Rp And The Rhodopsin Receptor

RP is a heterogeneous group of inherited disorders that lead to retinal degeneration [68] . Patients afflicted with RP show progressive night blindness, loss of central vision, rod cell degeneration often accompanied by subsequent cone loss, and progressive decreases in electroretinogram (ERG) potentials (for review, see Reference 69). The progressive death of rod and cone cells precipitates other pathological symptoms in the retina, including attenuation of retinal vasculature, a pale optic disk, and the accumulation of intra- retinal pigment deposits from which the disease derives its name (for review, see Reference 70). RP is estimated to affect 2 million people worldwide, with an incidence rate of approximately 1:3500 -71] . Mutations leading to RP have been found in over 20 different genes (for review, see References 69, 72-74]), with mutations in the rhodopsin gene representing the most common single cause of autosomal dominant RP (adRP) and accounting for up to 50% of all adRP cases (for review, see References 69, 75). To date, over 100 missense mutations and over 20 other insertion, deletion, nonsense, and splice site mutations that are associated with RP have been identified in the rhodopsin gene (http://www.sph.uth.tmc.edu/Retnet/disease.htm; http://www.retina-international.org/sci-news/rhomut.htm). P23H is the most common single mutant form of rhodopsin, accounting for up to 10% of all adRP cases [75] . Currently, there is no effective therapy for RP, though supplementation with vitamin A (the precursor of 11-ds-retinal; see below) has shown some efficacy in a subset of RP patients [76].

Rhodopsin is the dim light-activated photoreceptor located in vertebrate rod cells of the retina, where it functions to absorb light and initiate the visual photoexcitation response (for review, see Reference 77). Rhodopsin accounts for >70% of the total protein in the membranes of rod cell outer segment stacked disks. While rhodopsin is a member of the GPCR superfamily, it uniquely is comprised of two components: opsin, the membrane-bound polypeptide, and 11-cis-retinal, a chromophore component that is covalently bound to opsin via a protonated Schiff base. 11-Cis-retinal is derived from vitamin A and acts as an inverse agonist when occupying opsin 's binding pocket that yields a distinct UV-visible spectrum with a ^max of ~500nm (for review, see Reference 78). Once bound to opsin, 11-ds-retinal is stable in the absence of light. Rhodopsin captures photons of light to catalyze the isomerization of 11-ds-retinal to all-trans-retinal. Upon photoisomerization, both 11-ds-retinal and opsin undergo a series of conformational changes that result in the conversion of rhodopsin to metarhodopsin II, the active form of rhodopsin. Once activated by rhodopsin, G. (a) relieves inhibition of cyclic guanosine monophosphate (cGMP) phosphodiesterase, resulting in the hydrolysis of cGMP to 5'-GMP. The subsequent decrease in cytosolic cGMP triggers the closure of cGMP-gated calcium channels, lowering cytosolic calcium levels and leading to hyperpolarization of the cell, which is transmitted as a neural signal.

The gene for human rhodopsin is comprised of five exons, spans a total of -7000 bases, and codes for a glycoprotein of 348 amino acids with a molecular mass of approximately 40 kDa [79]. Nascent rhodopsin molecules are co-translationally inserted into the ER membrane of the rod cell inner segment, where they undergo ^-linked glycosylation and conformational maturation prior to export to the Golgi apparatus and vesicular transport to the stacked disk membranes in the rod cell outer segment (for review, see Reference 80). Like other GPCRs, rhodopsin is comprised of three domains: extracellular (intradiscal), TM, and intracellular (cytoplasmic). The integrity of these three domains is critical for proper function, as evidenced by adRP-associated mutations having been identified in all three regions. The X-ray crystal structure for bovine rhodopsin in the dark state has been solved at 2.8-Â resolution [81]. Mapping of point-mutated residues onto the structure for rhodopsin has been instrumental in providing a molecular understanding for many of the defects caused by mutations in rhodopsin's three domains.

The extracellular (intradiscal) domain is comprised of the amino-terminus (residues 1-33) as well as extracellular loops I (residues 101-105), II ("plug"

residues 173-198), and III (residues 277-285). The amino-terminal domain has two ^-linked glycosylation sites at Asn2 and Asn15, which are important for proper folding and trafficking of rhodopsin. Mutations in this region of the protein result in improper folding and poor 11-cis-retinal binding (for review, see Reference 82). Extracellular loop II contains a twisted p-hairpin and Cys187 that forms a disulfide bond with Cys110 in a-helix III of the TM domain; this is critical for proper folding, stability, and function [83-86]. Additionally, extracellular loop II forms a "plug" upon which 11-cis-retinal lies, with Glu181 and Cys187 in close proximity to the C12 of retinal -81, 86], Similarly, residues of the amino-terminus form a series of p-strands that are also an integral part of this amino-terminal plug [78], which keeps 11-cis-retinal in the proper position.

The TM domain, comprised of the seven 20-33 residue membrane-spanning a-helices (I-VII), is structurally coupled to the extracellular domain, such that mutations in the extracellular domain can impact opsin's ability to bind 11-cis-retinal in the TM domain on the e-amino group of Lys296 within a-helix VII [87-89]. Substitution of Lys296, as is found in some patients with severe adRP, abolishes 11-cis-retinal binding and leads to constitutive activity of opsin in the absence of light [90, 91]. Once bound to wild-type opsin, the 11-cis-retinal chromophore is further stabilized by salt bridge formation with negatively charged Glu113 of a-helix III -92, 93] ; other TM domain residues are also important for chromophore stabilization, photoisomerization efficiency, opsin conformational changes, and optimal wavelength absorption [94-96]. Charged residues on the cytoplasmic side of the TM domain, including the conserved Glu134-Arg135-Tyr136 triplet, are critical for interaction with, and activation of, G- (a) [97]. Lastly, mutations in the TM domain can lead to formation of an abnormal disulfide bond between Cys185 and Cys187 in the extracellular domain, preventing proper function and leading to irreversible protein mis-folding [84, 85, 96, 98, 99].

The intracellular (cytoplasmic) domain consists of intracellular loops I, II, and III, an additional a-helical loop formed between the end of membrane-spanning a-helix VII and the palmitoyl attachment sites at Cys322 and Cys323 [100, 101], as well as the carboxy-terminal tail. It is now clear that the intracel-lular loops are critical for Gt binding as mutations in loops II and III impair rhodopsin signaling -97, 102-104] - In addition, rhodopsin kinase associates with the carboxy-terminal tail and phosphorylates Ser334, Ser338, and Ser343 upon photoactivation [105-107], thus promoting interaction with visual arrestin and termination of signaling [108]. Mutations in this region may also result in impaired trafficking to the rod outer segment (see below).

P23H was the first point mutation identified within rhodopsin that was associated with adRP [109] and is the most common mutation found in adRP, accounting for up to 40% of the cases in the United States [ 110, 111]. As mentioned above, over 100 missense mutations and approximately 20 other insertion, deletion, nonsense, and splice site mutations that are associated with adRP have now been identified and are randomly distributed throughout the sequence of rhodopsin. Overexpression of mutant rhodopsins that have been identified in adRP patients show a number of molecular impairments, including protein folding, 11-cis-retinal chromophore binding, G protein coupling/ activation, and/or intracellular trafficking. On the basis of these biochemical defects, mutant rhodopsins have generally been classified into three categories [111 - 115] . In vitro, Class I mutants are similar to wild-type rhodopsin in that they can form functional photopigment upon reconstitution with 11-cis- retinal in the dark and show plasma membrane localization when expressed in HEK293 or COS cells [112-114] . Class I mutants typically cluster near the carboxy-terminal domain of rhodopsin and are defective in trafficking to the rod outer segment -n vivo [112, 116-119] . Some Class I mutants also inefficiently couple to Gt [82, 120, 121]. Class II mutants are the most prevalent, sh owing defects in 11- cis-retinal binding, glycosylation, and diminished cell surface localization due to various extents of ER retention [111, 114]. Compared to wild-type rhodopsin, several members of this mutant class (e.g., P23H, G188R) have extensive, long- -ived interactions with ER- resident molecular chaperones (including Grp78, Hsp60, and Grp94) [75, 122] and are substrates for degradation by the ubiquitin-proteasome system [123, 124]. In addition, Class II mutants have been further subdivided, with Class IIa mutants showing less cell surface expression than Class IIb mutants [112]. Mutations leading to the Class II phenotype are found in all three domains [111, 113, 114, 125, 126]. Lastly, Class III mutants (characterized by mutations at Arg135) show low levels of retinal binding, are hyperphosphorylated, and are constitutively internalized when expressed in HEK293 cells due to prolonged interaction with visual arrestin [115] - These rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions, suggesting that impaired endocytic activity may underlie the pathogenesis of adRP caused by Class III mutations [115].

Because more than 100 adRP mutations have been identified and these mutations are distributed throughout the different functional domains of opsin, it is likely that they lead to retinal degeneration through multiple pathways. Photoreceptor death in adRP is believed to occur by apoptosis, with sorting failures and prolonged rhodopsin activation having been postulated as key contributors to cell death in patients (for review, see Reference 127). In some cases, adRP and other neurodegenerative diseases appear to share a common etiology, having abundant production of folding-defective polypepetides that lead to overt inclusion bodies and ubiquitin immunoreactivity [123, 124, 128, 129]. The P23H rhodopsin is degraded by the ubiquitin-proteasome system and, unlike wild-type and the Class I mutant V345M rhodopsin, forms aggregates and results in a generalized impairment of the ubiquitin-proteasome system even when expressed at low levels. These data suggest that, in the case of Class II mutants, rhodopsin aggregation in the ER and Golgi may lead to a toxic gain of function, similar to other aggregation-prone proteins associated with neurodegenerative diseases. In the case of Class I mutants, which show attenuated targeting to the rod outer segment and subsequent accumulation in the plasma membrane, other possible mechanisms for the induction of apoptosis that are consistent with a sorting defect at or beyond the trans-Golgi network have been proposed - 130] . Mutant rhodopsin may overwhelm the normal vesicular machinery of the plasma membrane-bound pathway, interfering with the routing of legitimate cargo. In addition, the physical presence of high levels of mutant opsin in the plasma membrane could interfere with normal cellular processes, such as neurotransmitter release. Lastly, the source of photoreceptor damage could be related to the buildup of protein in the cell body, perhaps by producing an excessive metabolic burden associated with its destruction.

Based on -n vitro studies, it is now clear that the stability of opsin can be increased upon binding of 11-cis-retinal or other retinoids [131-134], and stability is a key property in the mechanism of a pharmacological chaperone. Thus, via stabilization and structural correction of less stable mutant forms of opsin, retinoids have the potential to act as pharmacological chaperones to increase the cellular content and cell surface localization of mutant opsins. To this end, the level of T17M rhodopsin isolated from cell membranes increased 10-fold when overexpressed in the presence of 11-cis-retinal [117]. Similarly, cellular expression levels and cell surface localization of P23H rhodopsin were increased when incubated with 9-cis-retinal, 11-cis-retinal, or a locked, non-photoisomerizable 7-membered ring form of retinal (11-cis-7-ring-retinal) [124, 135, 136]. Recent high-throughput screening efforts have identified additional molecules that can increase cell surface expression of P23H rhodopsin [137]. These molecules were shown to have low affinity for opsin and, as such, could be displaced with 11-cis-retinal after introduction to the culture media. Importantly, mutant rhodopsin was formed efficiently only when retinals were added during opsin expression, which facilitated trafficking of the mutant opsin through the secretory pathway to the cell surface [136], suggesting that the interaction and stabilization occur early in the biogenesis of opsin and most likely facilitate the folding and ER export of mutant forms of opsin. These data are supported by earlier studies showing that rhodopsin is present in the rough ER of rod cells, indicating that retinal binds to opsin in the inner segment early in its biogenesis [138] . Collectively, these studies demonstrate that retinoids can act as pharmacological chaperones to stabilize mutant forms of opsin, facilitating retinal binding and generating a functional rhodopsin. Furthermore, studies using the "locked" forms of retinals that are easily accepted into the binding site of opsin may provide additional information about contact sites that could be important to more efficiently facilitate folding and stabilization of mutant forms of rhodopsin [139, 140].

Several animal models of rhodopsin-associated RP have been generated. The rhodopsin knockout mouse, which lacks any functional rhodopsin, undergoes rapid retinal degeneration in the first 3 months of life, with reductions in the quantity of rod photoreceptors and thinning of the rod outer segment noted as early as postnatal day 24 [141]. More importantly, transgenic mouse lines that have different copy numbers of human P23H rhodopsin have been created on a wild-type background. The mice show a gene-dosing effect for disease severity that closely mimics the human condition, including diminished or absent ERG responses and profound loss of photoreceptor cells that begins in the central retina and progresses peripherally [125, 126] . A similar pattern of retinal degeneration is also seen in mice harboring a murine transgene containing P23H opsin as well as two other non-adRP-related point mutations located at the amino-terminus (V20G and P27L) [ 142] . Interestingly, overexpression of human wild-type rhodopsin at five times the normal levels also caused retinal degeneration, similar to that seen in mice carrying the mutant P23H transgene [125]. These studies indicate that photoreceptors, similar to HEK293 and COS cells, can express mutant forms of rhodopsin in vivo and that their expression can lead to disease, with loss- of-tunction, gain-of-function, and/or dominant-negative effects having been proposed. Other studies using transgenic mice harboring different mutant forms of human opsin (K296E, P347S, Q344X), as well as a transgenic rat model expressing Ser334X rhodopsin, have also shown gene -dosing effects, whereby the expression levels of the mutant opsins directly correlate with the rate of retinal degeneration [91, 116, 130, 143].

The utility of pharmacological chaperones in adRP was explored -n vivo using high-dose dietary vitamin A supplementation (40 times higher than obtained in a traditional rodent diet) in two transgenic mouse lines [117]. Mice harboring either T17M (amino-terminal domain) or P347S (carboxy-terminal domain) mutant forms of rhodopsin were chosen as representative of Class II and Class I mutants, respectively. High doses of vitamin A significantly reduced the rate of decline as measured by electroretinography in mice carrying a T17M mutant form of rhodopsin. Corresponding histological evaluation showed that the treatment was associated with significantly longer photoreceptor inner and outer segments, and a thicker outer nuclear layer. These effects were corroborated -n vitro - as inclusion of 11-cis-retinal in the culture media partially stabilized T17M mutant opsin expressed in HEK293 cells. No effect of vitamin A was seen in mice harboring P347S rhodopsin, though this mutant did show aberrant transport to the outer segments [143] - Similar observations have been made in pigs harboring a P347L opsin [ 144, 145] and in mice lacking the last five amino acids of opsin due to a premature stop codon (Q344X; - 116]- , pointing to a potentially important role of the carboxy-t erminus in rod outer segment targeting. Similar to the observations in mice, adult RP patients with the common forms of the disease that were administered oral vitamin A supplementation showed, on average, a slowing of the rate of retinal degeneration as measured by decline in ERG amplitude [76]; however, response was not correlated to genotype in this study. These data suggest that vitamin A supplementation may confer therapeutic benefit by stabilizing mutant opsins through increased availability of chromophore.

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