Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy

Abstract

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Figure 1. Responses of Hut-78 T cells to CCR4 ligands

(A) Hut78 cell migration to increasing concentrations of CCL17 and CCL22. CCR4 expression on Hut78 cells using 1G1 (B) and 10E4 (C) mAbs; shaded histograms are isotype controls. (D) CCR4 endocytosis on Hut78 cells following chemokine treatment measured using CCR4 mAbs. n=3, *p<0.05, **p<0.01 and ***p<0.001.

Figure 2. Comparison of staining of CEM-4 cells, CCR4 L1.2 transfectants and primary Th2 lymphocytes with the 10E4 and 1G1 antibodies.

Data are representative of at least three separate experiments.

Figure 3. Responses of human Th2 cells to CCR4 ligands

Homologous (A & B)/heterologous (C & D) desensitization of calcium responses following CCL17/CCL22 treatment. Arrows indicate addition of chemokine. Data are representative of three independent experiments. (E) Calcium release of Th2 cells after CCL17/CCL22 treatment. EC₅₀ values = 1.6 nM and 2.5 nM respectively. n=3.

Figure 4. CCL22 and CCL17 are conformationally selective ligands of CCR4

Displacement of ¹²⁵I-CCL17 (A & B) and ¹²⁵I-CCL22 (C & D) from CEM-4 cells (A & C) or CCR4-L1.2s (B & D) by increasing concentrations of unlabelled chemokine. n=3.

Figure 5. 10E4 blockade of CCL17 and CCL22 binding

Binding of ¹²⁵I-CCL17 (A) and ¹²⁵I-CCL22 (B) to CCR4 transfectants in the presence of unlabelled chemokine, an isotype control (IgG_2a) mAb or the CCR4-specific mAb 10E4. (B) and (C) - Migration of CCR4 transfectants to CCL17 (C) or CCL22 (D) following pre-treatment with either 10E4, 1G1, or both antibodies combined (closed symbols and dashed lines). Isotype treated cells are shown as open symbols and solid lines. Competitive staining of Hut78 cells with the 10E4 and 1G1 antibodies (E). n=3,*p<0.05, **p<0.01 and ***p<0.001.

Figure 6. A role for K310N in CCR4 activation by CCL17

Migration of WT CCR4-L1.2 or K310N CCR4-L1.2 to increasing concentrations of CCL17 (A) or CCL22 (B). Homologous displacement binding curves for ¹²⁵I-CCL17 (C) and ¹²⁵I-CCL22 (D). Staining of CCR4-L1.2 or K310N CCR4-L1.2 transfectants using 10E4, 1G1 or anti-HA mAbs (E). n=5, *p<0.05, **p<0.01 and ***p<0.001.

Figure 7. A two-state model for CCR4 activation

Cartoon of CCR4 showing two receptor conformations (R1 and R2) which are differentially bound and activated by CCL17 and CCL22. R1 is the major species as assessed by ligand binding assays and staining with the 10E4 antibody (and to a lesser extent by the 1G1 antibody in grey). The importance of residue K310 in CCR4 activation by CCL17 is highlighted in the R1 conformation, although this residue has no effect upon the 10E4 sensitive-conformation of the R1 state.

Figure 8. Putative salt bridge in CCR4 predicted by homology modelling

Crystal structures of bovine rhodopsin (blue N-terminus through red C-terminus), squid opsin (gold) and CXCR4 (magenta), and a CCR4 homology model (green). The cutout shows the residues corresponding to lysine 310 of CCR4, which is thought to form a salt bridge with a highly conserved aspartate residue in TMII.