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

• 成果类型: Article
• 署名作者: Sarma, Parishmita; Markova, Vendula Nagy; Dalal, Annu; Mishra, Sudha; Zaidi, Nashrah; Tiwari, Divyanshu; Yadav, Manish K.; Roy, Nabarun; Mahajan, Gargi; Miclea, Paul; Lazar, Josef; Shukla, Arun K.
• 署名单位: Indian Institute of Technology System (IIT System); Indian Institute of Technology (IIT) - Kanpur; Charles University Prague; Czech Academy of Sciences; Institute of Organic Chemistry & Biochemistry of the Czech Academy of Sciences; Charles University Prague
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11209
• DOI: 10.1073/pnas.2507384122
• 发表日期: 2025-09-16
• 关键词: protein-coupled receptors; biosensors reveal; biased agonism; phosphorylation; beta-arrestin2; TRAFFICKING; endocytosis; domain

摘要

Agonist-induced interaction of G protein-coupled receptors (GPCRs) with beta- arrestins (beta arrs) is a critical mechanism that regulates the spatiotemporal pattern of receptor localization and signaling. While the underlying mechanism governing GPCR-beta arr interaction is primarily conserved and involves receptor activation and phosphorylation, there are several examples of receptor-specific fine-tuning of beta arr-mediated functional outcomes. Considering the key contribution of conformational plasticity of beta arrs in driving receptor-specific functional responses, it is important to develop novel sensors capable of reporting distinct beta arr conformations in cellular context. Here, we design an intrabody version of a beta arr-recognizing nanobody (nanobody32), referred to as intrabody32 (Ib32), in NanoLuc enzyme complementation assay format and measure its ability to recognize beta arr1 and 2 in live cells upon activation of a broad set of GPCRs. Ib32 robustly recognizes activated beta arr1 and 2 in the plasma membrane and endosomes, and effectively mirrors beta arr 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 beta arr activation upon stimulation of selected GPCRs by natural and biased agonists. Taken together with a previously described sensor of beta arr1 activation, Ib32 underscores the inherent flexibility encoded in beta arrs 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 beta arr activation and highlights the structural diversity of beta arrs, which likely allows their ability to interact with, and regulate, a large repertoire of GPCRs.