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 most importantly, are the target of most pharmaceutical drugs. Our studies focus primarily on the GPCR rhodopsin and its affiliate proteins. Although knowledge of the proteins involved in visual signaling has been enormously advanced through recent crystallographic studies, the critical structural changes they undergo during activation and attenuation remain largely a matter of speculation. In particular, we lack even the most rudimentary information about the dynamic events that occur during the attenuation of rhodopsin signaling, namely, the mechanisms used to release retinal from the opsin-binding pocket, and how these processes affect arrestin binding and release. Understanding these processes is of fundamental importance for vision research, as the stability of the retinal linkage varies widely among different opsins and is a factor in some visual disease states. Furthermore, little is known regarding the mechanism and kinetics of arrestin release from activated rhodopsin.
In Aim I of this proposal we will determine the mechanisms through which rhodopsin controls the hydrolysis of its retinal Schiff base linkage.
In Aim II we will use our expertise in site-directed labeling methods to examine dynamic and structural changes occurring in the extracellular loop region of rhodopsin as it binds and releases retinal. Finally, in Aim III, we will utilize a new assay we have developed to study arrestin release from activated rhodopsin. Understanding what makes arrestin """"""""let go"""""""" after binding rhodopsin is fundamentally important, since stable rhodopsin-arrestin complexes have been suggested to be a contributing factor in apoptosis and autosomal dominant retinitis pigmentosa (ADRP). Furthermore, using our new assay, we find intriguing evidence that arrestin binds to and traps a post-Meta II photodecay product, possibly Meta III. Thus, arrestin may also 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). ? ? ?
|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|
|Jones Brunette, Amber M; Sinha, Abhinav; David, Larry et al. (2016) Evidence that the Rhodopsin Kinase (GRK1) N-Terminus and the Transducin G? C-Terminus Interact with the Same ""Hydrophobic Patch"" on Rhodopsin TM5. Biochemistry 55:3123-35|
|Tsukamoto, Hisao; Kubo, Yoshihiro; Farrens, David L et al. (2015) Retinal Attachment Instability Is Diversified among Mammalian Melanopsins. J Biol Chem 290:27176-87|
|Skjold-Jørgensen, 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|
|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|
|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|
|Alexiev, Ulrike; Farrens, David L (2014) Fluorescence spectroscopy of rhodopsins: insights and approaches. Biochim Biophys Acta 1837:694-709|
|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|
|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|
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