We haven’t done anisotropic, which is a good idea since labeling the arrestins isn’t much of an issue. We do observe differences between different receptors in terms of their recruitment of different orientations of oligomers.

Thanks!

We are doing some experiments now where we induce dimerization of beta arrestins before receptor activation. Depending on the orientation of the dimers, it can inhibit its function. But it looks like certain orientations function just as well as WT arrestins.

We think this finding may explain how beta-arrestins interact with a wide range of proteins and how they may organize signaling at the receptor. This is just the start! We are currently performing additional experiments and MD simulations to model these potential interactions.

Consistent with this, we found that a constitutionally active ADGRE1 (ADGRE1-b – lacking most of the extracellular domain but maintain the tethered agonist sequence) promoted condensates, unlike the apo receptor (ADGRE1 FL) or a receptor without tethered agonist (ADGRE-1 bT).

While the beta-arrestins in this 2:2 complex were not directly interacting, they were in a conformation that could promote an N-N interaction that was identified through metadynamics. This suggested that receptor activation could promote beta-arrestin oligomerization.

Serendipitously, Peng Xiao and Jinpeng Sun solved the structure of an adhesion receptor that forms tight complexes with beta-arrestins. Surprisingly, this structure displayed a 2:2 complex with the beta-arrestin at a unique angle compared to other GPCR:beta-arrestin structures.

Previous work from the Marullo, Benovic, and Gurevich labs identified specific sites in beta-arrestins that bind inositol hexaphosphate (IP6) to regulate beta-arrestin oligomerization. We found those mutations impacted beta-arrestin-dependent internalization and GPCR signaling.

We then used an optogenetic approach to test if these were consistent with condensates. When Cry2 undergoes light-induced oligomerization, the presence of specific sequences (such as IDRs) leads to condensate formation (see FUSN). Beta-arrestin 1 had a similar effect!

We performed FRAP that demonstrated that proteins in these puncta exchanged with solution, although with slightly slower kinetics than beta-arrestin 2 in the cytosol.

It has been known for decades that beta-arrestins oligomerize, but the relevance of this in a cellular context is unknown. Labeling beta-arrestin 2 with GFP results in a cytoplasmic pattern, while labeling with components of split GFP results in punctae, consistent with oligomers.

While useful in populations, another study clearly demonstrated that PRS in individuals is not useful. Would love to know your thoughts on how to reconcile these findings. jamanetwork.com/journals/jam...

This is usually the premise behind a horror movie…

Similar finding with resolution of chest pain after stress test or cath…