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
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
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
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
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
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
Arshavsky, Vadim Y; Burns, Marie E (2012) Photoreceptor signaling: supporting vision across a wide range of light intensities. J Biol Chem 287:1620-6
Kosloff, Mickey; Travis, Amanda M; Bosch, Dustin E et al. (2011) Integrating energy calculations with functional assays to decipher the specificity of G protein-RGS protein interactions. Nat Struct Mol Biol 18:846-53
Herrmann, Rolf; Lee, Bowa; Arshavsky, Vadim Y (2011) RGS9 knockout causes a short delay in light responses of ON-bipolar cells. PLoS One 6:e27573
Gospe 3rd, Sidney M; Baker, Sheila A; Kessler, Christopher et al. (2011) Membrane attachment is key to protecting transducin GTPase-activating complex from intracellular proteolysis in photoreceptors. J Neurosci 31:14660-8
Gospe 3rd, Sidney M; Baker, Sheila A; Arshavsky, Vadim Y (2010) Facilitative glucose transporter Glut1 is actively excluded from rod outer segments. J Cell Sci 123:3639-44

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