Pharmacological characterization of the chemokine receptor, hCCR1 in a stable transfectant and differentiated HL‐60 cells: antagonism of hCCR1 activation by MIP‐1β

Abstract

1 C-C chemokine receptor-1 (CCR1) has been implicated in mediating a variety of in¯ammatory conditions including multiple sclerosis and organ rejection. Although originally referred to as the MIP-1a/RANTES receptor, CCR1 is quite promiscuous and can be activated by numerous chemokines. 2 We used radioligand binding and [ 35 S]-GTPgS exchange assays in membranes from a cell line transfected to express CCR1 (Ba/F3-hCCR1) to characterize a panel of chemokines (HCC-1, MIP1a, MIP-1b, MIP-1d, MPIF-1, MCP-2, MCP-3, and RANTES) as CCR1 ligands. In this recombinant model, these chemokines displaced 125 I-MIP-1a with a wide range of potencies and, with the exception of MCP-2, acted as full agonists in stimulating [ 35 S]-GTPgS exchange. 3 We then assessed the utility of HL-60 cells cultured with known di erentiating agents (PMA, DMSO, dibutyryl-cAMP or retinoic acid) for investigating CCR1 pharmacology. In [ 35 S]-GTPgS exchange assays, membranes from cells cultured with retinoic acid (4 ± 6 days) were the most responsive to activation by MIP-1a and MPIF-1. FACS analysis and comparative pharmacology con®rmed that these activities were mediated by CCR1. HCC-1, haemo®ltrate CC chemokine 1; IP-10, interferon-inducible protein of 10 kDa; I-TAC, interferoninducible T cell a chemoattractant; IL-8, interleukin-8; MIP-1a, macrophage in¯ammatory protein-1a; MIP-1b, macrophage in¯ammatory protein-1b; MIP-1d, macrophage in¯ammatory protein-1d; MCP-1, MCP-2, MCP-3, monocyte chemotactic protein-1, -2 and -3; Met-RANTES, methionylated RANTES; Mig, monokine-induced by human interferon g; MPIF-1, myeloid progenitor inhibitor factor-1; NAP-2, neutrophil activating peptide-2; RANTES, regulated upon activation, normal T cell expressed and secreted; WGA-SPA, wheat germ agglutinin bead-scintillation proximity assay

Differential chemokine activation of CC chemokine receptor 1‐regulated pathways: ligand selective activation of Gα 14‐coupled pathways

“…We determined that MIP-1ˇhad an IC 50 value for CCR1/G § 14 /293 cells of approximately 60 nM (600 ng/ml; Fig. 6), and this is in good agreement with the value obtained by Chou et al [6]. In our IPformation assays the highest concentration of MIP-1ǔ sed was approximately 50 nM (500 ng/ml), which should result in the occupation of a high percentage of the receptors present in the system.…”

“…[21] demonstrated that MIP-1ˇhad a greater affinity than MIP-1 § for human neutrophils, 1.1 nM versus 3.2 nM. However, Chou et al [6] determined that in murine IL-3-dependent pro-B cells, MIP-1 § (IC 50 =56 pM) had a higher affinity than MIP-1ˇ(IC 50 =33 nM). In addition, they determined that MIP-1ˇwas a full agonist with an EC 50 value for stimulation of GTP + S binding to cell membranes of 39 nM.…”

“…A remarkable feature of the chemokine receptors is their relative lack of selectivity in ligand binding, with many chemokine receptors binding more than one chemokine with high affinity. For example, 11 chemokines are reported to bind to CC chemokine receptor (CCR) 1, including macrophage inflammatory protein (MIP)-1 § , MIP-1 g , MIP-1ˇ, regulated upon activation, normal T cell-expressed and secreted (RANTES), monocyte chemotactic peptide (MCP)-1, MCP-2, MCP-3, MCP-4, leukotactin-1 (Lkn-1), myeloid progenitor inhibitory factor (MPIF)-1, and hemofiltrate CC chemokine (HCC)-1 [4,6]. This promiscuity and the apparent redundancy of signaling pose many questions to the control of chemokine signaling in different tissues expressing different combinations of chemokines, receptors, and effectors.…”

CC Chemokine Receptors and Chronic Inflammation—Therapeutic Opportunities and Pharmacological Challenges

“…CCL23 (MPIF-1) . CCL5 $ CCL4 (Chou et al, 2002). Furthermore, CCL4 was found to antagonize responses to the other ligands, suggesting it could be an endogenous inhibitor of CCR1 activity.…”

Modulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) receptors in a human osteoclast model in vitro

“…One remarkable feature of these receptors is their apparent lack variety of selectivity in regards to ligand binding. For example, MIPlais one of eleven identified chemokines associated with CCR1 binding and activation (Chou et al, 2002). Likewise, while a receptor for MIP-la, CCR5 has received the majority of its publicity through the identification of its role as a co-receptor for HIV (Deng et al, 1997).…”