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Identification and characterisation of RP2 interacting proteins.

Evans, R.J.; (2007) Identification and characterisation of RP2 interacting proteins. Doctoral thesis , University of London. Green open access

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Abstract

Retinitis pigmentosa (RP) is a genetically and clinically heterogenous retinal degenerative disease, which is characterised by night blindness and constriction of visual fields in the early stages which progress to blindness. X-linked RP (XLRP) is the most severe form of the disease and 2 causative genes have been identified, RP2 and RPGR. RP2 encodes a ubiquitously expressed 350 amino acid protein with a tubulin folding cofactor C (TBCC) homology domain and a C-terminal NDK homology domain. Despite the ubiquitous expression of the RP2 protein, the disease pathogenesis in patients carrying RP2 gene mutations appears to be restricted to retina. The function of RP2 in retina, or other tissues, has yet to be determined. This study aims to increase the understanding of the pathobiology of RP2 by unravelling novel molecular pathways of RP2 function. The approach taken to elucidate the cellular roles of RP2 in retina was to identify potential interacting partner proteins from a retina library using a yeast-two-hybrid approach. The Sos Recruitment System (SRS) was exploited, as RP2 had previously been shown to be predominantly localised on the plasma membrane. Several candidate protein partners for RP2 were identified. ADP-ribosylation like factor 3 (Arl3) was the most common cDNA identified in the yeast-two-hybrid screen. The interaction between RP2 and Arl3 was further defined using site directed mutagenesis to model pathogenic mutations in RP2 and a series of deletion mutants. RP2 preferentially interacted with the active GTP-bound form of Arl3 via the RP2 TBCC-homology domain. An altered cellular localisation and behaviour of RP2 was observed when co-transfected with GTP-bound Ari3. Both Arl3 and RP2 exhibit partial relocalisation to the Golgi and increased intracellular transport of RP2-GFP. Arl2 is structurally very similar to Arl3, and was also identified as a potential interactor of RP2, however the physiological significance of the RP2-Ari2 interaction is unclear. Interestingly, TBCC did not bind Arl2 or Arl3 confirming that RP2 and TBCC have different cellular functions. Four other novel potential interactors were also identified from the yeast two-hybrid screen including another small GTPase, and their interaction mapped to the C-terminus of RP2. The cellular localisation and function of these novel interactors suggest RP2 may have multiple cellular functions.

Type: Thesis (Doctoral)
Title: Identification and characterisation of RP2 interacting proteins.
Identifier: PQ ETD:591494
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest. Third party copyright material has been removed from the ethesis
UCL classification:
URI: https://discovery.ucl.ac.uk/id/eprint/1444192
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