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 spatiotemporal pattern of receptor localization and 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 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. Ib32 robustly recognizes activated βarr1 and 2 in the plasma membrane and endosomes, and effectively mirrors βarr recruitment profile upon stimulation of selected GPCRs. We also design an Ib32 sensor for polarization microscopy with a change in linear dichroism as readout and demonstrate its utility for monitoring βarr activation upon stimulation of selected GPCRs by natural and biased agonists. Taken together with a previously described sensor of βarr1 activation, Ib32 underscores the inherent flexibility encoded in βarrs and conformational diversity imparted by different GPCRs, which is further corroborated using an orthogonal limited proteolysis assay. Our study presents Ib32 as a sensor of βarr activation and highlights the structural diversity of βarrs, which likely allows their ability to interact with, and regulate, a large repertoire of GPCRs.