Direct examination of peptide-G-protein coupled receptor conformation and trafficking using two new biophysical methods, PWR spectroscopy and two photon fluorescence laser microscopy

摘要

Integral membrane proteins, especially G-protein coupled receptors (GPCRs), are the targets of nearly 70% of all drugs, and yet little is known about the structural basis for information transduction, or the dynamics of the transduction process. This is due to the inherent difficulties in examining structure and structural change in highly anisotropic fluid environments such as proteolipid bilayers. Many of the endogenous ligands for these GPCRs are peptides, and as such offer unique opportunities due to their three-dimensional structures when bound to the receptor which allow them to be designed with specific 3-D structures and biological effects and to be modified at ancillary sites which do not interfere with peptide binding to the receptor or signal transduction. To help facilitate new structural and dynamic insights into the physical-chemical properties of these systems in relation to their biological effects we are developing two relatively new biophysical methods: plasmon waveguide resonance (PWR) spectroscopy and two photon fluorescence laser microscopy (TPFLM). These methods allow for the first time: direct studies of ligand-receptor interactions, and of the structural changes which accompany these interactions; receptor-G-protein interactions; the reciprocal affects of the ligands and G-proteins interacting with GPCRs; detailed examination of β-arrestin mediated internalization of GPCRs when bound to agonist ligands, and the mechanism of this internalization. These methods allow direct studies of receptor trafficking and provide new insights into receptor transduction.