR and DOR) are able to induce heterologous desensitization of CCR1, CCR2, CCR5, CXCR1, and CXCR2, but not CXCR4. On the other hand, CCR1, CCR2, CCR5, CCR7, CX3CR1, and CXCR4, but not CXCR1 or CXCR2, cross-desensitize MOR and DOR (Grimm et al., 1998, Honczarenko et al., 2002, Szabo et al., 2002, Szabo et al., 2003, Zhang et al., 2003). Based on these results we have suggested that MOR and DOR can be classified as “intermediate” in the cross-desensitization hierarchy, while CXCR4 appears to be relatively resistant to cross-desensitization (Szabo et al., 2002). Up to this point, little attention has been given to the cross-talk function of KOR, an opioid receptor which is known to exert significant effects on the function of the immune system (Alicea et al., 1996). Moreover, KOR agonists have been shown in several studies to exhibit effects on the immune response which are in opposition to effects induced by either MOR or DOR agonists (Alicea et al., 1996). For example, we have found that MOR appears to exert opposing effects on the expression of the critical chemokine receptors CCR5 and CXCR4 (Rogers and Peterson, 2003). In general, the function of KOR appears to be strongly anti-inflammatory, in contrast with MOR, and for this reason we chose to examine the capacity of KOR agonists to participate in cross-talk interactions with chemokine receptors.

In the present study, we examine the potential cross-talk between CXCR4 and KOR using both in vitro and in vivo analyses. We have found that there is profound desensitization between CXCR4 and KOR, and the cross-talk is bi-directional. Despite the fact that CXCR4 and KOR are relatively resistant to cross-desensitization, these receptors have the capacity to induce substantial cross-desensitization. Cross-desensitization of the opioid and chemokine receptors represents a level of regulatory control, which is only now becoming appreciated.

Section snippets

Chemicals

The selective KOR agonist trans-(−)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzene-acetamide hydrochloride (U50,488H) was purchased from Tocris Cookson Inc. (Ellisville, MO, USA) or obtained as a gift from the Upjohn Company, Kalamazoo, MI. 17,17′-(Dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-binorphinan-3,4′,14,14′-tetrol dihydrochloride (nor-Binaltrophimine dihydrochloride [nor-BNI]) was purchased from Tocris Cookson Inc. (Ellisville, MO, USA). Drugs were dissolved in 0.9%

Cross-desensitization of KOR- and CXCR4-induced chemotactic responses

We have previously reported results which show that activation of CXCR4 cross-desensitizes both MOR and DOR, but neither MOR or DOR are able to desensitize CXCR4 (Grimm et al., 1998, Honczarenko et al., 2002, Rogers et al., 2000, Szabo et al., 2002, Szabo et al., 2003). We have also observed that MOR and KOR induce distinct influences on the immune system, and exert opposing effects on the expression of CXCR4 (Rogers and Peterson, 2003). We examined the capacity of KOR to participate in

Discussion

We report results here which show that the chemokine receptor, CXCR4, is regulated by cross-talk with KOR. This result is apparent using either primary neutrophils, or stably-transfected cell lines. This finding is somewhat surprising given previous findings from this laboratory, and others, which showed that both MOR and DOR are able to cross-desensitize CCR1, CCR2, CCR5, CXCR1, CXCR2, but not CXCR4 (Grimm et al., 1998, Szabo et al., 2003, Zhang et al., 2004). This set of results illustrates

Acknowledgements

We thank Dr. Jodene Moore for assistance with flow cytometry and cell sorting.

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