Expression and characterization of the chemokine receptor CCR2B from rhesus monkey

• Published in: Biochemical pharmacology Vol. 66; no. 2; pp. 321 - 330
• Main Authors: Jin, Hong, Vicario, Pasquale P., Zweerink, Hans, Goyal, Shefali, Hanlon, William A., Dorn, Conrad P., Mills, Sander G., DeMartino, Julie A., Cascieri, Margaret A., Struthers, Mary
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 15-07-2003; Elsevier Science
• Subjects: Amino Acid Sequence; Animals; Antagonist; Biological and medical sciences; Calcium - metabolism; CCR2B; Chemokine; Chemokine CCL2 - metabolism; CHO Cells; Cloning, Molecular; Cricetinae; Female; Flow Cytometry; Humans; Macaca mulatta; MCP-1; Medical sciences; Molecular Sequence Data; Radioligand Assay; Receptor; Receptors, CCR2; Receptors, Chemokine - genetics; Receptors, Chemokine - metabolism; Rhesus monkey; Sequence Homology, Amino Acid; Transfection; MCP-1; RANTES; EAE; MIG; MDC; CCR2B; Chemokine; Rhesus monkey; GF/B; HIV; MIP; Receptor; FBS; TARC; Antagonist; COPD; Rhesus monkeys
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(03)00245-4

Species selectivity of chemokine receptor antagonists is a potential deterrent to making preclinical assessments
in vivo. To determine if rhesus monkey disease models could support these assessments, we pharmacologically and functionally characterized recombinant rhesus CCR2B receptor. For these studies we obtained the CCR2B coding region by PCR from genomic rhesus DNA and expressed the receptor as stable transfectants in Chinese Hamster Ovary cells. The surface expression of recombinant rhesus CCR2B was detected by flow cytometry using a commercially available monoclonal anti-hCCR2B antibody. This antibody was used to detect rhCCR2B on monocytes in peripheral blood mononuclear cell preparations from rhesus whole blood. The recombinantly expressed CCR2B exhibited similar high affinity binding to the CCR2 chemokine ligands from rhesus and human
125
I
-rhMCP-1 (
K
d
=433±14
pM) and
125
I
-hMCP-1 (
K
d
=550±256
pM). In competition binding, the receptor exhibited selective high affinity binding to the monocyte chemoattractant protein (MCP) family chemokines with little affinity for most other members of the CC family of chemokines. One exception was eotaxin, a high affinity ligand for CCR3, which bound to rhesus CCR2B receptor (
K
i
=1467±205
pM). Chemokines which exhibited binding affinity for the receptor were tested for their ability to induce intracellular calcium release. In these experiments the relative potencies of the MCP family of chemokines for rhCCR2B were similar to the observed binding affinities. In contrast, eotaxin was functionally inactive as an antagonist or agonist to this receptor. TAK-799 (
N,
N-dimethyl-
N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5
H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2
H-pyran-4-aminium chloride), a dual CCR2/CCR5 antagonist, demonstrated high affinity for the rhesus CCR2B in competition with
125
I
-hMCP-1 binding to the receptor (
K
i
=0.5
nM) and also potently blocked the MCP-1 induced calcium mobilization mediated through the receptor.