The long-term goal of the proposed research is to elucidate the molecular mechanisms by which mutations in genes that regulate rhodopsin trafficking and photoreceptor membrane renewal lead to retinal diseases. Mutations in rhodopsin that affect the C- terminal VxPx motif cause severe forms of autosomal dominant retinitis pigmentosa (ADRP). We identified and defined rhodopsin C-terminal VxPx motif as the conserved ciliary targeting signal. Furthermore, we identified a targeting complex that recognizes this signal and regulates rhodopsin sorting into post-Golgi transport carriers (RTCs). The targeting complex is assembled upon direct binding of the small GTPase Arf4 to the VxPx motif. As the VxPx motif is present in other ciliary membrane proteins, some of which bind Arf4, this leads us to propose that membrane targeting to the rod outer segments (ROS) is equivalent to ciliary targeting, and that the complex we identified is a ciliary targeting complex. We plan to study the role of this complex in rhodopsin trafficking and photoreceptor polarity. We propose: 1. To elucidate the architecture of the targeting complex, which is comprised of the small GTPases Arf4 and Rab11, the Arf GAP ASAP1, and the Arf/Rab11 effector FIP3, and determine its mode of interaction with rhodopsin at the trans-Golgi network (TGN), which is essential for ciliary targeting; 2. To determine whether the ASAP1/Rab11/FIP3 complex that remains associated with RTCs serves to recruit regulators of RTC fusion;and 3. To delineate the individual roles of the components of the complex in ciliary targeting of rhodopsin. To accomplish this, we will use our established cell-free system that reconstitutes rhodopsin trafficking in vitro and probe molecular interactions by biochemical assays, co-immunoprecipitation and pull-down experiments with recombinant and purified components of the complex, as well as by transgenic expression of inhibitory and dominant-negative mutant proteins, and by specific protein knockdowns and expression of mutant proteins in ciliated epithelial cells transfected with rhodopsin-eGFP-VxPx fusion protein. The product of our program will be the basic understanding of the molecular linkage between the regulatory machinery involved in the renewal of light-sensitive membranes through ciliary targeting and a wide range of systemic cilia disorders. This is expected to provide the foundation for improved therapeutic strategies for treatment of retina-specific and syndromic forms of photoreceptor loss.

Public Health Relevance

Blinding eye diseases afflicting human populations worldwide are often caused by mutations that inactivate the light receptor rhodopsin and associated proteins that are involved in the maintenance of healthy retinal rods. In some cases these proteins are also functional in other tissues and their failure results in syndromic diseases that affect eyes, kidneys and other organs. We propose to elucidate the role of such a group of proteins that are associated with rhodopsin. These studies will increase our understanding of the molecular underpinnings of inherited retinal diseases and provide therapeutic possibilities for future treatments.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012421-12
Application #
8247063
Study Section
Special Emphasis Panel (ZRG1-CB-G (90))
Program Officer
Neuhold, Lisa
Project Start
1999-01-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
12
Fiscal Year
2012
Total Cost
$362,400
Indirect Cost
$122,400
Name
University of New Mexico Health Sciences Center
Department
Surgery
Type
Schools of Medicine
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Wang, Jing; Deretic, Dusanka (2014) Molecular complexes that direct rhodopsin transport to primary cilia. Prog Retin Eye Res 38:1-19
Deretic, Dusanka (2013) Crosstalk of Arf and Rab GTPases en route to cilia. Small GTPases 4:70-7
Ward, Heather H; Brown-Glaberman, Ursa; Wang, Jing et al. (2011) A conserved signal and GTPase complex are required for the ciliary transport of polycystin-1. Mol Biol Cell 22:3289-305
Deretic, Dusanka; Mazelova, Jana (2009) Assay for in vitro budding of ciliary-targeted rhodopsin transport carriers. Methods Cell Biol 94:241-57
Mazelova, Jana; Astuto-Gribble, Lisa; Inoue, Hiroki et al. (2009) Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO J 28:183-92
Mazelova, Jana; Ransom, Nancy; Astuto-Gribble, Lisa et al. (2009) Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments. J Cell Sci 122:2003-13
Deretic, Dusanka (2006) A role for rhodopsin in a signal transduction cascade that regulates membrane trafficking and photoreceptor polarity. Vision Res 46:4427-33
Deretic, Dusanka; Williams, Andrew H; Ransom, Nancy et al. (2005) Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4). Proc Natl Acad Sci U S A 102:3301-6
Deretic, Dusanka; Traverso, Valerie; Parkins, Nilda et al. (2004) Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors. Mol Biol Cell 15:359-70
Lin, Dingbo; Boyle, Daniel L; Takemoto, Dolores J (2003) IGF-I-induced phosphorylation of connexin 43 by PKCgamma: regulation of gap junctions in rabbit lens epithelial cells. Invest Ophthalmol Vis Sci 44:1160-8

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