Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosis-associated mutations

Abstract

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 RPGRIP1lead to autosomal recessive congenital blindness [Leber congenital amaurosis (LCA)]. Most LCA-associated missense mutations in RPGRIP1 are located in a segment that encodes two C2 domains. Based on the C2 domain of novel protein kinase C (PKC ), we built a 3D-homology model for the C-terminal C2 domain of RPGRIP1. This model revealed a potential Ca 2؉ -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. Mutations in NPHP4 are associated with nephronophthisis and a combination of nephronophthisis 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 nephronophthisis or SLSN. Thus, we provide evidence for the involvement of this disrupted interaction in the retinal dystrophy of both SLSN and LCA patients.T he X-linked gene RPGR (retinitis pigmentosa GTPase regulator) is mutated in patients with retinitis pigmentosa type 3 (RP3) (1, 2), cone or cone-rod dystrophy (COD1) (3, 4), atrophic macular degeneration (5), and RP in combination with impaired hearing and sinorespiratory infections (6). All RP3-associated missense mutations in RPGR have been identified in the N-terminal RCC1-homologous domain, and they disrupt the interaction with the C-terminal domain of RPGR-interacting protein 1 (RPGRIP1) (7,8). Mutations in RPGRIP1 lead to Leber congenital amaurosis (LCA), a genetically heterogeneous recessive disorder that is regarded to be the earliest and most severe form of all retinal dystrophies (9, 10). LCA accounts for at least 5% of all retinal dystrophies and is one of the main causes for blindness in children (11). RPGR and RPGRIP1 isoforms have been found to colocalize at the connecting cilia as well as the outer segments of rod and cone photoreceptors (12, 13). Differential localization among species has also been reported (12,14), and different isoforms of RPGRIP1 have been described resulting from splicing variation (7,14,15). The distinct partitioning of a subset of RPGRIP1 isoforms between the nuclear and cytoplasmic compartments combined with the differential and limited proteolysis of RPGRIP1 among retinal neurons, and the impact of LCA-linked mutations in RPGRIP1 in these processes, support the involvement of nucleocytoplasmatic signaling processes mediated by RPGRIP1 and its interacting partners in the pathogenesis of LCA, RP3, and allied diseases (15, 16). The pres...

Evidence for RPGRIP1 gene as risk factor for primary open angle glaucoma

“…Gateway-adapted expression constructs were created using the Gateway cloning system (Invitrogen, Karlsruhe, Germany), and expression vectors used were described in Roepman et al 21 All PCR-generated fragments were verified by nucleotide sequencing. Mutations were introduced using the QuickChange site-directed mutagenesis kit (Stratagene, Amsterdam, Denmark).…”

“…21 The transformants were selected by growth on SD medium lacking Trp at 301C for 3-4 days. To test the expression of the LacZ gene, a colony lift filter assay was performed using X-GAL as a substrate.…”

“…21 Five of them indeed showed impaired interaction ( Table 1), suggesting that the variants are functionally relevant and not benign polymorphisms. When taking into account only these five variants (p.R598Q, p.A635G, p.T806I, p.A837G and p.I838V) detected in 10 patients and in none of the control subjects, the association remained significant (P¼0.001, Table 1).…”

“…Mutations located in RPGRIP1 C2 domain disrupt the RPGRIP1-NPHP4 complex and cause Leber congenital amaurosis (LCA). 21 Another known interaction partner of the RPGRIP1 C-terminal region (RID) is the X-linked RPGR gene, involved in X-linked retinitis pigmentosa type 3. 22 Furthermore, RPGRIP1 has been detected in the retina as a complex with the basal body protein CEP290 30 and in amacrine cells with the neuronal nucleoporin RANBP2.…”

“…21 To compare the functional relevance of the nonsynonymous variants found only in glaucoma patients with those GeneBank NP_065099) highlighting the location of the C2-domains region. All amino acids changes identified in the discovery group are listed upward; changes identified in the replication group are listed below the structure.…”