Rao Kollu N, Zhang Wei, Li Linjing, Anand Manisha, Khanna Hemant
Department of Ophthalmology, Horae Gene Therapy Center, UMASS Medical School, Worcester, MA, USA.
Hum Mol Genet. 2016 Oct 15;25(20):4533-4545. doi: 10.1093/hmg/ddw281.
Ciliary trafficking defects underlie the pathogenesis of severe human ciliopathies, including Joubert Syndrome (JBTS), Bardet-Biedl Syndrome, and some forms of retinitis pigmentosa (RP). Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are common causes of RP-associated photoreceptor degeneration worldwide. While previous work has suggested that the localization of RPGR to cilia is critical to its functions, the mechanism by which RPGR and its associated cargo are trafficked to the cilia is unclear. Using proteomic and biochemical approaches, we show that RPGR interacts with two JBTS-associated ciliary proteins: PDE6δ (delta subunit of phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E). We find that PDE6δ binds selectively to the C-terminus of RPGR and that this interaction is critical for RPGR’s localization to cilia. Furthermore, we show that INPP5E associates with the N-terminus of RPGR and trafficking of INPP5E to cilia is dependent upon the ciliary localization of RPGR. These results implicate prenylation of RPGR as a critical modification for its localization to cilia and, in turn suggest that trafficking of INPP5E to cilia depends upon the interaction of RPGR with PDE6δ. Finally, our results implicate INPP5E, a novel RPGR-interacting protein, in the pathogenesis of RPGR-associated ciliopathies.
纤毛运输缺陷是包括乔伯特综合征（JBTS）、巴德-比德尔综合征和某些形式的视网膜色素变性（RP）在内的严重人类纤毛病发病机制的基础。纤毛蛋白RPGR（视网膜色素变性GTP酶调节因子）的突变是全球RP相关光感受器退化的常见原因。虽然先前的研究表明RPGR定位于纤毛对其功能至关重要，但RPGR及其相关货物被运输到纤毛的机制尚不清楚。使用蛋白质组学和生化方法，我们发现RPGR与两种与JBTS相关的纤毛蛋白相互作用：PDE6δ（磷酸二酯酶的δ亚基；一种异戊二烯结合蛋白）和INPP5E（肌醇多磷酸-5-磷酸酶5E）。我们发现PDE6δ选择性地与RPGR的C末端结合，并且这种相互作用对于RPGR定位于纤毛至关重要。此外，我们表明INPP5E与RPGR的N末端相关联，并且INPP5E向纤毛的运输取决于RPGR的纤毛定位。这些结果表明RPGR的异戊二烯化是其定位于纤毛的关键修饰，进而表明INPP5E向纤毛的运输取决于RPGR与PDE6δ的相互作用。最后，我们的结果表明INPP5E是一种新型的与RPGR相互作用的蛋白，参与RPGR相关纤毛病的发病机制。
