Figure 4.

Identification of RvD1 GPCR. (A) Phylogenetic tree shows similarities in the amino acid sequences of human GPCRs closely related to LXA₄, RvE1, and LTB₄ receptors (left panel). Cluster was generated with the ClustalW2 software (www.ebi.ac.uk/Tools/clustalw2). Protein sequences were deduced from the NCBI database and receptor nomenclature followed the IUPAR classification for GPCR. (B) For functional screening and identification of RvD1 receptor, GPCRs were cloned in pcDNA3 vector and overexpressed in human HeLa cells cotransfected with pNF-κB luciferase plasmid. Cells were treated with RvD1 and TNF-α (see inset in the right panel). RvD1 reduces TNF-α-stimulated NF-κB activation in DRV1/GPR32 and ALX/FPR2 receptor-overexpressing cells, while cells transfected with other related GPCRs (e.g., BLT1, BLT2, CB1, GPR-1, FPR, and ChemR23) did not significantly inhibit TNF-α-induced NF-κB luciferase activity on addition of the ligand RvD1. The results illustrated are expressed in luminescence units subtracted from pNF-κB and pcDNA3 empty vector (*P < 0.05 vs. BLT2-transfected cells).