The conformational signature of β-arrestin2 predicts its trafficking and signalling functions

Abstract

Arrestins are cytosolic proteins that regulate G protein-coupled receptor (GPCR) desensitization, internalization, trafficking, and signaling1,2. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets them for internalization via clathrin-coated pits3,4. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G protein effectors into GPCR-based ‘signalsomes’5,6. While the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify divergent arrestin functions is not understood. Using a panel of intramolecular FlAsH-BRET reporters7 to monitor conformational changes in arrestin3, we show here that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor-arrestin complex and role of arrestin3 in activating or dampening downstream signaling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in arrestin3 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average arrestin3 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands8,9 and in identifying factors that dictate arrestin conformation and function.

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

“…This is in agreement with previous observation where JNK3 translocation to AT1R was detected (50). Interestingly, the kinetics of MEK1 and ERK2 recruitment were similar to the dynamics of receptor-β-arrestin2 interaction (Figure 8c), and did not follow that of F263 (Figure 5b), which was suggested previously to correlate with ERK1/2 activation (40). Strikingly, PMA stimulation led to a significant increase in the signal, although the amplitude was lower than that observed with AngII ( Figure 8b) and was proportional to the magnitude of β-arrestin2 recruitment (Figure 8c).…”

“…In this setup, F154 and F225 signals were abolished, whereas F263 and F410 signals were reversed upon AngII treatment (Figure 6c). Signal changes of F225 and F410 are in good agreement with the previous observation, that activation of these constructs correlates with the stability of the receptor-β-arrestin2 complex (40). Next, we constructed β-arrestin2-FlAsH sensors with K11A and K12A mutations (F139-K2A, F154-K2A, F225-K2A, F263-K2A and F410-K2A).…”

“…Upon stimulation with AngII, we detected BRET signal increase with F154, F410, and decrease with F139, F225, F263 ( Figure S3a). The direction of F139, F225, F263 and F410 signal changes were identical to those previously described using the similar FlAsH sensors with AT1R (40).…”

“…In these constructs, BRET signal is measured between two parts of the same protein, so that changes in the signal indicate conformational rearrangements within the molecule. The sensors have been designed in a way that the positions of the FlAsH binding cysteines match the previously published β-arrestin2 BRET and FRET sensors (40,41) (Figure 5a). CCPGCC binding motifs for the FlAsH were incorporated after residues 139, 154, 225, 263 or 410 (F139, F154, F225, F263 and F410 sensors, respectively).…”

“…Venus-Rab4, Venus-Rab7 and Venus-Rab11 were created by replacing EYFP to monomeric Venus in YFP-Rab4, YFP-Rab7 and YFP-Rab11 constructs (48). To generate Rluc8-β-arrestin2, the coding sequence of rat β-arrestin2 was subcloned from YFP-β-arrestin2 (72) rd or 410 th positions of β-arrestin2 using precise gene fusion PCR, the positions were selected based on earlier studies using similar sensors (40,41). The K2A-β-arrestin2 mutants were made analogously to bovine K2A-β-arrestin2 (32); K11A and K12A mutations were introduced to rat β-arrestin2 by precise gene fusion PCR to create K2A-β-arrestin2-Venus and Rluc8-K2A-β-arrestin2.…”