Structural Dynamics of GPCR Oligomerization: Impact on Localization and Signaling

ABSTRACT G protein-coupled receptors (GPCRs) are crucial in cellular signaling, transducing extracellular stimuli into intracellular responses. Predominantly located on the plasma membrane, GPCRs are vital in numerous physiological processes, making them key pharmacological targets. Emerging evidence also indicates roles for some GPCRs in various intracellular compartments, signaling in cooperation with soluble molecules or other cellular pathways. Significant advancements in structural biology have enabled the determination of over 350 GPCR structures in complexes with signaling partners, predominantly with G proteins (~96%), but also with β-arrestins (3%) and GPCR kinases (<1%). Nonetheless, our comprehension of their interactions with other regulatory proteins, such as chaperone proteins and membrane-associated proteins, and how these interactions influence GPCR localization and signaling diversity, remains poorly understood. This grant proposal aims to elucidate the structural and mechanistic basis of GPCR localization regulation and signaling diversity, focusing on two primary objectives. 1) Structural characterization of non-Class B GPCRs localization regulated by RAMPs. We propose a detailed structural analysis of a few non-Class B GPCRs in complex with Receptor Activity-Modifying Proteins (RAMPs), concentrating on their roles in GPCR cell surface localization, ligand recognition, and functional specificity. 2) Unraveling GPCR interactions with PDZ proteins and membrane-associated regulators in cell surface retention and signaling. Our objective is to dissect the structural and mechanistic aspects of GPCR interactions with PDZ domain-containing proteins and other membrane-associated regulators. These interactions are crucial in regulating GPCR cell surface retention, turnover dynamics, signal diversity, and spatial dynamics. We will intensify our efforts in optimizing and applying state-of-the-art technologies in structural characterizations (Cryo-EM, X-ray crystallography, MD simulations), as well as integrate mutagenesis studies, advanced fluorescence imaging techniques, and cell biology-based signaling measurements to provide a comprehensive understanding of these regulatory mechanisms. Our research seeks to develop a thorough understanding of GPCR-regulator complexes, examining their spatial and functional dynamics. We aim to uncover novel insights into the molecular mechanisms regulating GPCR localization and signaling diversity, applicable to various GPCR families. This will not only enhance our multifaceted understanding of GPCR biology, but may also pave the way for novel approaches in therapeutics targeting the impaired trafficking and functioning of GPCRs.