POLÍTICAS DE CONTROL

1.6.1 In vitro studies

To increase our understanding of the molecular mechanisms of rhodopsin RP the biochemical differences between wild-type (WT) and the adRP mutants of rhodopsin were investigated using cell culture models. The seminal work by Sung and co-workers in 1991 and 1993 revealed differences in the biochemical properties of the RP mutants of human rhodopsin expressed in cultured cells (Sung at a/., 1991b, Sung at a/., 1993); which included variations in the ability of mutants to regenerate with 11-c/s-retinal to form functional rhodopsin and also variations in yield and cell surface expression of the receptor. The correct tertiary structure of the mutant opsins was assessed by the ability to bind 11-c/s-retinal and produce pigments with the normal WT rhodopsin absorbance spectra, protein yield and cellular localisation. Two distinct classes of mutants emerged form this work: class I mutants resemble WT in yield, regeneration with 11-c/s-retinal and cellular localisation i.e. plasma membrane staining. The majority of rhodopsin mutations in class I lie in the C-terminus of the protein and do not effect the folding of the protein (eg. Q344ter, V345M, P347S). Class II mutants gave lower yields, regenerated with 11-c/s- retinal to variable extents or not at all, and show diminished plasma membrane expression. A further distinction was made within class II mutants between those mutants that showed predominantly intracellular localisation (Class lia) and those that also show significant cell surface localisation (Class lib). Rhodopsin mutations characterised as class II were found in the intradiscal domain, the transmembrane domains and the cytoplasmic domain of the protein. The analysis of rhodopsin adRP mutations in cell culture by Sung at a!., 1991b and Sung at a!., 1993 and other investigators (Kaushal and Khorana, 1994) has revealed that the majority of rhodospin mutations result in a protein that cannot attain its native conformation. In addition to these mutants three particular mutations that are responsible for adRP result in amino acid substitution of Lysine-296

(Keen et al., 1991, Gal et al., 1997, Sohocki et al., 2001) the site of attachment of 11-c/s- retinal, thus preventing its binding to opsin. K296E rod opsin purified from cultured cells was found to activate transducin (from bovine retinas) in the absence of light with activities comparable to that of light activated WT rhodopsin, revealing a novel phenotype for this particular mutant (Robinson et a i, 1992). However, K296E rhodopsin is inactive in vivo as this mutant protein was found in a stable complex with arrestin and was not, as previously thought, constitutively active (Li et a i, 1995).

1.6.2 Transgenic models of Rhodopsin RP

Research in to the phenotypic differences of rhodopsin RP mutants has continued using transgenic mouse technology to produce mouse models of retinal degeneration. These models revealed that RP rhodopsin transgene expression in mice lead to photoreceptor cell death by apoptosis (Portera-Cailliau et al., 1994) and in addition Drosophila mutant rhodopsin causes photoreceptor degeneration by apoptosis (Davidson and Steller. 1998). Transgenic mice have been created with a number of rhodospin RP mutants including class I and class II mutants. Of the class I mutants, mice expressing, P347S (Huang et a i, 1993, Weiss et a i, 1994, Li et a i, 1996), Q344ter (Sung et a i, 1994) and swine expressing P347L (Petters et a i, 1997, Tso et ai, 1997, Li et ai, 1998) were engineered. Of the class II mutants P23H (Olsson et a i, 1992, Roof et a i, 1994) K296E (Li et al 1995) and T17M (Li et a i, 1998) were expressed in mice. There appears to be no early developmental defect involving the retina in RP mouse models but retinal abnormalities (including abnormal ERGs, shorter than nonrial rod outer segments and rod and cone cell loss) are detected approximately 10 days after birth.

A number of Class I adRP mutants of rhodopsin are found within the rhodopsin C-terminal sequence QVAPA (QVSPA in frog and fish) which are the last 5 amino acids of the polypeptide and is highly conserved among different species. Amino acid substitutions of proline and valine along with a Q344 termination in this conserved sequence have been associated with adRP (Table 1). The carboxy-terminus of rhodopsin together with the palmitoylation of cysteines 322/323 are important for efficient rhodopsin transport and sorting to the outer segement (Tam et a i, 2000). The wild type carboxy-terminus binds to Tctex-1 protein, a widely expressed dynein light chain which is abundant in photoreceptor inner segments and is thought to guide the transport of rhodopsin laden post Golgi vesicles to the rod outer segment (Tai et a i, 1999). However, palmitoylation of cysteine residues 322 and 323 which provide a point of membrane attachment is also required for

efficient targeting of rhodopsin to outer segments in addition to the distal amino acids of the carboxy-terminus (Tam et al., 2000). The C-terminal RP mutants of rhodopsin just described fail to bind to Tctex-1 and in transgenic animals these mutants accumulate in the cell body of rod cells and show defective intracellular transport. For example, one class I mutant (Q344ter) showed inefficient outer segment localisation of the mutant opsin but not of the endogenous wild type opsin (Sung et al., 1994) and a second mutant (P347S) induces accumulation of vesicles at the base of the outer segment (Li et a/., 1996).

Of particular relevance to my work are the transgenic mice expressing the adRP mutants of rhodopsin P23H and K296E. The P23H transgenic mice revealed that P23H rod opsin accumulates in the inner segments and mislocalises to the synapses of rod cells at the outer plexiform layer but also translocates to the rod outer segment (Olsson et a/., 1992, Roof et a!., 1994). Furthermore increased expression of the P23H transgene led to a more rapid degeneration of the retina suggesting a gene dosage effect (Olsson et a/., 1992). K296E rhodopsin constitutively activates transducin in vitro (Robinson et ai., 1992) and causes RP in vivo (Keen et ai., 1991, Vaithinathan et ai., 1994). It has been suggested that this mutant causes disease by constitutive activation of the phototransduction cascade. Support for this hypothesis not only comes from the finding that K296E rhodopsin constitutively activates transducin in vitro but also from observations that constant light exposure damages photoreceptor cells (Lavail, 1980, Rapp and Williams, 1980) and opsin apoprotein without ligand was reported to activate phototransduction without light, albeit at six orders of magnitude lower efficiency (Fain and Cornwall, 1993). Surprisingly however, K296E in transgenic mice was found to be inactivated by phosphorylation and the binding of arrestin (Li et ai., 1995) thus showing that the K296E mutant does not cause photoreceptor degeneration by continuous activation of phototransduction.

Two classes of disease expression were found in patients with different mutations in the rhodopsin gene and disease type was found to be allele specific (Cideciyan et ai., 1998). Class A mutants (R135G, R135L, R135W, V345L and P347L) led to severely abnormal rod function across the retina early in life, in comparison class B mutants (T17M, P23H, T58R, V87D) were compatible with normal rods in adult life in some retinal regions or throughout the retina and there was a slow stereotypical disease sequence (Cideciyan et ai., 1998). Class A families reported onset of night blindness in early life where as class B families reported little or no night vision symptoms and rod function was relatively

preserved. Some class A disease mutants (V345L and P347L) have been described as class I mutants in vitro i.e. attain the native conformation and bind 11-c/s-retinal. However, three mutants in class A disease type involve R135G, R135L, R135W (arginine-135 is highly conserved in members of the rhodopsin GPCR family) and in vitro studies of this mutant showed abnormal folding and poor regeneration with 11-c/s-retinal (Sung et al., 1991, Sung et al., 1993). Most class B disease mutants show folding abnormalities in vitro. Thus the relationship between in vitro classification of rhodopsin mutants and the human disease is obviously complex.