RESUMEN
Retinitis pigmentosa 2 (RP2) is the causative gene for a form of X-linked retinal degeneration. RP2 was previously shown to have GTPase-activating protein (GAP) activity towards the small GTPase ARL3 via its N-terminus, but the function of the C-terminus remains elusive. Here, we report a novel interaction between RP2 and osteoclast-stimulating factor 1 (OSTF1), an intracellular protein that indirectly enhances osteoclast formation and activity and is a negative regulator of cell motility. Moreover, this interaction is abolished by a human pathogenic mutation in RP2. We utilized a structure-based approach to pinpoint the binding interface to a strictly conserved cluster of residues on the surface of RP2 that spans both the C- and N-terminal domains of the protein, and which is structurally distinct from the ARL3-binding site. In addition, we show that RP2 is a positive regulator of cell motility in vitro, recruiting OSTF1 to the cell membrane and preventing its interaction with the migration regulator Myo1E.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Actinas/genética , Proteínas del Ojo/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas/genética , Retinitis Pigmentosa/genética , Factores de Ribosilacion-ADP/química , Actinas/química , Sitios de Unión/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/genética , Cilios/genética , Cilios/metabolismo , Proteínas del Ojo/química , Proteínas de Unión al GTP , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Simulación del Acoplamiento Molecular , Miosina Tipo I/química , Miosina Tipo I/genética , Unión Proteica/genética , Conformación Proteica , Dominios Proteicos/genética , Estructura Terciaria de Proteína , Proteínas/química , Retina/metabolismo , Retina/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patologíaRESUMEN
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.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Cilios/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lipoproteínas/metabolismo , Animales , Ratones , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de ProteínasRESUMEN
Cilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone.