Pharmacological Profiling of Lysophosphatidylinositol Species at GPR55 Using xCELLigence Cellular Impedance Analysis

Abstract

L-α-lysophosphatidylinositol (LPI) is the endogenous ligand for GPR55, a Gα13-coupled GPCR. The most abundant endogenous LPI species are stearoyl-LPI (16:0 LPI), palmitoyl-LPI (18:0 LPI) and arachidonoyl-LPI (20:4 LPI). The pharmacology of ligands at GPR55 is contentious therefore further studies are required to elucidate the pharmacology of GPR55 in native cells. This study aimed to elucidate the signaling profiles of LPI species in stably expressing GPR55-HEK293 cells and human osteoclasts using xCELLigence cellular impedance in order to validate this approach as a screening tool for GPR55 ligands. In GPR55-HEK293 cells, all LPI species produced a dose dependent decrease in cellular impedance that was absent in HEK293 cells and antagonised by GPR55 antagonists CID1261822 and CID16020046. Immunocytochemical staining confirmed that the decrease in impedance was attributable to cellular retraction. GPR55 mediated signaling via Gαi, Gαs, Gαq/11 or Gβγ subunits was excluded due to lack of inhibition by pre-treatment with pertussis toxin, SQ22536, U73122 and gallein respectively. Responses to all species were completely inhibited by Y27632 and H89 suggesting that the main pathways contributing to the decrease in impedance are Rho and PKA. Interestingly, the response to 20:4 LPI was also significantly attenuated by the PKC inhibitor Bisindolylmaleimide II, indicative of biased agonism. Treatment with all LPI species in human osteoclasts produced a dose dependant decrease in cellular impedance attributed to Rho, ERK and PKC as well as Gi and Gβγ, reflective of more diverse signaling. These experiments validate use of xCELLigence technology to screen novel GPR55 ligands in native cells.