How membranes shape the β-arrestin conformational landscape to provide spatiotemporal control of GPCR desensitization and trafficking

Abstract ID 99322 Poster Board 558 G protein-coupled receptors (GPCRs) are a family of seven transmembrane proteins that function to transduce extracellular signals, such as hormones and neurotransmitters, into intracellular signals. To achieve temporal regulation of signaling, a family of proteins known as ß-arrestins are responsible for terminating G protein-mediated signaling and desensitizing receptors. These ß-arrestin proteins are also responsible for mediating the internalization of most GPCRs, and thus dictating their recycling and re-sensitization behavior. For a ß-arrestin to engage a GPCR it must transition from a basally autoinhibited state to an active state. The prevailing mechanism for ß-arrestin activation involves displacement of its autoinhibitory C-terminal tail (C-tail) by the phosphorylated C-terminus of a GPCR. However, this mechanism poses several challenges. First, it would require a ß-arrestin protein to embark on a 3D-search to identify the target GPCR and second it fails to address how GPCRs with little or no C-terminal phosphorylation are still able to activate -arrestins. Recently we showed that membrane phosphoinositides, such as PI(4,5)P2, induce conformational changes in ß-arrestin1 and represent an integral component of the GPCR-ß-arrestin interface. We believed the involvement of phosphoinositides in GPCR-ß-arrestin complex assembly allows for spatiotemporal control of complex assembly. Here we use a combination of biophysical techniques, including hydrogen-deuterium exchange mass spectrometry (HDX-MS) and single-molecule Förster resonance energy transfer (smFRET), to investigate the assembly and dynamics of GPCR-ß-arrestin complexes. We find that the -arrestin C-tail is intrinsically dynamic, even in its autoinhibited state. Mutations to the C-tail that compromise the inactive state show faster complex assembly in cells and spend an increased proportion of time in an intermediate "active-like" state (henceforth primed state) as seen by smFRET. Interestingly, PI(4,5)P2 acts as an allosteric modulator of ß-arrestin dynamics, shifting its equilibrium towards the primed state. In the context of GPCR engagement, our data suggest a mechanism for -arrestin engagement where conformational changes induced by membrane phosphoinositides may be necessary for GPCR core engagement. Ongoing work investigating the integration of phosphoinositide inputs with other -arrestin inputs (e.g., GPCR phosphorylation, GPCR conformation) will also be described. Together, this work provides valuable mechanistic insights into the process of ß-arrestin activation and the assembly of GPCR--arrestin complexes, a process of crucial importance for regulating diverse physiological processes.