Swing Up utilizando regulaci´ on de energ´ıa

The genetic defects that cause RP are heterogeneous and many of the genes involved encode proteins with roles in phototransduction or photoreceptor outer segment maintenance (Chapter 1). RP2 differs from these retinitis pigmentosa genes as it is present at similar levels in all tissues examined, yet the pathology associated with RP2 mutations is restricted to the eye. Another XLRP gene, RP3, is also expressed outside of the eye and encodes the retinitis pigmentosa GTPase regulator (RPGR)(Meindl et al., 1996; Roepman et al., 1996). The specific role of RPGR in the retina remains to be determined, but may have a function in cellular transport essential to the long-term maintenance of photoreceptor viability (Reviewed in Vervoort et al., 2002). RPGR has been shown to localise to the connecting cilia of photoreceptors in mouse retina (Hong

et al., 2000; Mavlyutov et al., 2002; Hong et al., 2002), to rod outer segments in bovine retina (Mavlyutov et al., 2002) and to rod and cone outer segments in human retina (Mavlyutov et al., 2002). Another study localised RPGR to rod, but not cone, outer segments and connecting cilia in human and bovine retina (Roepman et al., 2000). Investigations into RPGR transcription have suggested that levels of RPGR mRNA are lower in the retina than in other tissues (Meindl et al., 1996; Kirschner et al., 1999; Zeiss et al., 2000), raising the possibility of retina-specific regulation of RPGR mRNA. Furthermore, the observation that the alternatively spliced 0RF15 transcript is preferentially expressed in the retina and testis of bovine tissues (Vervoort et al., 2000), coupled with the high frequency of causative mutations in the ORF15 exon (Vervoort et al., 2002) suggests an essential retinal-specific role for ORFIS-containing transcripts. Collectively, these data suggest that the relationship between the ubiquitous RPGR expression and retinal disease pathogenesis is not a simple one. This may also be true for ubiquitously expressed RP2 and so the detailed study of the localisation and function of RP2 and RP2 interacting proteins will be necessary not only for the retina, but also in a wide variety of cells and tissues.

It has been demonstrated that RP2 is targeted to the plasma membrane by an N- terminal Met-Gly-Cys-X-Phe-Ser motif that is necessary and sufficient for the dual acylation of the protein (Chappie et al., 2002a). A different study has suggested however, that RP2 may not be targeted to the plasma membrane in all cell types (Schwahn et al., 2001). To further investigate the plasma membrane association of RP2, I exploited the ubiquitous expression of RP2 to examine the subcellular

Chapter 3 - Expression and subcellular localisation of RP2

localisation of endogenous RP2 in a variety of cultured human cells types. RP2 was targeted to the plasma membrane in each of the cell types examined, including lymphoblastoid cells. The plasma membrane association of RP2 in lymphoblastoid cells was confirmed by the presence of the protein in fractions of a sucrose gradient associated with plasma membrane proteins.

The ubiquitous expression of the acyl modification machinery predicts that RP2 would be targeted to the plasma membrane in most cell types and the data presented in this chapter confirms this hypothesis. Possible reasons for the reported differences in localisation seem likely to reflect variations in experimental conditions. In this chapter I have studied the endogenous expression of RP2, whereas the other study used cells transfected with GFP-fusion constructs (Schwahn et al., 2001). Furthermore, in the study by Schwahn and colleagues (Schwahn et a/., 2001), RP2-GFP fusions were localised to the cytoplasm in transfected COS-7 cells, but recent results obtained in collaboration with Dr J. Paul Chappie have demonstrated that RP2-GFP fusions can be efficiently targeted to the plasma membrane in COS-7 cells (Chappie et a!., 2002b). The differences in localisation are, therefore, likely to be due to high levels of heterologous protein expression or levels of endogenous acylated proteins, rather than cell type variation in the myristoylation and palmitoyiation machinery. As the association of RP2 with the plasma membrane may be dynamic it is also possible that RP2 localisation could be regulated by other cellular factors in addition to N-myristoyl transferase (NMT) and palmitoyl acyl transferase (PAT).

RP2 is ubiquitously expressed, but there are significant variations in the levels of protein expression between cell types and during cellular differentiation. With the exception of HeLa cells, the highest levels of RP2 were observed in ARPE19 cells. These cells have been used extensively as a model for studies of the RPE (Dunn et a/., 1996). The RPE fulfils many essential functions in the retina, including trophic support, metabolite delivery and the phagocytosis of photoreceptor outer segments. The site of the primary pathological deficit for retinitis pigmentosa caused by mutations in RP2 is not clear from the protein's localisation in the retina (Chapter 5)(Grayson et a/., 2002b). RP2 is expressed in the RPE in vivo (Chapter 5)(Grayson et ai., 2002b). Therefore, it remains possible that mutations in RP2 could cause retinal disease through a disruption of RPE function, and at least seven genes expressed in the RPE have been associated with photoreceptor degeneration to date (Clarke et ai., 2000).

Chapter 3 - Expression and subcellular localisation of RP2

The data presented in this chapter has also demonstrated that RP2 does not partition into either the apical or basolateral domains of polarised epithelial cells, both in vitro

and in vivo. Polarity is a fundamental property of eukaryotic cells that requires the asymmetrical distribution of many plasma membrane associated proteins. Dually- acylated proteins have been associated with preferential sorting to the apical region of polarised cells (Simons et al., 2000). One such protein, Gtk, is found in the apical membrane of gut columnar epithelial cells (Sunitha et al., 1994). RP2 localisation was examined in intestinal epithelial cells both in vitro and in vivo, but unlike Gtk, partitioning of RP2 to a specific membrane macro-domain was not observed. It is possible that RP2 could have a cell type specific sorting signal. A recent study has suggested that RP2 localises to photoreceptor synapses in the human retina (Breuer et ai., 2001), which would be consistent with an apical localisation. The data presented in this chapter, however, indicates that the polarised sorting of RP2 to a specific membrane macro-domain does not occur in epithelial cells or in the retina (Chapter 5)(Grayson et ai., 2002b). In this chapter it was also observed that there was an increase in the level of RP2 expression in Caco-2 cells as they polarised, suggesting a possible function for RP2 relating to cellular differentiation such as specialised protein transport or signalling.

Although the data described in this chapter highlights the importance of the plasma membrane association of RP2, the function of RP2 at the plasma membrane remains unclear. The next important step will be to delineate the dynamics of RP2 membrane association and to identify the interacting partners of RP2 on the membrane. Once this has been established, the cellular pathways under the control of these proteins in the retina will enable a better understanding of the retinitis pigmentosa disease pathogenesis caused by mutations in RP2.

The next chapter describes the use of a lymphoblastoid cell model to investigate a potential drug-mediated therapy for treating patients with a specific mutation in RP2.

The work described in this chapter has been submitted for publication (Chappie et al.,