Mechanisms Underlying Gαi and β-Arrestin Complex Formation at GPCRs

Abstract

G protein-coupled receptors (GPCRs) are targets for almost a third of all FDA-approved drugs. Canonically, GPCRs are known to signal through both heterotrimeric G proteins and β-arrestins. Traditionally these pathways were viewed as largely separable since G proteins primarily initiate downstream signaling while β-arrestins function to regulate receptor desensitization and trafficking in addition to modulating their own signaling. However more recent studies have provided evidence for an integrated role of G protein and β-arrestins in GPCRs signaling through the formation of G protein:β-arrestin:Receptor “megaplexes”. Additionally, G proteins and βarrs were found to associate across a panel of receptors. This association was surprisingly specific for inhibitory G protein (Gαi), even among receptors which do not classically signal through Gαi and suggested that this may be a novel non-canonical method of GPCR signaling. The mechanism required for such interaction and the biochemical and physiological significance remains unknown. To elucidate how this novel complex forms downstream of GPCRs, this dissertation focuses on uncovering mechanisms associated with Gαi: β-arrestin association. Utilizing biochemical and biosensor-based approaches, we show a large portion of selectivity for Gαi in these complexes lies within the N-terminal helical domain. Additionally, we detail the requirements for complex formation where β-arrestin translocation to the plasma membrane is sufficient for complex assembly and, surprisingly, G protein activation and recruitment via GPCRs is dispensable. These findings delineate a novel mechanism for GPCR signaling through β-arrestin recruitment to the plasma membrane that results in complex formation and signaling through novel signaling cascades.