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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012859-15
Application #
8423374
Study Section
Special Emphasis Panel (ZRG1-CB-G (90))
Program Officer
Neuhold, Lisa
Project Start
2000-02-07
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
15
Fiscal Year
2013
Total Cost
$511,912
Indirect Cost
$183,763
Name
Duke University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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
Arshavsky, Vadim Y; Burns, Marie E (2014) Current understanding of signal amplification in phototransduction. Cell Logist 4:e29390
Salinas, Raquel Y; Baker, Sheila A; Gospe 3rd, Sidney M et al. (2013) A single valine residue plays an essential role in peripherin/rds targeting to photoreceptor outer segments. PLoS One 8:e54292
Pearring, Jillian N; Salinas, Raquel Y; Baker, Sheila A et al. (2013) Protein sorting, targeting and trafficking in photoreceptor cells. Prog Retin Eye Res 36:24-51
Skiba, Nikolai P; Spencer, William J; Salinas, Raquel Y et al. (2013) Proteomic identification of unique photoreceptor disc components reveals the presence of PRCD, a protein linked to retinal degeneration. J Proteome Res 12:3010-8
Arshavsky, Vadim Y; Wensel, Theodore G (2013) Timing is everything: GTPase regulation in phototransduction. Invest Ophthalmol Vis Sci 54:7725-33

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