title: Expression and characterization of the chemokine receptor CCR2B from rhesus monkey.
abstract: 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 125I-rhMCP-1 (K(d)=433+/-14 pM) and 125I-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-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]tetrahydro-2H-pyran-4-aminium chloride), a dual CCR2/CCR5 antagonist, demonstrated high affinity for the rhesus CCR2B in competition with 125I-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.
authors.affiliation: Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Jin
authors.forename: Pasquale P
authors.initials: PP
authors.lastname: Vicario
authors.forename: Hans
authors.initials: H
authors.lastname: Zweerink
authors.forename: Shefali
authors.initials: S
authors.lastname: Goyal
authors.forename: William A
authors.initials: WA
authors.lastname: Hanlon
authors.forename: Conrad P
authors.initials: CP
authors.lastname: Dorn
authors.forename: Sander G
authors.initials: SG
authors.lastname: Mills
authors.forename: Julie A
authors.initials: JA
authors.lastname: DeMartino
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Cascieri
authors.forename: Mary
authors.initials: M
authors.lastname: Struthers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D018932:Chemokine CCL2 / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D054390:Receptors, CCR2; D019707:Receptors, Chemokine / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: C515334:CCR2 protein, human; D018932:Chemokine CCL2; D054390:Receptors, CCR2; D019707:Receptors, Chemokine; D002118:Calcium