Modulation of cannabinoid CB₁ receptor signalling via the interacting protein CRIP₁ᴀ and via biased agonism

Cannabinoid CB₁ receptors are widely expressed G protein coupled receptors (GPCRs) and given their many biological roles in the brain, the CB₁ receptor presents an intriguing target for the treatment of central nervous system disorders (Mackie, 2006; Zhang et al., 2009). Although multiple exogenous cannabinoid ligands have been developed, their therapeutic applications are limited due to adverse effects. Selective activation of CB₁ receptor-mediated signalling events that mediate desired therapeutic effects at the expense of those that mediate adverse effects may overcome the unwanted side effects. This may be achieved via interacting proteins and ligand-biased signalling. Cannabinoid CB₁ receptor signalling is mediated by Gi/o proteins and leads to the inhibition of adenylyl cyclase, regulation of ion channels, and activation of members of the mitogen-activated protein kinase (MAPK) family (Howlett, 2005). A novel protein, Cannabinoid Receptor Interacting Protein 1a (CRIP₁a) binds to the distal C-terminus of the CB₁ receptor and alters CB₁ receptor-mediated neuronal function (Niehaus et al., 2007; Stauffer et al., 2011). However the mechanisms by which CRIP₁a regulates CB₁ receptor signalling remain largely unknown. This study initially established and validated two complimentary and robust models to study CRIP₁a in a gain-of-function and loss-of-function context. These models were used to investigate the effect of CRIP₁a on agonist-mediated CB₁ receptor signalling and furthermore, to detect and quantify biased agonism at the CB₁ receptor in the absence and presence of CRIP₁a. We show for the first time that CRIP₁a is able to modulate Gα protein-coupling in recombinant HEK293-CB₁-TREx CRIP₁a cells when examined over time using kinetic BRET studies. Importantly, over-expression of CRIP₁a attenuated Gαo and Gαi₁ protein-coupling in response to the endocannabinoids, 2-AG and methanandamide. Knockdown of CRIP₁a in AtT-20 cells inherently expressing CRIP₁a was found to potentiate endocannabinoid-mediated GIRK channel activation whilst overexpression of CRIP₁a had no effect on cannabinoid-mediated cAMP inhibition or pERK1/2 activation in recombinant HEK293 cells. Biased agonism at the CB₁ receptor was investigated to establish potential CB₁ receptor bias profiles that may guide structure-activity studies and the drug discovery process towards the development of selective therapeutics with reduced adverse effects. Our results show that the endocannabinoids, 2-AG and methanandamide, display different bias profiles. 2-AG showed an overall preference for G protein activation over pERK1/2 activation, with bias factors of over 10 for 2-AG-mediated Gαo and Gαi₃ protein activation and of over 25 for Gαi₁ protein activation. In contrast, methanandamide displayed a bias factor of over 15 for Gαio protein activation over pERK1/2 activation, whilst it was found to not couple efficiently to Gαi₁₋₃ proteins in HEK293 cells. Exogenous cannabinoids also displayed distinct biased profiles however, CRIP₁a was found to have no effect on the bias profile of any of the cannabinoid ligands tested in HEK293 cells. Thus, this thesis reveals various cellular mechanisms by which the CB₁ receptor interacting protein, CRIP₁a, modulates CB₁ receptor signalling, highlighting the cell line-dependent effects of CRIP₁a, and in addition, provides quantitative insights into biased agonism at the CB₁ receptor. These novel insights may contribute to the development of selective CB₁ receptor-targeted therapies.