G-protein coupled receptors (GPCR) are seven-helix transmembrane proteins that participate in numerous physiological functions. They play an essential role in vision, brain function, and immune-system regulation, and are the largest protein family targeted by marketed drugs. Rhodopsin is a prototypical class A GPCR found in high densities at the outer segment of rod cells in the retina. It plays a central role in vision a the photoreceptor protein in rod cells. Rhodopsin mutations are implicated in a wide range of visual disorders, many of which are irreversible and untreatable. While isolated rhodopsin is functional as a monomer, there is overwhelming evidence that it dimerizes within rod outer segment (ROS) membranes. The functional relevance of rhodopsin dimers has been demonstrated through in vitro assays of purified rhodopsin, but the structural connection between dimerization and physiological function in native membranes has not been established due to the lack of experimental tools that can resolve molecular-scale interactions under physiological conditions and with sufficient time resolution to measure real-time receptor activation events. To investigate these interactions, we will use a quantitative imaging approach, including pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), to determine opsin oligomerization and dynamics in the live-cell plasma membrane. Our hypothesis is that opsin dimerization is prevalent in cell membranes, structurally selective, and active in signaling. To test that hypothesis, this proposal specifically aims to (1) investigate th prevalence and dynamic stability of opsin (rod, cone and melanopsin) protein dimers in live-cell membranes, (2) measure the dimerization equilibrium of several prototypical class A GPCRs including dopamine receptors, protease-activated receptors, and beta-adrenergic receptors, and (3) develop a platform to investigate dimerization in light-activated rhodopsin and melanopsin and measure the effect of dimerization on signal transduction. Support for the existence of rhodopsin dimers is abundant, but missing from the current body of literature is a full characterization of the thermodynamics of opsin dimerization and the way in which dimerization plays an active role in cell signaling events. This is significant because it will advance the fiel from a debate about the existence of rhodopsin dimers to a comprehensive understanding of the chemical equilibria that govern dimerization and the properties of the rhodopsin complexes. By determining the molecular-level details of opsin oligomerization, we will obtain insight into th mechanisms that drive GPCR oligomerization. Successful completion of the aims of this proposal will have a direct impact on the growing field of GPCR oligomerization and will advance our understanding of this important class of membrane receptors.
Photo-induced isomerization of retinal within opsin is the basis of vision and the first in a series of molecular events leading to phototransduction. Resolving the molecular-level details of rod and cone opsin organization and molecular interactions will contribute to a better understanding of the mechanism of visual function and will provide new avenues for the treatment of blinding diseases.
