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ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting

Nov 27, 2012

Melissa C. Humbert, Katie Weihbrecht, Charles C. Searby, Yalan Li, Robert M. Pope, Val C. Sheffield, Seongjin Seo | Proceedings of the National Academy of Sciences of the United States of America (PNAS) | 27 Nov 2012 | 109 (48) | 19691-19696 | doi.org/10.1073/pnas.1210916109


Abstract

Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed "ciliopathies." Recent protein-protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein-protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D) -dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B-INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.


Primary cilia are microtubule-based cell surface projections that emanate from the centrosome. This subcellular organelle functions as an antenna, sensing and transducing extracellular signals into the cell, and plays an essential role in regulating multiple cellular processes including the cell cycle, embryonic development, and tissue homeostasis (1–3). Mutations affecting ciliary and centrosomal components underlie a group of related human disorders such as Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and Bardet-Biedl syndrome (BBS), collectively termed ciliopathies (1–3). Recent protein–protein interaction studies have identified several functional modules or networks involved in these ciliopathies (4). For example, BBS proteins and intraflagellar transport (IFT) proteins form multiprotein complexes, the BBSome and the IFT complexes, respectively, and these complexes are involved in transporting ciliary proteins. Likewise, NPHP and MKS proteins form a distinct modular complex at the transition zone of primary cilia and regulate ciliary membrane compositions (5–9). However, there are many ciliary and centrosomal proteins [e.g., inositol polyphosphate-5-phosphatase E (INPP5E) and ADP-ribosylation factor-like 13B (ARL13B)] that have not been linked to any of the known functional networks and their precise functions remain to be elucidated.


INPP5E encodes an enzyme that hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 and localizes to primary cilia. Mutations in this gene cause JBTS in humans (10, 11). In mice, loss of Inpp5e activity results in cystic kidney, bilateral anophthalmia, polydactyly, skeletal defects, cleft palate, and cerebral developmental defects (11). Inactivation of Inpp5e in adult mice results in obesity and photoreceptor degeneration. Interestingly, many proteins that localize to cilia, including INPP5E, RPGR, PDE6 α and β subunits, GRK1 (Rhodopsin kinase), and GNGT1 (Transducin γ chain), are prenylated (either farnesylated or geranylgeranylated), and mutations in these genes or genes involved in their prenylation (e.g., AIPL1 and RCE1) lead to photoreceptor degeneration in vertebrates and humans (12–18). Understanding the molecular mechanisms by which prenylated proteins are transported to primary cilia would have significant ramifications in developing therapeutic strategies to treat blindness and other ciliopathy phenotypes associated with these genes.


In this study, we sought to determine the functional network of INPP5E and the mechanisms by which INPP5E is targeted to primary cilia. We determined the ciliary targeting sequence (CTS) of INPP5E, the small GTPase responsible for its ciliary targeting, and an interaction network that connects INPP5E to other known ciliopathy genes and new candidates.



 

Reference

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