Biased Signalling Might be the Answer to the Inconsistent Pharmacology of GPR55

GPR55 has been proposed to be a third cannabinoid receptor. However, its pharmacology, including its potential ligands and signaling is uncertain. Here, GPR55 signaling was studied in HEK293 cells stably-transfected with human GPR55. Intracellular Ca2+ ([Ca2+]i) was increased by both lysophosphatidylinositol (LPI; F/F0max = 2.79) and virodhamine (Vdh; F/F0max = 4.44). The responses were blocked by CID16020046, a GPR55 antagonist. However, the agonists activated different signaling pathways. In the presence of the Rho protein inhibitor, C3 toxin (20 µM), the Vdh-induced increase in [Ca2+]i was reduced by 66% (F/F0max = 1.8). A pull down assay for activated Rho protein confirmed the role of Rho in Vdh signaling by GPR55. Also, inhibition of ROCK (a downstream signaling molecule to Rho) with 50µM Y27632, did not affect LPI responses while those to Vdh were reduced by 18% (F/F0max = 3.6). Gα protein knockdown with siRNA showed that Vdh activated Gα13 (70% reduction in the [Ca2+]i signal (F/F0max = 1.3) but not Gαq (3% reduction). In contrast, LPI was more dependent on Gαq (61% reduction in [Ca2+]i signal, F/ F0max = 1.1). siRNA knockdown of PLC subtypes showed that PLCε, the direct effector of Rho protein, largely affected [Ca2+]i responses to Vdh (F/F0max = 1.5), but PLCβ, the main subtype of PLC activated by Gαq, was involved in LPI activation of GPR55 (F/F0max = 1.5). Thus while both agonists activated GPR55 and evoked [Ca2+]i elevation, Vdh was largely dependent on the G13-Rho-PLCε pathway, but LPI seemed to activate Gq and PLCβ. Biased agonism at GPR55 may help explain the inconsistencies in GPR55 pharmacology seen with an array of ligands.