Activation of G Protein-Coupled Receptors and Heterotrimeric G Proteins.

Abstract

G protein-coupled receptors (GPCRs) are an important class of cell-surface transmembrane receptors that pass an activation signal to the interior of the cell through heterotrimeric G proteins. In this work, we study the human beta-2-adrenergic receptor (B2AR) and stimulatory G protein (Gs) as examples in order to understand the molecular basis of this signal transfer event. We solved a 3.2 angstrom crystal structure of B2AR and Gs in a nucleotide-free, intermediate signaling complex, revealing the interaction between the proteins at atomic resolution. The structure was consistent with previous biochemical knowledge, but also revealed several previously unknown features of the activation process. We used deuterium/hydrogen exchange and electron microscopy in order to find regions in the complex that change conformation during the activation process. These regions are highly conserved within the GPCR and G protein families, and his work shows the central role that they play in the process of GPCR signal transduction. The binding of drugs to the receptor in the fully activated state, as seen in the B2AR-Gs complex, was also characterized by radioligand and antibody fragment binding. A full kinetic model was developed for drug binding to the activated receptor which demonstrated how the ligand is held very tightly in the receptor binding pocket. This tight ligand binding can be relieved by the addition of GDP, demonstrating a direct allosteric link between the G protein nucleotide binding site and the receptor ligand binding site. Overall, this work demonstrates how the GPCR signal transduction machinery operates in high-resolution structural, kinetic, and pharmacological detail. It advances our understanding of how GPCRs and G proteins pass a signal across the cellular membrane.