Research Project
10276224
PROJECT SUMMARY/ABSTRACT The overall goal of my research project is to understand how the dynamic behavior of human G protein-coupled receptors (GPCRs) drives the assembly of GPCR complexes with drugs and partner signaling proteins at a single-molecule level. GPCRs are sensory membrane proteins that recognize a wide array of hormones, drugs, and neurotransmitters, representing the largest class of proteins targeted by FDA-approved therapeutics. The energy landscape of GPCRs is complex and populated by multiple conformers with distinct functions and structures. While the structures of some GPCR conformers have been characterized by x-ray crystallography and cryo-EM, the lifetimes of these different conformations and their rates of exchange are mostly unknown. We aim to map the energy landscape of GPCR complexes using single-molecule fluorescence (SMF), which enables the investigation of GPCR dynamics in real-time and in environments that recapitulate the cellular milieu. In the long term, we aim to apply this information to improve our understanding of how disease-associated mutations alter these energy landscapes, which may ultimately guide the design of new therapeutics. This proposal aims to apply SMF to map the energy landscapes of two human GPCRs. First, we will investigate the conformational dynamics of a representative class A human GPCR, the A2A adenosine receptor (A2AAR). A2AAR provides an important benchmark for single-molecule fluorescence studies and will enable us to compare our experimental measurements of dynamics to computational predictions. Our studies will reveal both similarities and differences in mechanisms of signaling between different class A GPCRs and will also show for the first time how lipids in the bilayer membrane can act as allosteric modulators of GPCR function. In the second direction, we will use SMF to study the conformational dynamics of the human glucagon receptor (GCGR). GCGR is a hormone- binding class B GPCR that is activated by one of the central metabolites, glucagon. GCGR is critical to glucose homeostasis and is a validated drug target for type 2 diabetes therapy. Crystal and cryo-EM structures of GCGR have shown that the large extracellular domains appear to act in concert with the transmembrane domain to bind hormones and small molecules, but the dynamics of ligand binding are as yet not understood. Our studies of GCGR will reveal in real-time the mechanisms of ligand recognition by the extracellular domain and quantify function-related dynamic fluctuations of the transmembrane domains. These studies will allow us to compare the dynamics of complex formation with hormones and with small molecules to understand the role of the extracellular domain in ligand recognition. This will help us understand how ligands with different chemical structures and pharmacological efficacies affect the receptor activation pathways and will ultimately aid in the design and screening of GPCR-targeted drugs with tailored pharmacological responses.
