The RPGRIP1 Assembly Complex Mediates Multiple Biological Processes That Are Vital for Retinal Function

Abstract

Purpose: : RPGR interacting protein 1 (RPGRIP1) is a key component of cone and rod photoreceptor cells, where it interacts with RPGR (Retinitis Pigmentosa GTPase regulator). Mutations in RPGRIP1 lead to autosomal recessive congenital blindness (Leber congenital amaurosis – LCA). Most LCA–associated missense mutations in RPGRIP1 are located in an alternatively spliced segment that encodes two C2 domains. We have investigated the functional relevance of this domain by identifying novel interacting proteins and analyzing the effects of the mutations on the protein–protein interactions.

Methods: : A homology model of the C–terminal C2 domain of RPGRIP1 was built using the programs WHAT IF, SCWRL and YASARA. Novel interacting proteins were identified by yeast two–hybrid screening of retinal cDNA libraries. Interactions were confirmed by GST pull–down analysis and coimmunoprecipitation assays. Colocalization was analyzed using GFP fused variants in mammalian cell lines or by immunohistochemistry of retinal cryosections.

Results: : The C2–C homology model revealed a potential Ca²⁺–binding site that was predicted to be disrupted by a missense mutation in RPGRIP1, which was previously identified in an LCA patient. Through yeast two–hybrid screening of a retinal cDNA library, we found this C2 domain to specifically bind to nephrocystin–4, encoded by NPHP4, and other proteins linking to different cellular pathways. Mutations in NPHP4 are associated with nephronophthisis (NPHP), and a combination of NPHP and retinitis pigmentosa called Senior–Løken syndrome (SLSN). We show that RPGRIP1 and nephrocystin–4 interact strongly in vitro and in vivo, and that they colocalize in the retina, matching the panretinal localization pattern of specific RPGRIP1 isoforms. Their interaction is disrupted by either mutations in RPGRIP1, found in patients with LCA, or by mutations in NPHP4, found in patients with NPHP or SLSN.

Conclusions: : We provide evidence for the involvement of a disrupted interaction with the C2 domain of RPGRIP1 in the retinal dystrophy of both SLSN as well as LCA patients. The identification of additional interactors builds evidence for an emerging protein complex with a pleiotropic and dynamic behaviour in the (patho–)physiology of the retina and other tissues.

Keywords: retina • retinal degenerations: hereditary • retinal degenerations: cell biology