The overall goal of this proposal is to integrate our mechanistic understanding of the signaling processes that take place in the outer segment of photoreceptor cells with the framework of the cellular processes responsible for maintenance of the protein composition of this organelle. To this end, Aim 1 will continue our longstanding investigation of the GTPase activating protein complex for transducin with an emphasis on the interplay between the cellular and catalytic aspects of its function.
In Aims 2 and 3 a similar interplay will be analyzed for rhodopsin, which has three distinct roles in photoreceptor cells: signaling (activation of the phototransduction cascade), structural (building material of the disc membranes) and targeting (directing the flow of vesicles transporting rhodopsin and likely other proteins, e.g. the GTPase activating complex, to the outer segment). We will first test whether the role of rhodopsin in directing outer segment vesicular transport can be dissociated from its other major functions and then determine whether the structural function of rhodopsin can be replaced by other membrane proteins made to localize to the photoreceptor discs. Finally, Aim 4 will elucidate trafficking pathways responsible for the delivery of other proteins functioning in the outer segment. We will focus on a photoreceptor disc rim protein, peripherin-2/RDS, which is the most likely known candidate for using an intracellular targeting mechanism alternative to rhodopsin. The proposed experiments are relevant to understanding the most basic issues in photoreceptor cell biology and are key for understanding the causes of many types of photoreceptor degeneration associated with defects in protein signaling, targeting and trafficking.

Public Health Relevance

The studies proposed in this application address the molecular and cellular mechanisms responsible for the functioning of the light-sensitive compartment of the photoreceptor cells, the outer segment. Because of adverse effects of daily light exposure, the building materials of the outer segment have to be replaced approximately every ten days, which requires an enormous flow of highly organized protein trafficking from the intracellular biosynthetic machinery to this compartment. Dysfunction of these pathways causes some of the most severe types of inherited degenerative diseases of the retina, highlighting the importance of understanding the mechanisms underlying protein signaling, trafficking and assembly into large functional complexes. Elucidating these mechanisms is essential for developing strategies for disease prevention and future therapeutic interventions.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-G (90))
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Neuhold, Lisa
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Duke University
Schools of Medicine
United States
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Sharif, Ali S; Yu, Dongmei; Loertscher, Stuart et al. (2018) C8ORF37 Is Required for Photoreceptor Outer Segment Disc Morphogenesis by Maintaining Outer Segment Membrane Protein Homeostasis. J Neurosci 38:3160-3176
Salinas, Raquel Y; Pearring, Jillian N; Ding, Jin-Dong et al. (2017) Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release. J Cell Biol 216:1489-1499
Pearring, Jillian N; San Agustin, Jovenal T; Lobanova, Ekaterina S et al. (2017) Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration. PLoS Genet 13:e1006740
Spencer, William J; Pearring, Jillian N; Salinas, Raquel Y et al. (2016) Progressive Rod-Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability. Biochemistry 55:5028-37
Ploier, Birgit; Caro, Lydia N; Morizumi, Takefumi et al. (2016) Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants. Nat Commun 7:12832
Ding, Jin-Dong; Salinas, Raquel Y; Arshavsky, Vadim Y (2015) Discs of mammalian rod photoreceptors form through the membrane evagination mechanism. J Cell Biol 211:495-502
Pearring, Jillian N; Spencer, William J; Lieu, Eric C et al. (2015) Guanylate cyclase 1 relies on rhodopsin for intracellular stability and ciliary trafficking. Elife 4:
Pearring, Jillian N; Lieu, Eric C; Winter, Joan R et al. (2014) R9AP targeting to rod outer segments is independent of rhodopsin and is guided by the SNARE homology domain. Mol Biol Cell 25:2644-9
Srinivasan, Pratul P; Heflin, Stephanie J; Izatt, Joseph A et al. (2014) Automatic segmentation of up to ten layer boundaries in SD-OCT images of the mouse retina with and without missing layers due to pathology. Biomed Opt Express 5:348-65
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