Recent Assays in GPCR Deorphanization

Recent advances clearly show that not all receptor actions rely on the activation of heterotrimeric G proteins but rather can be promoted via activation of G protein - independent and/or P-arrestin-dependent pathways [45]. Two orphans that do not signal through G protein-dependent pathways are GPCR C5L2 (also known as GPR77) [46, 47] and the D6 chemokine receptor [48] . Whereas the first binds to C5a anaphylotoxin, the latter binds to chemokines. No evidence has been found for the activation of intracellular signaling pathways upon ligand binding to these receptors, which are also called decoy receptors as the ligands are captured and cointernalize with the receptor inside the cell.

GPCR-Ga Fusion Proteins Construction of fusion proteins between GPCRs and Ga subunits was reported more than 10 years ago [49]. The same strategy has more recently been applied to the deorphanization of orphan 7TM proteins [50, 51] . Typically, orphan 7TM proteins are fused to the promiscuous Ga16 protein, and receptor activation is monitored by measuring [35S] GTPyS binding in membranes prepared from Sf9 cells expressing the fusion protein [52]. Sf9 insect cells provide an excellent high-level expression system that is nearly devoid of mammalian endogenous G proteins. Using this approach, the 5- oxo- eicosatetraenoic acid was successfully identified as the ligand for GPCR48 [53, 54] followed by the deorphanization of GPR87 [55] and GPR120 [56].

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