A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins

Abstract

Small G-proteins of the ADP-ribosylation-factor-like (Arl) subfamily have been shown to be crucial to ciliogenesis and cilia maintenance. Active Arl3 is involved in targeting and releasing lipidated cargo proteins from their carriers PDE6δ and UNC119a/b to the cilium. However, the guanine nucleotide exchange factor (GEF) which activates Arl3 is unknown. Here we show that the ciliary G-protein Arl13B mutated in Joubert syndrome is the GEF for Arl3, and its function is conserved in evolution. The GEF activity of Arl13B is mediated by the G-domain plus an additional C-terminal helix. The switch regions of Arl13B are involved in the interaction with Arl3. Overexpression of Arl13B in mammalian cell lines leads to an increased Arl3·GTP level, whereas Arl13B Joubert-Syndrome patient mutations impair GEF activity and thus Arl3 activation. We anticipate that through Arl13B’s exclusive ciliary localization, Arl3 activation is spatially restricted and thereby an Arl3·GTP compartment generated where ciliary cargo is specifically released.DOI: http://dx.doi.org/10.7554/eLife.11859.001

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

“…Recently, Gotthardt et al (2015) reported that ARL13B serves as a GEF for ARL3, which in turn binds to and stimulates release of PDE6D from C-terminally prenylated proteins, including INPP5E, which are then allowed to enter cilia (Fansa et al, 2016;Ismail et al, 2011). Furthermore, Fansa et al reported that treatment of cells with ARL3 siRNA caused substantial loss of the dominant ciliary localization of GFP-tagged INPP5E and its redistribution to between the cilia and the plasma membrane (Fansa et al, 2016).…”

“…ARL13B has been shown to interact with and participate in the ciliary targeting of another JBTS protein INPP5E (the gene encoding this protein is also known as JBTS1) (Humbert et al, 2012), which is a phosphoinositide 5-phosphatase. Recently, ARL13B has been reported to serve as a guanine nucleotide exchange factor (GEF) for ARL3 (Gotthardt et al, 2015), which interacts with PDE6D (Linari et al, 1999;Van Valkenburgh et al, 2001), another JBTS protein (the gene encoding this protein is also known as JBTS22). PDE6D is known to bind to C-terminally prenylated proteins, including INPP5E, and ARL3 stimulates release of PDE6D from prenylated proteins (Fansa et al, 2016;Ismail et al, 2011).…”

Sorting of lipidated cargo by the Arl2/Arl3 system

“…The structure shows that Arl13B contacts Arl3 via its switch regions, which explains the finding that the GEF activity of Arl13B is dependent on its nucleotide state. Although the structure could not delineate the exact mechanism of how the nucleotide is kicked out, biochemical experiments suggest that an extended helical end (not visible in the structure) could directly reach into and interfere with the nucleotide binding site of Arl3, 48 as demonstrated previously for many other GEFs.…”

“…Such an Arl3-GEF was indeed identified as Arl13B. 48 Arl13B is a GTP-binding protein itself, which in addition to the G domain contains a coiled-coil and a proline-rich domain and localizes exclusively inside the cilium. 49 Surprisingly the G domain and a part of the CC domain are responsible for the GEF activity.…”

Shuttling and sorting lipid-modified cargo into the cilia

“…Indeed both PDE6D and INPP5E are disease genes in Joubert syndrome, a neurodevelopmental ciliopathy clinically defined as the presence of a "molar tooth sign" on axial MRI alongside one or more classic ciliopathy presentations [11,12]. Mutations in INPP5E have been reported in a number of JBTS patients [11,13] throughout the gene; widely in the phosphatase domain, but also in the CAAX domain, required for interaction with PDE6D, and the SH3 domain, required for interaction with Arl13b [13][14][15] suggesting the importance of GSF trafficking in healthy ciliogenesis.…”