The molecular cloning and functional expression of the dog CCR5.
Activation of CCR5 by specific chemokines is involved in the regulation of the immunological response of leukocytes at sites of inflammation. In addition, CCR5 serves as a fusion co-factor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). Consequently, several CCR5 antagonists are currently in development for the treatment of HIV-1 infection. The dog CCR5 gene was cloned in order to characterise the chemokine binding site of the dog receptor for comparison across species. The deduced amino acid sequence of the dog CCR5 has close homology to the human receptor (80% identity). A HEK-293 cell line expressing the dog recombinant receptor was generated and immunoblot analysis with an anti-human CCR5 antibody revealed a 58kDa band in the cell lysate. In functional calcium signalling assays, the CCR5 endogenous ligands MIP-1alpha, MIP-1beta and RANTES evoked a robust response in the dog recombinant CCR5 cells. In a CRE-Luc (cAMP response element-luciferase) reporter gene assay, MIP-1beta (0.01-30nM) produced concentration-dependent inhibition of forskolin induced elevation in cAMP levels, and was equipotent in dog, human and macaque recombinant CCR5 cells (EC(50) 0.4, 0.21 and 0.47nM, respectively). These data suggest that chemokine signalling is conserved in the dog CCR5.
Mosley
Michael
M
Pfizer Global Research & Development, Discovery Biology, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK. michael.mosley@pfizer.com
Pullen
Sarah
S
Botham
Andrew
A
Gray
Andrew
A
Napier
Carolyn
C
Mansfield
Roy
R
Holbrook
Mark
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D002460:Cell Line; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5 / Q000276:immunology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000276:immunology; D004285:Dogs / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000276:immunology; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D014162:Transfection / Q000662:veterinary
D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018946:Chemokine CCL5; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D011994:Recombinant Proteins; D012313:RNA; D000242:Cyclic AMP