Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

doi:10.1016/j.bcp.2007.06.032

Biochemical Pharmacology

Volume 74, Issue 6, 15 September 2007, Pages 891-897

https://doi.org/10.1016/j.bcp.2007.06.032 Get rights and content

Abstract

Antagonists of the chemokine receptor, CCR5, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCR5 (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [³⁵S]GTPγS binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2)-stimulated [³⁵S]GTPγS binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (∼15%) in cell surface CCR5 was detected.

Introduction

The G protein-coupled chemokine receptor, CCR5, was originally characterised as a receptor responding functionally to the CC-chemokines CCL3 (MIP-1α), CCL4 (MIP-1β) and CCL5 (RANTES) [1]. CCR5 was subsequently described as the primary co-receptor for macrophage tropic human immunodeficiency virus type 1 (HIV-1) [2], [3], [4], [5].

Chemokine interaction with CCR5 initiates several events. Activated receptor associates with G proteins, leading to activation of signalling processes, e.g. changes in Ca²⁺ followed by receptor phosphorylation by G protein coupled receptor kinases (GRKs) [6]. This results in association of β-arrestin with the receptor [7] and desensitisation via uncoupling of receptor and G protein. A number of CC chemokines (CCL3, CCL4, CCL5, CCL8, CCL13 and CCL7) bind to CCR5 with different affinities and abilities to activate the receptor [8]. These chemokines can be divided in two subgroups based on amino acid sequence identity [9], [10]. CCL3, CCL4 and CCL5 form one subgroup and are full agonists, whereas CCL7, CCL8 and CCL13 form a second subgroup, which share ∼60% amino acid identity within the group and ∼30% identity with CCL3, CCL4 and CCL5. The two groups of chemokine exhibit differential abilities to activate signalling systems. For example, whereas CCL5 and CCL8 can both stimulate [³⁵S]GTPγS binding, CCL5 will cause internalisation of CCR5 but CCL8 is unable to do so [7], [11], suggesting that the two groups of chemokines may stabilise different conformations of CCR5.

The role of CCR5 as a co-receptor for the entry of HIV-1 into cells has prompted the development of small molecule antagonists of CCR5 as a potential therapy for preventing infection by HIV-1 [12]. Several such compounds have been described including 873140 (aplaviroc) [13], UK 427,857 (maraviroc) [14], SCH 351125 (vicriviroc) [15] and TAK 779 [16] and some biochemical studies on these compounds have been reported. The emerging data suggest that the compounds may act as allosteric inhibitors of chemokine binding and function at CCR5 and some differential effects of compounds on the actions of chemokines have been reported. For example, 873140 inhibited CCL3 and CCL5-induced Ca²⁺ responses via CCR5 but did not inhibit CCL5 binding to CCR5 [13]. These small molecule antagonists may also stabilise different conformations of CCR5 accounting for the differential antiviral spectrum of different antagonists [14] and the lack of cross-resistance seen for these different antagonists [17].

In order to probe this further we have analysed the interaction of two small molecule antagonists directed at CCR5 (UK-396,794, UK-438,235, Fig. 1) for their effects on the actions of two chemokines (CCL5 and CCL8) which have previously been shown to interact with CCR5 in a different manner [7], [11]. We have examined the interactions between the small molecule drugs and chemokines at CCR5 by determining effects on chemokine-stimulated [³⁵S]GTPγS binding to membranes of CHO cells expressing CCR5 (CHO-CCR5 cells) and by determining effects on chemokine-induced internalisation of CCR5 in CHO-CCR5 cells.

Section snippets

Cells and materials

CHO cells stably expressing CCR5 (CHO·CCR5) were grown as described previously [7], [11]. [³⁵S]GTPγS (∼37 TBq mmol⁻¹) was from Amersham Biosciences (Buckinghamshire, UK). CCL5 and CCL8 were purchased from PeproTech (Rocky Hill, NJ). Secondary antibodies were obtained from Sigma (Poole, UK), anti-CCR5 antibodies HEK/1/85a/7a have been described previously [7], [11] and all other chemicals were from Sigma (Poole, UK).

Membrane preparation

Membranes were prepared from confluent cells. Cells were harvested in ice-cold

Effects of UK-396,794 and UK-438,235 on agonist-independent [³⁵S]GTPγS binding

Fig. 2 shows the effects of UK-396,794 and UK-438,235 on [³⁵S]GTPγS binding to membranes of CHO cells expressing CCR5 (CHO-CCR5 cells). The two compounds were both potent inverse agonists (pIC₅₀ ± S.E.M. UK-438,235 8.71 ± 0.05, UK-396,794 8.93 ± 0.09) with UK-396,794 giving a slightly larger inhibition of agonist-independent [³⁵S]GTPγS binding in this preparation (UK-438,235 −27.9 ± 0.6%, UK-396,794 −30.4 ± 1.5%, P < 0.05).

Effects of UK-396,794 and UK-438,235 on CCL5- and CCL8-stimulated [³⁵S]GTPγS binding

Next we analysed the effects of different concentrations of the compounds on the

Discussion

In this study we have examined the effects of two small molecule inhibitors of the chemokine receptor CCR5. Both compounds were potent inverse agonists, inhibiting agonist-independent activation of CCR5 by about 30% in this preparation as assessed by [³⁵S]GTPγS binding. This shows that CCR5 is constitutively active in this preparation assessed using this assay system. We examined this apparent constitutive activity further by carrying out chronic treatment of CHO-CCR5 cells with the two

Acknowledgement

We thank Pfizer GRD-Sandwich Laboratories for financial support and for supplying reagents.

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Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Abstract

Introduction

Section snippets

Cells and materials

Membrane preparation

Effects of UK-396,794 and UK-438,235 on agonist-independent [35S]GTPγS binding

Effects of UK-396,794 and UK-438,235 on CCL5- and CCL8-stimulated [35S]GTPγS binding

Discussion

Acknowledgement

Cell

Cell

Blood

Blood

Immunol Today

J Biol Chem

Biochem Pharmacol

Trends Pharmacol Sci

Molecular cloning and functional expression of a new human CC-chemokine receptor gene

Biochemistry

CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1

Science