The long-term goal of the proposed research is to improve the understanding of 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 its targeting motif cause severe forms of autosomal dominant retinitis pigmentosa (ADRP). In the course of our research, we defined the rhodopsin C-terminal VxPx motif as a conserved ciliary targeting signal. Furthermore, we identified a ciliary targeting complex that recognizes this signal and regulates sorting into post-Golgi rhodopsin transport carriers (RTCs). Our previous studies revealed that membrane targeting to the rod outer segments (ROS) is a conserved form of ciliary targeting, and that the complex we identified is a conserved complex that targets sensory receptors to primary cilia through intricate functional networks of small GTPases and their regulators that are exquisitely sensitive to mutations causing retinal degenerations and ciliopathies. We now propose to define the function of the Rab8 GEF Rabin8, and determine whether and how its phosphorylation by NDR2/STK38L kinase, a product of (erd) retinal degeneration gene, controls rhodopsin trafficking and ciliogenesis. We will also delineate the molecular mechanisms regulating Rabin8 interactions with the R-SNARE VAMP7, and determine how their dysfunction affects ROS membrane trafficking. To accomplish this, we will use our established cell-free system that reconstitutes rhodopsin trafficking in vitro and probe molecular interactions by kinase assays, and pulldown experiments with recombinant and purified components?as well as by transgenic expression of mutant and phosphomimetic protein constructs, confocal microscopy, Correlative Light and Electron Microscopy (CLEM), Super Resolution (SR) direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging, and immunoprecipitation from transgenic retinas with anti-GFP antibody followed by LC-MS/MS analysis. The basic understanding of the molecular linkage between the regulatory machineries involved in the renewal of light-sensitive membranes through ciliary targeting and a wide range of systemic cilia disorders is expected to provide the foundation for improved therapeutic strategies to treat retina-specific and syndromic forms of photoreceptor loss.

Public Health Relevance

Blinding diseases affecting millions worldwide are often caused by mutations in the light receptor rhodopsin and associated proteins that are involved in the maintenance of healthy retinal rods. In some cases, these proteins are functional in other tissues and their failure results in syndromic diseases that affect eyes, kidneys and other organs. We study 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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Biology of the Visual System Study Section (BVS)
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Neuhold, Lisa
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University of New Mexico Health Sciences Center
Schools of Medicine
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Wang, Jing; Fresquez, Theresa; Kandachar, Vasundhara et al. (2017) The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking. J Cell Sci 130:3975-3987
Wang, Jing; Deretic, Dusanka (2015) The Arf and Rab11 effector FIP3 acts synergistically with ASAP1 to direct Rabin8 in ciliary receptor targeting. J Cell Sci 128:1375-85
Vetter, Melanie; Wang, Jing; Lorentzen, Esben et al. (2015) Novel topography of the Rab11-effector interaction network within a ciliary membrane targeting complex. Small GTPases 6:165-73
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
Wang, Jing; Morita, Yoshiko; Mazelova, Jana et al. (2012) The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting. EMBO J 31:4057-71
Lu, Rong-Wen; Curcio, Christine A; Zhang, Youwen et al. (2012) Investigation of the hyper-reflective inner/outer segment band in optical coherence tomography of living frog retina. J Biomed Opt 17:060504
Deretic, Dusanka; Wang, Jing (2012) Molecular assemblies that control rhodopsin transport to the cilia. Vision Res 75:5-10
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
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

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