Rpgr ORF15 connects to the usher protein network through direct interactions with multiple whirlin isoforms

Abstract

PURPOSE. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are a frequent cause of X-linked retinitis pigmentosa. The RPGR transcript undergoes complex alternative splicing to express both constitutive (Rpgr ex1-19) and Rpgr ORF15 variants. Because functional studies of Rpgr suggest a role in intracellular protein trafficking through the connecting cilia, the goal of this study was to identify potential binding partners for Rpgr ORF15 and to identify the domains on whirlin necessary for Rpgr binding. METHODS. The C-terminus of mouse Rpgr ORF15 was used as bait in a yeast two-hybrid system. Whirlin expression was analyzed using RT-PCR and Western blot analysis. Protein-protein interactions were confirmed using in vitro binding assays and coimmunoprecipitation. Subcellular colocalization was analyzed using immunohistochemistry on retinal cryosections. RESULTS. Yeast two-hybrid analysis identified whirlin, a PDZscaffold protein, as a putative binding partner for Rpgr ORF15. The RPGR ORF15-whirlin interaction was confirmed using in vitro binding assays and coimmunoprecipitation from retinal tissue, and both proteins were shown to colocalize in the photoreceptor connecting cilia in vivo. Results from RTPCR, Western blot analysis, and immunocytochemistry demonstrated that whirlin expressed multiple isoforms in photoreceptors with variable subcellular localization. CONCLUSIONS. Whirlin expression has been reported in photoreceptors and cochlear hair cells, and mutations in whirlin cause Usher syndrome (USH2D) and nonsyndromic congenital deafness (DFNB31). Because mutations in the 5' end of whirlin are associated with the syndromic phenotype associated with USH2D, the identification of novel N-terminal isoforms in the retina and a novel RPGR ORF15-whirlin interaction provide a potential mechanism for the retinal phenotype observed in USH2D. © 2012 The Association for Research in Vision and Ophthalmology, Inc.