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

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 ciliopathyrelated 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.photoreceptor degeneration | retinitis pigmentosa | leber congenital amaurosis | polydactyly | cystic kidney P rimary 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 proteinprotein 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 ...

PDE 6D binds to the C‐terminus of RPGR in a prenylation‐dependent manner

“…When we compared the amino acid sequences of verified PDE6D-binding and non-binding prenylated proteins [1,3] (and references therein), Ser was found to be the most preferred amino acid at the À3 position among PDE6D-binding prenylated proteins (Fig 1D). Polar or positively charged residues (Asn and Lys) were also found.…”

“…Polar or positively charged residues (Asn and Lys) were also found. In contrast, hydrophobic residues were found in RAB8A, RAB8B, and RAB11A, which are geranylgeranylated but do not bind to PDE6D [3]. To test whether the Ser residue at the À3 position plays an important role in PDE6D binding in other proteins, we mutated the Ser residue at the À3 position in INPP5E, a prenylated protein that binds to PDE6D and localizes to cilia [3,4], to a hydrophobic amino acid Ala and examined its interaction with PDE6D.…”

The role of primary cilia in the development and disease of the retina

“…71 Human mutations are associated with several syndromic retinal dystrophies including mental retardation, obesity, retinal dystrophy and micropenis (MORM) syndrome, JBTS and CORS. 70,72 Trafficking of INPP5E to the cilium depends on PDE6D, in concert with ARL13B and CEP164, 73 which when mutated in humans also lead to syndromic retinal dystrophies. 74,75 The Tectonic proteins TCTN1 and TCTN2 play a role in targeting GPCRs to the cilium membrane, as well as other components of G-protein signaling such as the downstream effector adenylyl cyclase III (ACIII) and the putative channel protein polycystin-2 76 .…”

Nedd9 restrains renal cystogenesis in Pkd1 −/− mice

“…These results suggested the possibility of a defect in ciliary trafficking. We examined localization of a number of ciliary proteins, including intraflagellar transport protein IFT88, a component of the IFT-B complex (26), INPP5E and ARL13B, membrane-associated proteins associated with the ciliopathy Joubert's syndrome (27), and the ciliary protein adenylate cyclase III (ACIII), which catalyzes the production of cAMP, is abnormally regulated in ADPKD (28), and is excluded from the cilia of fibroblasts from individuals with familial nephronophthisis-like cystic syndromes (29). IFT88, INPP5E, and ARL13B were comparably localized in cilia regardless of Pkd1 or Nedd9 genotype (Fig.…”