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 RPGRIP1 lead 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 epsilon (PKC epsilon), we built a 3D-homology model for the C-terminal C2 domain of RPGRIP1. This 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. 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.

Fig. 1.

Structure of RPGRIP1 and 3D-homology model of its C2-C domain. (A) Structure of human RPGRIP1 (GenBank NP_065099). (B) 3D-homology model of human RPGRIP1^C2-C (Right), with the amino acid positions of residues that correspond to the mutations analyzed in this study. The part of the peptide chain colored yellow indicates the portion that is truncated in the RPGRIP1 p.V857fs mutant. (B Left) Close-up of the residues that are involved in the predicted Ca²⁺ binding.

Fig. 2.

RPGRIP1 interaction with nephrocystin-4. (A) GST-RPGRIP1^C2-C efficiently pulled down in vitro-translated N4-I but not N-RPGR (lane 3). Unfused GST (lane 2) pulled down neither, whereas, as a control, GST-RPGRIP1^RID pulled down N-RPGR (lane 4). Lane 1 shows 20% of the in vitro-translated protein input (20% IVT). (B and C) Immunoprecipitation (IP) of nephrocystin-4 and RPGRIP1. (B) The immunoblot (IB) in Top shows that HA-nephrocystin-4^f-l (160 kDa, input shown in Middle) coimmunoprecipitated with FLAG-RPGRIP1^C2-N+C2-C (lane 1) and with FLAG-RPGRIP1-b (lane 3), but not with STRAD-FLAG (lane 2) or mock-transfected cell lysate (lane 4). The anti-FLAG immunoprecipitates are shown in Bottom. (C) FLAG-RPGRIP1^C2-N+C2-C (lane 1) and FLAG-RPGRIP1-b (lane 2) coimmunoprecipitated with the HA-tagged nephrocystin-4-I fragment (HA-N4-I, 58 kDa). Protein inputs are shown in Middle; anti-HA immunoprecipitates are shown in Bottom.

Fig. 3.

Nephrocystin-4 colocalizes with RPGRIP1 upon overexpression in COS-1 cells. (A and E) DAPI staining of the cell nuclei (blue signal). (B) eCFP-nephrocystin-4 (green signal) localized in the cytoplasm around, but not in, the cell nucleus (top cell) when singly transfected. (C) eYFP-RPGRIP1 (red signal) localized only in the nucleus (bottom cell) when singly transfected. (D) overlay of A–C. (F–H) When both proteins are expressed in the same cell (F, eCFP-nephrocystin-4; G, eYFP-RPGRIP1), they colocalize in the cytoplasm (H, overlay of E–G, yellow signal).

Fig. 4.

Immunolocalization of nephrocystin-4 and RPGRIP1 proteins in the retina and Western blot analysis of nephrocystin-4. (A–O) Localization of nephrocystin-4 and RPGRIP1 proteins in the retina. Focal-plane images of radial retinal cryosections shown in A–C are, respectively, murine retinal sections immunostained with MCW3 Ab against the C2 domain of RPGRIP1, Ab#6 against nephrocystin-4, and an overlay of A and B. The C2-containing RPGRIP1 isoform(s) colocalized perfectly with nephrocystin-4 throughout the retina. D–F are, respectively, murine retinal sections immunostained with Ab39 against the RID domain of RPGRIP1, N4#6 against nephrocystin-4, and an overlay of D and E. G–I are, respectively, the photoreceptor layer of a bovine retinal section immunostained with MCW4 Ab against the C2 domain of RPGRIP1, Ab#6 against nephrocystin-4, and an overlay of G and H. J–L, are respectively, the photoreceptors layer of a murine retinal section immunostained with MCW4 Ab against the C2 domain of RPGRIP1, Ab#6 against nephrocystin-4, and an overlay of J and K. M–O are, respectively, the photoreceptors layer of a murine retinal section immunostained with Ab39 against the RID domain of RPGRIP1, Ab#6 against nephrocystin-4, and an overlay of M and N. (Scale bars: J and M,10 μm; A and D,50 μm.) ROS, rod outer segments; RIS, rod inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; CC, connecting cilium. (P) Immunoblot of tissue homogenates from mouse and bovine, showing that anti-nephrocystin-4 antibody N4#6 specifically detects a single protein (indicated by an arrow) in the retinal extracts of mouse and bovine. (Q) Recombinant HA-nephrocystin-4 was detected by antibody N4#6 (second lane) as well as by anti-HA (fourth lane) on immunoblots of COS-1 cell lysates. In lysates from mock-transfected cells, only the N4#6 antibody detects a specific signal of lower intensity but of exactly the same size (indicated by an arrow).

Fig. 5.

RPGRIP1 and NPHP4 mutations disrupt the interaction of nephrocystin-4 with RPGRIP1. (A) Exon structure of RPGRIP1 and mutations identified in this gene. The sequence variants that were analyzed in the yeast two-hybrid system in this study are annotated; the other missense mutations that have been reported (9, 29, 31) are indicated by filled circles. (B) Wild-type and mutated human RPGRIP1^C2-C proteins (fused to GAL4-AD) were assessed for interaction with nephrocystin-4-I, fused to the GAL4-BD domain. An ONPG assay was used to quantify the β-galactosidase activity of the yeast cells (in arbitrary units), which is indicated by the black bars. pBD-LaminC/pAD-pSV40 (pLC) was used as a negative control, indicating the somewhat leaky activation of this reporter gene without selection for transactivation. (C) Exon structure of NPHP4 and mutations identified in this gene. The sequence variants that were analyzed in the yeast two-hybrid system in this study are annotated; the other missense mutations that have been reported (32) are indicated by filled circles. (D) Wild-type and mutated human nephrocystin-4-I proteins (N4, fused to GAL4-BD) were assessed for interaction with RPGRIP1^C2-C, fused to the GAL4-AD domain. (E) Immunoprecipitation (IP) of wild-type and mutated HA-nephrocystin-4^f-l and FLAG-RPGRIP1-b. Coimmunoprecipitation is shown in Top, protein inputs are shown in Middle, and anti-FLAG immunoprecipitates are shown in Bottom. The sizes (in kDa) of the proteins corresponding with the specific antibody signals are indicated. Cross-reacting signals, present in all samples, are marked with an asterisk.