A long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. GPCRs are the largest family in the human genome, and the target of most pharmaceutical drugs. One exception has been rhodopsin ? although the first GPCR discovered, it has so far been refractory to direct pharmacological treatments. Here the Kliger and Farrens lab join forces to define the dynamic events and mechanisms involved in the photo-activation of human rhodopsin and cone photopsins, determine how rhodopsin interacts with its ligand, retinal, determine how its function changes with mutations responsible for retinal diseases and determine how these interactions enable, and are modulated by, interactions with its affiliate protein arrestin. Although the structures of retinal, rhodopsin, and arrestin are now known, the dynamic processes that enable them to interact with each other are not. Thus, the types of studies we propose here are required.
Specific Aim 1 will determine the photoactivation kinetics of human red and green cone pigments, determine how the activation of human rhodopsin is short-circuited by mutations associated with ADRP, and test how these kinetics are effected by small molecule chaperones used to treat and stabilize misfolded opsins.
Specific Aim 2 will determine what role novel receptor conformations play in the process of retinal uptake and release, test if a previously unidentified receptor conformation enables binding of 11-cis retinal (11CR), and expand on our discovery that opsin can transiently linger in an active-like state after releasing all-trans retinal (ATR). Finally, Specific Aim 3 will determine if arrestin binding enables ATR to bind photobleached rhodopsin in equilibrium, and define what effect arrestin binding to rhodopsin dimers has on this phenomenon. Understanding what regulates the process of rhodopsin photoactivation, and retinal uptake and release, and how arrestin regulates these actions is critically important from a health perspective. The retina must accommodate huge variations in these events as it adapts to widely different light conditions, yet aberrations in this process over time are thought to result in the formation of oxidative retinal adducts that promote diseases like atrophic age-related macular degeneration (AMD). Thus, it appears that arrestin must walk a fine line ? on the one hand controlling the amount of free retinal released under varying light conditions, and on the other releasing retinal and itself from the receptor at the appropriate time to avoid forming stable rhodopsin-arrestin complexes that can contribute to apoptosis and some forms of retinitis pigmentosa. The work here complements our recent discovery that ATR can exchange in equilibrium with some rhodopsin photoproducts, and recent discoveries by others of non-retinal ligands that bind and stabilize misfolded opsins. These findings dramatically increase the possibility that drugs can be developed to either compete with or enhance retinal binding, thus opening the door for treating this key photoreceptor with pharmacological agents.

Public Health Relevance

Given the importance of rhodopsin activation and the retinal cycle in vision, we propose to define the molecular events involved in the photoactivation of human rhodopsin and cone photopsins, and the key residues involved, as well as define novel conformational states involved in retinal binding and release to rhodopsin, guided by our discovery that the agonist, all-trans retinal (ATR) can bind in equilibrium to active receptor. Our goals are to determine when and why such equilibrium binding occurs, define the role novel receptor conformations play in this process, and determine how interactions with the protein arrestin affect these activities. Addressing these fundamental issues will help elucidate underlying mechanisms for several retinal diseases, yield insights into why retinal binding and release varies so much between rod and cone rhodopsin, and provide key insights needed for developing drugs targeting photoreceptors.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY029343-02
Application #
9920141
Study Section
Biology of the Visual System Study Section (BVS)
Program Officer
Neuhold, Lisa
Project Start
2019-05-01
Project End
2024-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239