Dr. Dusanka Deretic Lab
Our research focuses on the understanding of the molecular mechanisms by which mutations in rhodopsin, and in genes that regulate its trafficking, lead to retinal diseases. Rhodopsin is a major constituent of the light-sensing membranes of retinal rod photoreceptor cells, where it functions as a light sensing GPCR that initiates phototransduction cascade. Rhodopsin is also a central regulator of photoreceptor health and subject to over 150 different mutations that cause retinitis pigmentosa. Mutations that affect rhodopsin C-terminus 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 (CTS). We identified the constituents of the ciliary targeting complex that recognizes this signal to regulate rhodopsin trafficking and retinal rod photoreceptor membrane renewal.
The foundation for the optimal function of rod photoreceptors is the strict compartmentalization of the rhodopsin-laden photosensitive membranes to the uniquely modified primary cilium that forms the rod outer segment (ROS). Rod photoreceptor homeostasis is maintained by continuous replenishment of light-damaged ROS membranes and efficient sequestration of ROS proteins and lipids involved in phototransduction, away from those engaged in diverse cellular functions taking place in the adjacent cell body named the rod inner segment (RIS), or at the rod synaptic terminal. Golgi and other biosynthetic organelles are localized in the RIS, in the myoid region (M). Polarized membrane trafficking in retinal rods involves synthesis, sorting and transport, through the RIS, of prodigious quantity of Golgi-to-cilia-directed rhodopsin transport carriers (RTCs). RTCs traverse the ellipsoid region (E) filled with mitochondria and fuse with the RIS plasma membrane in the proximity of the cilium. Newly synthesized membranes are then delivered to the ROS (Figure 1).
Sorting into RTCs is regulated by the small GTPases of the Rab and Arf families that play a central role in organizing intracellular membrane trafficking as well as membrane delivery to primary cilia. Proteins involved in cilia formation and maintenance are encoded by approximately 25% of inherited retinal disease genes, with mutations causing retinal degeneration, cystic kidneys, obesity and neural tube defects in a broad range of genetic disorders, collectively known as ciliopathies.
The small GTPase Arf4 recognizes and directly binds the rhodopsin C-terminal VxPx CTS. Arf4, activated at the photoreceptor Golgi by the Arf guanine nucleotide exchange factor (GEF) GBF1, initiates a stepwise assembly of the targeting nexus centered on the Arf GTPase-activating protein (GAP) ASAP1, which mediates GTP hydrolysis on Arf4, and the Rab11a-FIP3-Rabin8 dual effector complex. This complex controls the assembly of the highly conserved Rab11a-Rabin8-Rab8 targeting module that directly recruits the R-SNARE VAMP7 onto RTCs to regulate their fusion at the ciliary base, via VAMP7 pairing with the cognate plasma membrane SNAREs syntaxin 3 and SNAP-25 (Figure 2).
At the crux of the Rab11a-Rabin8-Rab8 ciliary cascade is the Rab8 GEF Rabin8, a multifunctional scaffold protein that interacts with select ciliary proteins, such as the TRAPPII trafficking complex and the BBSome, suggesting a central role in ciliary pathways of sensory receptors. Its function is affected by mutations in NDR2 kinase (STK38L), encoded by the canine early retinal degeneration (erd) gene corresponding to human ciliopathy Leber congenital amaurosis (LCA). Our current study shows that human GFP-Rabin8 expressed in transgenic X. laevis colocalizes with endogenous Rabin8 and rhodopsin at the Golgi and on RTCs, paving the way for the future studies on the role of Rabin8 in membrane progression along the ciliary pathway, which is potentially disrupted in inherited retinal degenerative diseases.
Collectively, our studies revealed that membrane targeting to ROS is a conserved form of ciliary targeting. The VxPx motif is present in other ciliary membrane proteins. The conserved Arf4-based targeting complex 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.
Deretic D., Lorentzen E, and Fresquez T. (2019). The ins and outs of the Arf4-based ciliary membrane-targeting complex. Small GTPases. Small GTPases. 2019 May 9:1-12. doi: 10.1080/21541248.2019.1616355. [Epub ahead of print]
Kandachar V, Tam BM, Moritz OL and Deretic D. (2018) An interaction Network between the SNARE VAMP7 and Rab-GTPases within a ciliary membrane-targeting complex. J Cell Sci. (2018) J Cell Sci. 2018 Dec 10;131(24). pii: jcs222034. doi: 10.1242/jcs.222034.
Wang J., Fresquez T. Kandachar V. and Deretic D (2017). The Arf GEF GBF1 and Arf4 synergize with the sensory receptor cargo, rhodopsin, to regulate ciliary membrane trafficking. J Cell Sci. J Cell Sci. 2017 Dec 1;130(23):3975-3987. doi: 10.1242/jcs.205492. Epub 2017 Oct 12.
Vetter M, Wang J, Lorentzen E, and Deretic D. (2015) Novel topography of the Rab11-effector interaction network within a ciliary membrane-targeting complex. Small GTPases. 2015 Oct 2;6(4):165-73. doi: 10.1080/21541248.2015.1091539. Epub 2015 Sep 23.
Wang J, and Deretic D. (2015) The Arf and Rab11 effector FIP3 acts synergistically with the Arf GAP ASAP1 to direct Rabin8 in ciliary receptor targeting. J Cell Sci. J Cell Sci jcs.162925; Advance Online Article February 11, 2015, doi:10.1242/jcs.162925 (2015).
Wang J. and Deretic D (2014). Molecular complexes that direct rhodopsin transport to primary cilia. Prog Retin Eye Res. 2014 Jan;38:1-19. doi: 10.1016/j.preteyeres.2013.08.004. Epub 2013 Oct 14
Wang J, Morita Y, Mazelova J and Deretic D. (2012). The Arf GAP ASAP1 provides a platform to regulate Arf4-and Rab11-Rab8 mediated ciliary receptor targeting. EMBO J 31, 4057-4071.
Mazelova J, Ransom N, Astuto-Gribble L, Wilson MC and Deretic D. (2009) Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid (DHA), controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments. J. Cell Sci. 122, 2003-2013.
Mazelova J, Astuto-Gribble L, Inoue H, Tam BM, Schonteich E, Prekeris R, Moritz OL, Randazzo PA and Deretic D. (2009) Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4. EMBO J. 28, 183-192.
Deretic D, Williams AH, Ransom N, Morel V, Hargrave PA and Arendt A. (2005) Rhodopsin C-terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ARF4. Proc. Natl. Acad. Sci. USA. 102:3301-3306.