Ciliary dysfunction is a frequent cause of severe renal cystic diseases. In this work, we have sought to identify the function of the RP2 protein with specific emphasis on its role in renal primary cilia. We have demonstrated that RP2 is enriched in the ascending loop of the kidney nephron. Furthermore, we show that RP2 localizes along the length of cilia in renal epithelial cells. This localization is in addition to the previously described localization of RP2 to apical and basolateral membrane domains (27
) and to centrioles/basal bodies (24
). Furthermore, we have demonstrated that this localization requires the acylation of the amino-terminus of RP2. This is in agreement with recent work demonstrating that acylation of RP2 is necessary for targeting of RP2 to the centrosome (24
). This is highly analogous to Cystin1
, the gene mutated in the cpk
mouse model of polycystic kidney disease (42
). Like RP2, the cystin protein localizes to cilia and is myristoylated at its amino-terminus and mutation of the cystin myristoylation site blocks its entry into the primary cilium. In addition, we demonstrate not only an interaction between RP2 and polycystin 2, but that polycystin 2 trafficking in the cilium may also be regulated by RP2. Interestingly, RP has been observed occasionally in ADPKD patients (43
) and in a polycystin 2 mutant rat model (47
), suggesting that in RP2 patients polycystin 2 dysfunction may have relevance for the disease process.
It is not clear how RP2 may regulate polycystin 2 trafficking within the cilium. The observed swellings and accumulation of polycystin 2 at the distal region of cilia are strongly indicative of a retrograde trafficking defect. However, we failed to see accumulation of IFT88 or Crb3 to this region. RP2 may be involved in specifically regulating the retrograde transport of a specific subset of ciliary cargoes such as polycystin 2. Recent work has shown that Caenorhabditis elegans
mutants of Arl13 demonstrate abnormal cilia morphology and accumulation of PKD2 within the cilium (48
). Interestingly, a very recent report has suggested that Arl13b and Arl3 may regulate IFT in a coordinated fashion (49
). Since RP2 has GAP activity towards Arl3, the loss of RP2 may result in dysregulation of Arl3 and Arl13 and subsequent impairment of IFT.
Previous work has demonstrated that the loss of RP2 in retinal pigmented epithelia results in Golgi fragmentation and dispersion of IFT20 from its Golgi localization (24
). However, similar to our data, the loss of RP2 expression in RPE cells does not perturb ciliogenesis. Since the loss of IFT20 results in impaired ciliogenesis, further work is required to better understand the precise mechanism by which RP2 regulates cilia trafficking and the function of IFT20 in relation to this process (50
). It was previously observed that loss of RP2 results in Golgi fragmentation and defects in post-Golgi traffic in RPE cells (24
). We observed that knockdown of RP2 in renal epithelial cells (MDCK) had no effect on the ability of the cells to correctly polarize and form lumen-containing cysts. Since cell polarity is dependent on correct intracellular trafficking, more study is necessary to determine the exact pathways in the Golgi that are impacted by RP2.
We have demonstrated that both RP2 and polycystin 2 are secreted from the apical side of MDCK cells. In addition, it appears that this process is regulated by RP2 as depletion of RP2 results in reduction in polycystin 2 secretion. This process appears to be cilia dependent as inhibition of cilia formation by IFT88 knockdown results in reduced polycystin 2 secretion. However, IFT88 may be regulating this process at the level of exocytosis as it has been recently demonstrated that IFT proteins, namely IFT20, are also required for membrane trafficking (51
). Of particular interest was our observation that knockdown of RP2 leads to swelling of distal tips of cilia that phenotypically resemble those seen in BBS knockout and aln mutant mice (53
). Since polycystin 2 was observed to accumulate in malformed cilia following knockdown of RP2, we hypothesize that RP2 can potentially regulate cilia-dependent polycystin 2 secretion. Interestingly, vesicle budding has been observed from the tips of Chlamydomonas
). Consideration of the structure and function of the photoreceptor outer segment (a modified cilium) suggests this secretion may be a function common to certain cilia. Photoreceptor outer segments contain numerous rhodopsin-containing membranous discs that mediate light-induced signaling. At the distal portion of the outer segment, these discs are shed and subsequently engulfed by the overlying retinal pigmented epithelium. Perturbations in outer segment disc shedding are associated with severe retinal degenerative diseases (10
). Hence, if RP2 is intrinsically involved in cilia-based secretion, the loss or mutation of RP2 could adversely affect the shedding of membrane discs from the distal end of the photoreceptor outer segment leading to retinal degeneration. Further studies are being carried out to understand this process.
To date, no extra-retinal phenotype has been reported in RP2
patients. Due to the low frequency of RP2 mutations (5–10% of all XLRP cases), we surmise that these may have been overlooked. Our study suggests that the loss of RP2 function in humans may result in a more severe and syndromic phenotype or lethality. It is, therefore, possible that RP2 mutations reported so far may not be complete null and perhaps retain partial function (hypomorphic alleles). Given the high trafficking demands of photoreceptors, subtle changes in RP2 function due to hypomorphic mutations may result in deleterious effects in photoreceptors while sparing other tissues. Our genetic interactions further suggest that hypomorphic RP2 mutations may act as modifiers of kidney phenotype in NPHP or PKD patients. Moreover, recent reports demonstrated that silencing of the other XLRP protein RPGR can also result in developmental disorders and renal cysts (41
). These phenotypes have not been reported in XLRP patients thus far. However, combined knockdown of rpgr and its interacting cystic protein NPHP6 results in increased observation of renal cysts. This and other reports in conjunction with our data highlight the growing phenomenon of both genetic and physical interactions between ciliopathy proteins. Further work is being undertaken to examine how RP2 function may intercede with other known ciliopathy pathways.