The mammalian retina comprises two types of photoreceptors: rods and cones. Each is unique in morphology, light sensitivity, recovery rate, thermal stability, outer segment shedding time, and resistance to apoptotic cell death. Under low light or scotopic conditions, 95% of photoreceptors in humans are rods containing the light sensitive visual pigment, rhodopsin. In contrast, cones are responsible for high acuity color vision initiating in the S, M, or L pigment, cone opsins. All opsins are G-protein coupled receptor (GPCR) and each shares a similar mechanism for the initiation of phototransduction: photons are absorbed by pigment molecules (i.e. rhodopsin &S, L, M opsins) leading to the closure of cGMP-gated sodium channels in the outer membrane via activation of the phosphodiesterase (PDE) resulting in membrane hyperpolarization. Once GPCRs are initiated the visual system also requires a way to shut off transduction, which is terminated by GRK1 phosphorylation, followed by the subsequent binding of either Santigen/arrestinl (SAG) or cone arrestin/arrestin4 (CAR). Although modulating phototransduction shutoff is well established for SAG, deciphering the functions of CAR is ongoing. To address the aspects inherent to the cone phototransduction pathway and to accomplish our goals, experiments are designed to explore the function(s) of CAR, its targeted cone GPCRs and other relevant protein partners in the cone synapse. Our hypothesis is that CAR modulates recovery of cone phototransduction and interacts with other proteins to control cone synaptic transmission.
The specific aims i nclude 1) identify interacting partners for CAR from the Nrl knockout mouse retina by yeast two-hybrid (Y2H) screen in parallel to immunoprecipitation """"""""pulldowns"""""""" assays;2) confirm the physical interaction of CAR and determine the functional domains with these proteins with in vitro binding and antibody pull-down assays;and 3) verify the functional relevance of these interactions in vivo using molecular, biochemical and cell biology techniques. The significance of visual cycle proteins is evident by the prevalence of visual impairment, such as age related macular degeneration. In part, visual loss develops as a result of genetic mutations encoding proteins essential for transduction. To preserve high acuity vision and to provide a basis for diagnosis, prevention, and treatment, we must decipher the underlying phototransduction mechanisms. Understanding the mechanisms of initiation and termination of the phototransduction cascade are imperative to cure currently untreatable forms of blindness and to preserve healthy vision.
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| Zuniga, Freddi I; Craft, Cheryl M (2010) Deciphering the structure and function of Als2cr4 in the mouse retina. Invest Ophthalmol Vis Sci 51:4407-15 |
