A genetically-encoded nanobody sensor reveals conformational diversity in β-arrestins orchestrated by distinct seven transmembrane receptors

Agonist-induced interaction of G protein-coupled receptors (GPCRs) with β-arrestins (βarrs) is a critical mechanism that regulates the spatio-temporal pattern of receptor localization and downstream signaling. While the underlying mechanism governing GPCR-βarr interaction is primarily conserved and involves receptor activation and phosphorylation, there are several examples of receptor-specific fine-tuning of βarr-mediated functional outcomes. Considering the key contribution of conformational plasticity of βarrs in driving receptor-specific functional responses, it is important to develop and characterize novel sensors capable of reporting distinct βarr conformations in cellular context. Here, we design an intrabody version of a βarr-recognizing nanobody (nanobody32), referred to as intrabody32 (Ib32), in NanoLuc enzyme complementation assay format, and measure its ability to recognize βarr1 and 2 in live cells upon activation of a broad set of GPCRs. We discover that Ib32 robustly recognizes activated βarr1 and 2 in the plasma membrane as well as in the endosomes, and effectively mirrors βarr recruitment profile upon stimulation of GPCRs. We also design an Ib32 sensor for single-photon polarization microscopy with a change in linear dichroism as readout and demonstrate its utility for monitoring βarr activation upon stimulation of angiotensin receptor by its natural and biased agonists. Interestingly, when used side-by-side with a previously described sensor of βarr1 conformation known as Ib30, Ib32 uncovers distinct conformational signatures imparted on βarrs by different GPCRs, which is further corroborated using an orthogonal limited proteolysis assay. Taken together, our study presents Ib32 as a novel sensor to monitor βarr activation and leverages it to uncover conformational diversity encoded in the GPCR-βarr system with direct implications for improving the current understanding of GPCR signaling and regulatory paradigms.