The long-term goal of our research is to understand the molecular mechanisms through which G-protein coupled receptors (GPCRs) are activated and attenuated. These receptors represent the largest family in the human genome, and they are the target of most pharmaceutical drugs. We focus our studies primarily on the GPCR rhodopsin and its affiliate proteins. Although crystal structures of key proteins involved in visual signaling are now known, most of the critical structural changes these proteins undergo during their activation and attenuation remain largely a matter of speculation. In particular, we lack even rudimentary information about the dynamic events involved in attenuating rhodopsin signaling, namely, the mechanisms through which retinal is released from the opsin-binding pocket, and how retinal binding and release affects arrestin binding and release. Understanding these processes is of fundamental importance for vision research - the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, although much is known about the mechanism and kinetics of arrestin binding to rhodopsin, little is known about what makes arrestin release after binding, and how this release is related to the status of the retinal chromophore.
In Aim I of this proposal we will determine how rhodopsin controls the hydrolysis of its retinal Schiff base linkage.
In Aim II we will examine how retinal uptake and release occurs in rhodopsin, using the recent structure of opsin to guide our studies. Finally, in Aim III, we will use our novel methods to follow up on a discovery we made during the last funding period - that arrestin can bind to MIII rhodopsin, thus trapping and preventing retinal release. Understanding how arrestin regulates retinal release is fundamentally important to health, as arrestin may serve to limit the release of free retinal under bright light conditions, and thus help limit the formation of oxidative retinal adducts that can contribute to diseases like atrophic age-related macular degeneration (AMD). Similarly, understanding what makes arrestin """"""""let go"""""""" after binding rhodopsin is also crucial - stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP).
The proposed research will define the molecular events involved in the binding and release of retinal. We will investigate how retinal gets into and out of the binding pocket in rhodopsin, what makes it stay there (i.e., how the Schiff-base linkage forms and hydrolyzes), and how the protein arrestin affects these processes. Answering these fundamental questions will help shed light on more general questions in vision, such as why rates of retinal binding and release vary so greatly between Rod and Cone rhodopsins, and help elucidate underlying mechanisms for several retinal diseases.
|Schafer, Christopher T; Fay, Jonathan F; Janz, Jay M et al. (2016) Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state. Proc Natl Acad Sci U S A 113:11961-11966|
|Schafer, Christopher T; Farrens, David L (2015) Conformational selection and equilibrium governs the ability of retinals to bind opsin. J Biol Chem 290:4304-18|
|Skjold-JÃ¸rgensen, Jakob; Bhatia, Vikram K; Vind, Jesper et al. (2015) The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study. Biochemistry 54:4186-96|
|Fay, Jonathan F; Farrens, David L (2015) Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1. Proc Natl Acad Sci U S A 112:8469-74|
|Tsukamoto, Hisao; Kubo, Yoshihiro; Farrens, David L et al. (2015) Retinal Attachment Instability Is Diversified among Mammalian Melanopsins. J Biol Chem 290:27176-87|
|Jones Brunette, Amber M; Farrens, David L (2014) Distance mapping in proteins using fluorescence spectroscopy: tyrosine, like tryptophan, quenches bimane fluorescence in a distance-dependent manner. Biochemistry 53:6290-301|
|Sinha, Abhinav; Jones Brunette, Amber M; Fay, Jonathan F et al. (2014) Rhodopsin TM6 can interact with two separate and distinct sites on arrestin: evidence for structural plasticity and multiple docking modes in arrestin-rhodopsin binding. Biochemistry 53:3294-307|
|Alexiev, Ulrike; Farrens, David L (2014) Fluorescence spectroscopy of rhodopsins: insights and approaches. Biochim Biophys Acta 1837:694-709|
|Fay, Jonathan F; Farrens, David L (2013) The membrane proximal region of the cannabinoid receptor CB1 N-terminus can allosterically modulate ligand affinity. Biochemistry 52:8286-94|
|Tsukamoto, Hisao; Farrens, David L (2013) A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor. J Biol Chem 288:28207-16|
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