Identification of CCR8, the receptor for the human CC chemokine I-309.

Abstract

The nucleotide sequence for a putative chemokine receptor, termed TER1, ChemR1, or CKR-L1, was recently obtained by a polymerase chain reaction-based cloning technique. It encodes a protein of 355 amino acids that shows 32-45% sequence identity with human chemokine receptors. The gene was localized on human chromosome 3p21-24, the site for the genes for the five known CC chemokine receptors, suggesting that the natural ligand may be a CC chemokine. We have stably expressed this receptor in murine pre-B cells 300-19 and have tested their responsiveness to 20 human chemokines and some other potential agonists. The CC chemokine I-309 was the only agonist that selectively induced intracellular Ca2+ mobilization and chemotaxis in receptor-transfected 300-19 cells. Stromal cell-derived factor 1, which binds to murine CXCR4 expressed in parental as well as transfected 300-19 cells, served as positive control in the functional screening. The interaction of I-309 with TER1 was of high affinity as shown by 125I-I-309 binding (Kd of 1.2 nM) and transient [Ca2+]i changes at subnanomolar concentrations of agonist. Migration responses in receptor-transfected 300-19 cells was typically bimodal with maximal activity at 10 nM of I-309. These data demonstrate that TER1 (ChemR1 or CKR-L1) is the receptor for I-309, and we propose to call this receptor CCR8 in agreement with the current nomenclature for chemokine receptors. The expression of CCR8 in blood leukocytes and lymphocytes was analyzed by Northern blot. No transcripts were found in RNA from freshly isolated blood neutrophils, monocytes, cultured macrophages, and phytohemagglutinin-stimulated T lymphocytes, and a faint hybridization signal corresponding to the RNA species of 4 kb was obtained only with RNA from interleukin-2-treated T lymphocytes. CCR8 is unusual for its selectivity for a single chemokine, previously shown only for CXCR1 and CXCR4, which bind interleukin-8 and stromal cell-derived factor 1, respectively. Identification of the receptor for I-309 represents a significant progress in determining the function of I-309 in inflammation and disease.