Arrestin C-tail dynamics regulate activation and GPCR engagement.

Abstract

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 called beta-arrestins function to terminate G protein-mediated signaling and desensitizing receptors. beta-arrestins also mediate the internalization of most GPCRs, and thus dictating their recycling and re-sensitization behavior. For a beta-arrestin to engage a GPCR it must transition from a basally autoinhibited state to an active state. The prevailing mechanism for beta-arrestin activation involves displacement of the beta-arrestin autoinhibitory C-terminal tail (C-tail) by the phosphorylated C-terminus of a GPCR. However, this mechanism raises questions as to how GPCRs which exhibit little C-terminal phosphorylation are still able to activate beta-arrestins. Using a combination of biophysical techniques, from in-cell proximity assays to hydrogen-deuterium exchange mass spectrometry (HDX-MS) and single molecule Förster resonance energy transfer (smFRET), we investigated the assembly and dynamics of GPCR-beta-arrestin complexes. We find that the beta-arrestin C-tail is intrinsically dynamic, but largely occupies the autoinhibited state. However, mutations to the C-tail which compromise the inactive state show faster complex assembly in cells and spend an increased proportion of time in an intermediate “active-like” state as seen by smFRET. Further, we find that membrane phosphoinositides act as allosteric modulators of beta-arrestin dynamics, altering the behavior of the C-tail through binding to a distal region of the beta-arrestin. In the context of GPCR engagement, our data show that membrane-derived allosteric inputs function in concert with canonical beta-arrestin inputs (e.g., GPCR phosphorylation, GPCR conformation) to regulate GPCR-beta-arrestin complex assembly. Together, our studies provide valuable mechanistic insights into the process of beta-arrestin activation and the assembly of GPCR-beta-arrestin complexes, a process of crucial importance for regulating diverse physiological processes.