Pleiotropic Signaling in the Endocannabinoid System: The role of the G protein γ3 subunit

The Cannabinoid 1 (Cb1) receptor is increasingly recognized as being involved in numerous pathological and physiological processes. Classical Cb1 receptor signaling occurs at the pre-synaptic terminal, where it couples to Gαi/o proteins to inhibit adenylyl cyclase and mediate retrograde inhibition. It is now well established that Cb1 signaling is pleiotropic and occurs in many different cell types through the actions of different G-protein alpha (α), beta (β) and gamma (γ) subunits. While Gβγ has been identified as having specific roles in this signal transduction process, the unique roles that individual Gγ subunits perform remains elusive. To explore these roles, we transiently overexpressed Cb1 and Gγ3 (Gng3) in a CHO-K1 cell line and measured cAMP accumulation following receptor activation. We hypothesized that overexpression of Gng3 would result in preferential coupling of Gγ3 into the Gαβγ heterotrimer and result in an altered response when compared to cells only expressing Cb1. However, lack of statistical significance and the high variation between trials lead us to reject this hypothesis. Similarly, we used this overexpression model to measure intracellular calcium levels in cells expressing Cb1 or Cb1+gng3. We found that stimulation of Cb1 with Anandamide had no effect on intracellular calcium in either group. Next, we used a CRISPR-Cas9 gene targeting approach against Gg3 in developing Danio rerio (zebrafish) embryos. Our approach at targeting the gng3 allele was successful, with target efficiency of up to 95% in the F0 progeny. Finally, we utilized a series of behavioral assays to measure Visual and Acoustic startle responses in wild type Zebrafish. Herein, we report on these experiments and how additional troubleshooting of these assays are needed before any claims can be made on the mutant phenotype.