Thursday, April 25, 2024
4/25/2024
Project Summary G protein-coupled receptors are important conduits to relay extracellular signals to downstream intracellular signal transduction pathways. Their central role in intercellular communication together with the shear magnitude of the gene family (>800 genes) have therefore made GPCRs superb therapeutic targets. Understanding the mechanism of hormone action on GPCRs and understanding how drugs modulate their behavior is an important fundamental endeavor but also an important mission for health scientists. The primary goal of this ongoing research program is to study the mechanism of GPCR regulation of their primary signaling partners, G proteins. In this renewal we will use biochemical and biophysical approaches to delineate the mechanism of GPCR·G protein (R·G) interactions to try to resolve the extraordinary selectivity of G protein isoforms for specific members of the GPCR superfamily. We will focus on a narrow but representative collection of GPCRs (b2AR, M2 & M3AChR, µOR and NTSR1) and their coupling to different G protein isoforms (Gs, Gi/o, Gq/11 and G12/13). Our major goal is to gain insight into the structural and dynamic bases underlying R·G specificity by determining how these family members couple to and activate specific G protein isoforms. In the previous funding cycle we made several breakthroughs by solving the structures of 6 different R·G complexes: µ opioid receptor (µOR)·Gi1, neurotensin receptor subtype 1 (NTSR1)·Gi1, cannabinoid receptor subtype 1 (CB1)·Gi1, muscarinic M2AChR·GoA, muscarinic M1AChR·G11, and glucagon receptor (GCGR)·Gs. These structures reveal key regions on the receptors and G proteins that we suspect confers receptor and G protein isoform selectivity. In this renewal we propose to apply a spectrum of biochemical and biophysical approaches to interrogate the interaction sites revealed in the R·G structures. In addition, our recent studies suggests various conformational states of the R·G complex, strongly suggesting the existence of intermediate states. In this renewal we propose to examine these intermediate states and probe their potential to contribute toward R·G specificity, and toward receptor-catalyzed nucleotide exchange. We will utilize cutting-edge approaches including cryo-electron microscopy (CryoEM), double electron-electron resonance (DEER) spectroscopy, fluorescence resonance energy transfer (FRET), single molecule spectroscopy (SMS) and interferometry to study these R-G interactions. We will study the nature of the R-G specificity, whether the underlying mechanism may be at the pre- association (perhaps through an intermediate state), or at the coupling stage. We feel that with our expertise, the generation of innovative reagents, the incorporation of cutting edge biophysical approaches and the generation of strong preliminary data together make this proposal tractable.
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