Not So Hot Rods: Mutations of in Regards to J. Bade, C. Czerniak, S. Daley, A. Kellicut, J. Killoren, E. Krupski, L. Semler, C. Wilkins Teacher: Mark Arnholt Mentor: Melissa Wilk, B.S., Medical College of Wisconsin

Abstract Structure of Rhodopsin Kinase Oguchi Disease

The average person’s eyes adapt to darkness within minutes. For those with Oguchi disease, Oguchi Disease is caused by mutations in rhodopsin kinase which result in adaptation can be slowed to several hours. Oguchi disease is an autosomal recessive greatly slowed adaptation of the rods of the retina. For a person living with disorder that results in greatly slowed phototransduction. Phototransduction is a cascade this disease, the adaption to the dark will take several hours, whereas a reaction beginning with a photon activating rhodopsin in the rod and leading to person without the disease, adaption will only take several minutes. This makes everyday tasks such as driving at night, navigating a dark room, hyperpolarization of the cell. Oguchi disease is caused by mutations in rhodopsin kinase and going into a dimly lit restaurant difficult. Patients with Oguchi Disease which prevent the phosphorylation of rhodopsin, lowering rhodopsin’s affinity for . This have a distinct fundus appearance, with a golden sheen under light reduced ability to bind arrestin decreases the speed in which rhodopsin is deactivated and adapted states (left) that disappears with dark adaption (right). prepped to reactivate. After a long period in a dark environment, the rhodopsin is eventually deactivated by arrestin, allowing it to be recycled. The Hartford Union High School SMART (Students Modelling a Research Topic) Team has designed a model of rhodopsin kinase to investigate its structure-function relationship. Oguchi disease can be caused by two different mutations in rhodopsin kinase: large deletion or point mutation. In our 3D model, we will highlight the complete deletion of exon five, the partial deletion at the C-terminus, and point mutations in the catalytic domain (Val380Asp and Pro391His) that cause Oguchi disease. Understanding the structure-function relationships of rhodopsin kinase could shed more light on night blindness. This program is supported by a grant from NIH and CTSA. Model of Rhodopsin Kinase based on file 3C51.pdb

● A phosphate binding loop is formed by the Glycine-rich β1-β2 turn. It directly interacts with the triphosphate tail of ATP and helps stabilize the phosphorylation transition state. Function of Rhodopsin Kinase ● The N-terminal amino acid residues 8-17 of rhodopsin kinase appear to be critical for rhodopsin phosphorylation and binding. Summary ● Point mutations Val380Asp and Pro391His in catalytic domain, as well as a deletion of exon 5, disrupt the When a photon of light enters the eye, phototransduction, the process enabling us to normal function of rhodopsin kinase, resulting in slowed dark adaptation. One of three mutations in the rhodopsin kinase gene cause Oguchi see, begins. Within the rod photoreceptors, the cells responsible for night vision, the disease: a point mutation, two point mutations in the catalytic domain, or a photon activates rhodopsin, stimulating a cascade of neural events resulting in the complete deletion of exon 5. PCR can be used to identify the exon 5 deletion of this . Rhodopsin kinase is involved in the process of recognition of light. Following activation of rhodospin, a protein called rhodopsin phototransduction in the rods of the retina where it works to prepare the kinase is responsible for deactivating rhodopsin so that it can be recycled for another Mutations in Rhodopsin Kinase rod to accept another photon of light. The specific mutations alter the round of activation. During phototransduction, calcium levels decrease, allowing shape of rhodopsin kinase and ultimately slow down the process of [1] recoverin, a calcium-binding protein, to release rhodopsin kinase. Rhodopsin kinase adaptation to a lack of light by nine times . Oguchi disease is the condition of slowed adaptation to darkness. Understanding the underlying then uses ATP to phosphorylate the activated rhodopsin. Once phosphorylated, cause of Oguchi disease may help in the development of future treatments arrestin binds the rhodopsin, fully deactivating it and allowing for the recycling of for this debilitating disease. retinal within the rhodopsin. Without the rhodopsin kinase, the rhodopsin cannot be recycled properly, resulting in slowed dark adaptation.

References

1. Cideyciyan, A.V., Zhao, X., Nielsen, L., Khani, S.C., Jacobson, S.G., Palcaewski, K. (1997). Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man. Oguchi disease is typically caused by one of three mutations in the rhodopsin kinase gene: point mutations in Proc. Natl. Acad. Sci. USA, 95(1): 328-333. the Val380Asp or Pro391His, or a total deletion of exon 5. Cideciyan and other used polymerase chain reaction (PCR) to amplify the rhodopsin kinase gene with and without an exon 5 deletion. Different primers were used to 1. Baylor, D.A., Burns, M.E. (1998). Control of rhodopsin activity in pinpoint the exons, then the DNA fragments were run through the gel. In gel electrophoresis, strands that travel further toward the negative end are shorter, and strands that stay closer to the positive end are longer. The vision. Eye, 12(Pt 3b): 521-525. results of the gel electrophoresis show a complete absence of the exon 5 sequence in lane one. 1. Singh, P., Wang, B., Maeda, T., Palczewski, K., Tesmer, J.J.G. (2008). Structures of rhodopsin kinase in different ligand states reveal key elements involved in G protein-coupled receptor kinase activation. J The largest mutation that causes Oguchi disease is the complete deletion of exon Biol Chem, 283(20): 14053-14062. 5, a 1.7 kb deletion that removes 42 amino acids from the protein. This greatly alters the shape of the protein, making it inert and therefore slowing down the rate of rhodopsin deactivation.

There are also two point mutations in the catalytic domain that can cause Oguchi disease, Val380Asp and Pro391His. These mutations reside in exon 5, and are involved in phosphorylating rhodopsin to help deactivate it. SMART Teams are supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR0C0055. Another point mutation, at amino acid 334, not in the catalytic domain, has been Its contents are solely the responsibility of the author and do not necessarily represent the known to also cause Oguchi disease. official views of the NIH.