Structural Basis of GPCR-Transducer Signaling

dc.contributor.advisor

Lefkowitz, Robert J

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Nguyen, Anthony

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2021-05-19T18:08:11Z

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2023-05-17T08:17:07Z

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2021

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Biochemistry

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G protein-coupled receptors (GPCRs) are a class of ubiquitous cell-surface receptors involved in the regulation of virtually every physiological processes. GPCRs typically signal through either G proteins or beta-arrestins (βarrs), each of which activate distinct cellular pathways that leads to divergent physiological consequences. Therefore, not only are GPCRs attractive therapeutic targets, but the ability to selectively ‘bias’ GPCR signaling either through orthosteric or allosteric ligands represents a significant goal in both academic and industrial settings. While receptor-mediated G protein signaling has been extensively studied (due to many decades of investigation prior to, and concurrent with the study of receptors), βarrs has emerged relatively recently as a signal transducer in its own right. Originally discovered as a desensitizer of receptor-mediated G protein signaling, βarrs have been proposed to compete with G protein for binding to the receptor intracellular core, thereby sterically blocking further G protein binding and activation. A large body of literature has demonstrated that productive G protein coupling (i.e., receptor-G protein coupling that leads to G protein activation and second messenger propagation) to receptors are subtype-specific and high affinity. Conversely, little is known about the ability of βarrs to couple to GPCRs. Given that there are over 800 GPCRs encoded by the human genome, and that they all virtually interact with βarrs, a key question in the field remains: through what mechanism does βarrs promiscuously interact with these receptors? Secondly, while βarrs has been shown to mediate G protein desensitization, as well as signal independently of G protein, we and others have reported that for some GPCRs, βarrs enhances G protein signaling from within endosomes. This newly-appreciated phenomenon of ‘sustained signaling’ stands in stark contrast with our ‘classical’ understanding of GPCR signaling, which dictates that (1) βarrs serve as a desensitizer of G protein signaling, and that (2) G protein signaling occurs exclusively at the plasma membrane. Recently, our lab has demonstrated that sustained signaling is potentially mediated by a GPCR-G protein-βarr megacomplex, a novel entity that is emblematic of the many unknowns regarding sustained signaling. My work aims to investigate these phenomena through structural analysis. Our ability to study membrane complexes has been greatly facilitated by the emergence of high-resolution cryo-electron microscopy (cryo-EM), and my goal has been to stabilize and isolate receptor-βarr complexes, obtain their high-resolution structures in an attempt to answer the two questions above. In the last few years, we have successfully obtained high resolution structures of a prototypical chimeric receptor, the β2V2R, bound to βarr1 as well as the β2V2R–G protein–βarr1 megaplex. From these structures, we show that (1) within a megaplex, βarr1 binds to the receptor by its phosphorylated tail, leaving open the receptor intracellular core to which a G protein binds. This allows for the simultaneous binding of both G protein and βarr to the same receptor, enabling G protein activation while the receptor is being internalized by βarr. (2) Through structures of three different states of a stabilized β2V2R–βarr1 complex, we show that the ability of βarr to couple to the receptor intracellular core relies on the plasticity of an unstructured loop called the ‘finger loop.’ The finger loop is capable of adopting a number of different conformations as it interacts with the GPCR, and relies heavily on Van der Waals interaction for such coupling. Overall, the plasticity of the finger loop as well as the importance of hydrophobic interactions in receptor–βarr coupling accounts for the ability for βarr to promiscuously couple to hundreds of different GPCRs.

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https://hdl.handle.net/10161/23025

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Biochemistry

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Structural Basis of GPCR-Transducer Signaling

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Dissertation

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23.9013698630137

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