Primary or sensory cilia are microtubule-based extensions of the plasma membrane that are found in almost all cell types [1
]. They regulate diverse cellular processes, including signal transduction, sensory perception, and protein trafficking [3
]. Assembly of cilia occurs by a conserved process called intraflagellar transport (IFT) [4
]. During IFT, precursor moieties are assembled at the base of the cilia (basal bodies) and transported in anterograde and retrograde directions by the kinesin-II and dynein motor subunits. The transport is facilitated by the multiple IFT polypeptides. Defects in cilia assembly or function are associated with multi-systemic disorders, including Senior-Løken syndrome (SLSN), Bardet-Biedl syndrome, Joubert syndrome (JBTS), and Meckel-Gruber syndrome [5
Retinitis pigmentosa (RP; OMIM 268000
), a degenerative disease of the photoreceptors, is frequently associated with ciliary dysfunction. RP is a clinically and genetically heterogeneous group of disorders characterized by severe vision loss and blindness [7
]. X-linked RP (XLRP) is a common form of RP, accounting for 10%–15% of all RP cases [8
]. Clinical manifestations of XLRP usually include night blindness due to rod photoreceptor dysfunction, followed by loss of cone function and blindness by the fifth decade of life [8
]. Some female carriers also exhibit severe retinal defects.
RP3, a major locus for XLRP, encodes for the retinitis pigmentosa GTPase regulator (RPGR
) gene [10
]. Mutations in RPGR
account for 70%–80% of XLRP and ~20% of simplex RP cases [12
]. Some RPGR
patients exhibit extra-retinal phenotypes, including hearing dysfunction, sperm defects, respiratory infections, and primary cilia dyskinesia [14
]. The RPGR
gene undergoes extensive alternative splicing and expresses multiple protein isoforms in the retina [17
]. Most RPGR isoforms contain a common N-terminal domain encoded by exons 1–15, which encompass an RCC1-like domain (RLD; encoded by exons 2–11). The originally described constitutive isoforms of RPGR are encoded by exons 1–19 and account for ~20% of XLRP patients with no known mutations in exons 16–19. Later studies revealed another isoform of RPGR that contains an alternative terminal exon ORF15 (encompasses part of intron 15). Mutations in exon ORF15 account for additional 50%–60% of XLRP patients.
The RLD of RPGR is thought to be the functional domain based on its homology to RCC1 and its involvement in interaction with other proteins. RPGR and RPGR-RLD predominantly localize to primary cilia and photoreceptor connecting cilium (CC) [19
], which is a conduit for trafficking of proteins from the inner segment to the photosensitive outer segment [22
]. An Rpgr
-ko; deletion of exons 4–6 in RLD) mouse mutant [23
], which was later shown to carry a hypomorphic allele of Rpgr
], exhibits delayed onset photoreceptor degeneration and mistrafficking of cone opsins. In addition, two canine models of RPGR mutation have been reported [24
]. These animal models exhibit disparate phenotypes depending upon the type of mutation. Despite extensive efforts, it is still not clear how RPGR regulates photoreceptor function or how mutations in RPGR cause retinal degenerative disease.
Identification of RPGR-interacting proteins has played a key role in understanding its function. RPGR interacts with several ciliary and transport proteins in the retina, including intraflagellar transport protein IFT88/Polaris and RPGR-interacting protein 1 (RPGRIP1) [19
]. In addition, RPGR associates with NPHP proteins mutated in renal retinal syndromes, including SLSN and JBTS [26
]. For example, RPGR exists in complex with NPHP5 (or IQ domain containing calmodulin binding protein [IQCB1]; SLSN), centrosomal protein of 290 kDa (CEP290)/NPHP6 (Leber congenital amaurosis, SLSN, JBTS), and NPHP8/RPGRIP1-like (RPGRIP1L; mutated in JBTS and Meckel-Gruber syndrome) in the retina [30
]. Notably, hypomorphic mutations in NPHP6 and NPHP8, which are associated with relatively early-onset photoreceptor degeneration [26
], disrupt their association with RPGR [30
]. Based on these observations, we hypothesize that RPGR-containing multiprotein complexes play a key role in facilitating photoreceptor protein trafficking.
To elucidate the precise role of RPGR in regulating ciliary transport, it is important to identify and characterize the components of the RPGR-interaction network in the retina. Using co-immunoprecipitation (IP) and mass spectrometry analysis, here we report that RPGR binds to NPHP1 and NPHP4. Using serial immunodepletion, we also found that the RPGR-NPHP interaction network can be divided into at least two distinct complexes: the first complex constitutes NPHP1, NPHP2, and NPHP5, while the second complex consists of NPHP4, NPHP6, and NPHP8.