regeneration, whereas deletions in the first and second intradiskal loops caused regenerationincompetent misfolding, with segments 171–182 and 189–192 being structurally essential. Single-point mutations utilized to investigate the relative contribution of residues in the deleted segments along the second intradiskal loop showed that most individual changes resembled wild-type regeneration. Deletion of 189–190 resulted in dramatic retention of the mutant protein in the endoplasmic reticulum. This region is now known to contribute to the formation of a structurally critical salt bridge between D190 and R177 [97], linking the ends of the second intradiskal loop. Although interaction of this ion pair has no effect on solvent exposure or signaling, this interaction appears conserved as R or K and E or D pairings in most GPCRs and is critical for stabilization of the dark state of rhodopsin. The stabilizing role of this salt bridge is highlighted by its proximity to the disulfide bond between C110 and C187 in the intradiskal region. Formation of this disulfide bridge is critical to receptor structure and is highly conserved throughout the GPCR superfamily. RP mutants directly modifying one of these cysteines, C110F and C110Y or C187Y, cause abnormal disulfide formation of C185:C187 or C110:C185, respectively [26]. Surprisingly, additional RP mutants (G89D, L125R, A164V, and H211P) influence this structural bridge, through disruption of normal helical packing, promoting formation of the abnormal C185:C187 disulfide bridge [15].

IMPLICATIONS OF RECEPTOR MISFOLDING

The dominant impact of misfolded rhodopsin is demonstrated through coexpression of wild-type rhodopsin and a misfolded mutant, for which intracellular retention of the mutant results in a corresponding retention of wild-type receptor, decreasing surface expression and signaling [79]. Numerous structural elements are ultimately responsible for maintenance of the rhodopsin structure, including membrane lipid interactions [98, 99], salt bridges, and both hydrophobic and polar contacts within the transmembrane regions. However, no single interaction has demonstrated as much significance in stabilization of the overall receptor as the conserved disulfide bond located at the intradiskal–transmembrane interface.

Evaluating the rate of vision decline among 140RP patients from 1975 to 2000 showed C-terminal rhodopsin mutations to decline most rapidly [100]. Considering the various classifications of rhodopsin mutations eliciting RP, it seems intuitive that significant variability in disease severity and rate of progression would arise between the various classes. Indeed, adRP mutations in rhodopsin show astounding variability within a given mutation class [85], even when considering only a single-point mutation, such as P23H. The progression model included age, gender, baseline function, and affected region but could only account for 20–34% of the variation. Predominant theories behind individual variability in disease progression focus on complementary genetic and nongenetic contributions. Complementary genetic contributions may include any polygenic interaction affecting retina function, including rhodopsin folding, trafficking, degradation, ion homeostasis, RPE integrity, ROS structure, and vitamin A processing. Detailed analyses involving polygenic interactions in RP are becoming more accessible with advances in techniques to identify single-nucleotide polymorphisms and perform haplotype analysis, with findings anticipated to have considerable impact on disease severity and progression.

Acknowledging differences between the various forms of RP may lead to interesting implications in the prospect of treatments. Misfolding and aberrant disulfide formation, for example, may be alleviated through traditional pharmacological intervention with retinoid-mimetic folding chaperones. Transgenic mice expressing the misfolding T17M mutation and receiving dietary supplementation with high-dose vitamin A showed significant reduction in degeneration symptoms [101]. No such effect was observed in transgenic mice expressing the trafficking-impaired P347S variant, suggesting that such a therapeutic approach would indeed be specific to misfolding. In vitro, similar receptor rescue improves purification yield of severely misfolded RP mutant A164V using increased concentrations of inverse agonist [66]. The most predominant rhodopsin RP mutation, P23H, is also a misfolding mutation, resulting in intracellular accumulation [102]. Use of retinal-based structural chaperones 9-cis retinal, 11-cis retinal, and 11-cis 7-ring retinal [103] improved the ability of the photoreceptor to reach the plasma membrane [104], although not necessarily resulting in functional protein production [105]. Demethylation at C1 or C5 of the retinal ring produces partial agonist activity, shifting conformational equilibrium from MII to inactive MI by interfering with E134mediated proton transfer [106]. Thus, pharmacotherapy for rhodopsin RP mutations may require a degree of individualization based on specific structural defects imparted by the mutations. Simple in vitro assays may be of potential use in predicting in vivo responsiveness.

NONGENETIC CONTRIBUTIONS TO RP

Nongenetic contributions are also being elucidated. Suggested factors affecting the clinical course of RP focus primarily on diet, general health, and light exposure. The observation that light exposure exacerbates retinal degeneration in particular RP subtypes provides an interesting and complicating factor in understanding disease progression. One explanation for this phenomenon relates to cellular damage incurred by ultraviolet radiation, potentially damaging the rod cell, the RPE, or other surrounding supportive cell types. An alternative, and potentially additive, hypothesis suggests that ligand, released on photoactivation, becomes unable to stabilize mutant opsin, allowing the receptor to collapse and encouraging the misfolded-degeneration process.

The theory of exacerbated misfolding/instability due to loss of ligand is indirectly supported by the only common treatment available for RP patients, vitamin A supplementation. As a required precursor to 11-cis retinal formation, which acts as a structural stabilizing agent for rhodopsin, treatment with vitamin A provides support for mutations affecting protein folding. However, effectiveness is, again, highly variable. One likely explanation for the variability of effectiveness with vitamin A supplementation in RP patients is the diversity of causal defects, as described with rhodopsin mutation classifications, which would be expected to produce various responses. Precisely such a difference in effectiveness was demonstrated through comparison of vitamin A effectiveness in transgenic mice expressing either a trafficking-impaired mutant (P347S) or a foldingimpaired mutant (T17M) [101]. As anticipated, based on the stabilizing effect of ligand binding, vitamin A improved histologic morphology and decreased the rate of decline for the misfolding mutant but not for the trafficking-impaired mutant.

Circumstantial evidence points to another supplement with potential impact on disease progression. Association of zinc deficiency with a number of clinical manifestations of RP, impaired dark adaptation [107], decreased rhodopsin regeneration [108], and degeneration of ROS [109], has suggested possible involvement of the essential trace metal in both native functional and pathological roles. Treatment of bullfrog eyes with low-level zinc resulted in elevated dark-adapted electroretinogram (ERG) thresholds, increased peak ERG amplitudes, and accelerated rhodopsin regeneration [110]. As a critical component of the retina, zinc concentrations in ROS extracts suggest that it may be fortified in ROS disks [111], and radionuclide 65Zn has shown direct binding to purified rhodopsin [112]. Recent evidence also supports direct association of zinc at a high-affinity coordination site near H211 and E122 [113] as well as concentration-dependent alteration of rhodopsin thermostability [114]. Crystallographic data also suggest the presence of such a site [20], although this is not reflected in some later structures as the resolved metal ions were manually replaced with waters during processing [52]. Although solid-state NMR data suggest a lack of direct binding between H211 and zinc, the observed chemical shifts are consistent with the presence of Zn2+ within that region [115]. Direct interactions between divalent cations and GPCRs are not unprecedented as a diverse list of receptors, including β2-adrenergic [116, 117], dopaminergic [118], melanocortin MC1 and MC4 [119, 120], and olfactory receptors [121], have demonstrated direct and specific interactions with zinc. Evaluation of zinc and other trace metals in the context of RP has remained suggestive, although inconclusive [122–124], likely due to a focus on serum levels and a lack of stratification based on different RP mechanisms.

CONCLUSION

The development of powerful biochemical and biophysical techniques to study rhodopsin has allowed for considerable advances and understanding of this fascinating photoreceptor protein. The heterogeneous RP mutations have provided additional insights into rhodopsin structure/function. In vitro biochemical and biophysical assays will provide valuable information on potential therapy at the molecular level. Improvements with vitamin A treatment could be predicted through 11-cis retinal regeneration assays, and the effects of trace metals on folding and stability could also be evaluated. Comprehensive biochemical correlation to clinical disease is a necessary direction for future studies. One can envision as new mutations are discovered that such simple characterization of the mutants will determine appropriate therapy and may predict disease progress and prognosis.

REFERENCES

1.Okano T, Kojima D, Fukada Y, Shichida Y, Yoshizawa T. Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments. Proc Natl Acad Sci U S A 1992;89:5932–5936.

2.Marmor MF, Martin LJ. 100 years of the visual cycle. Survey Ophthalmol 1978;22:279–285.

3.Wald G. Molecular basis of visual excitation. Science 1968;162:230–239.

4.Nathans J, Hogness D. Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 1983;34:807–814.

5.Dryja TP, McGee TL, Reichel E, et al. A point mutation of the rhodopsin gene in one form of retinitis pigmentosa. Nature 1990;343:364–366.

6.Fung BK, Hurley JB, Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A 1981;78:152–156.

7.Bennett N, Dupont Y. The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides. J Biol Chem 1985;260:4156–4168.

8.Ridge KD, Marino JP, Ngo T, Ramon E, Brabazon DM, Abdulaev NG. NMR analysis of rhodopsin-transducin interactions. Vision Res 2006;45:4482–4492.

9.Herrmann R, Heck M, Henklein P, Hofmann KP, Ernst OP. Signal transfer from GPCRs to G Proteins: role of the G{alpha} N-terminal region in rhodopsin-transducin coupling. J Biol Chem 2006;281:30234–30241.

10.Wilden U, Wust E, Weyand I, Kuhn H. Rapid affinity purification of retinal arrestin (48 kDa protein) via its light-dependent binding to phosphorylated rhodopsin. FEBS Lett 1986;207:292–295.

11.Konig B, Arendt A, McDowell JH, Kahlert M, Hargrave PA, Hofmann KP. Three cytoplasmic loops of rhodopsin interact with transducin. Proc Natl Acad Sci U S A 1989;86:6878–6882.

12.Farahbakhsh ZT, Ridge KD, Khorana HG, Hubbell WL. Mapping light-dependent structural changes in the cytoplasmic loop connecting helixes c and d in rhodopsin: a site-directed spin labeling study. Biochemistry 1995;34:8812–8819.

13.Sakmar TP, Franke RR, Khorana HG. Glutamic acid-113 serves as the retinylidene schiff base counterion in bovine rhodopsin. Proc Natl Acad Sci U S A 1989;86:8309–8313.

14.Karnik SS, Sakmar TP, Chen HB, Khorana HG. Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci U S A 1988;85:8459–8463.

15.Hwa J, Klein-Seetharaman J, Khorana HG. Structure and function in rhodopsin: mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants. Proc Natl Acad Sci U S A 2001;98:4872–4876.

16.Unger VM, Schertler GF. Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy. Biophys J 1995;68:1776–1786.

17.Unger VM, Hargrave PA, Baldwin JM, Schertler GFX. Arrangement of rhodopsin transmembrane [alpha]-helices. Nature 1997;389:203–206.

18.Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 1996;274: 768–770.

19.Sheikh SP, Zvyaga TA, Lichtarge O, Sakmar TP, Bourne HR. Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature 1996;383: 347–350.

20.Palczewski K, Kumasaka T, Hori T, et al. Crystal Structure of rhodopsin: a g protein-coupled receptor. Science 2000;289:739–745.

21.Downs MA, Arimoto R, Marshall GR, Kisselev OG. G-protein alpha and beta-gamma subunits interact with conformationally distinct signaling states of rhodopsin. Vision Res 2006;46:442–448.

22.Vogel R, Siebert F, Yan ECY, Sakmar TP, Hirshfeld A, Sheves M. Modulating rho-

dopsin receptor activation by altering the pka of the retinal Schiff base. J Am Chem Soc 2006;128:10503–10512.

23.Liu X, Garriga P, Khorana HG. Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa. Proc Natl Acad Sci U S A 1996;93:4554–4559.

24.Garriga P, Liu X, Khorana HG. Structure and function in rhodopsin: correct folding and

misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125→Arg in the transmembrane helix C. Proc Natl Acad Sci U S A 1996;93: 4560–4564.

25.Hwa J, Garriga P, Liu X, Khorana HG. Structure and function in rhodopsin: packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled. Proc Natl Acad Sci U S A 1997;94:10571–10576.

26.Hwa J, Reeves PJ, Klein-Seetharaman J, Davidson F, Khorana HG. Structure and function in rhodopsin: further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function. Proc Natl Acad Sci U S A 1999;96:1932–1935.

27.Hwa J, Klein-Seetharaman J, Khorana HG. Structure and function in rhodopsin: mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants. Proc Natl Acad Sci U S A 2001;98:4872–4876.

28.Ramon E, del Valle LJ, Garriga P. Unusual thermal and conformational properties of the rhodopsin congenital night blindness mutant Thr-94→Ile. J Biol Chem 2003;278:6427–6432.

29.Stojanovic A, Hwang I, Khorana HG, Hwa J. Retinitis pigmentosa rhodopsin mutations L125R and A164V perturb critical interhelical interactions: new insights through compensatory mutations and crystal structure analysis. J Biol Chem 2003;278:39020–39028.

30.Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 2003;421:127–128.

31.Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A, Palczewski K. The G proteincoupled receptor rhodopsin in the native membrane. FEBS Lett Struct Dyn Funct Proteins Biol Membr 2004;564:281–288.

32.Liang Y, Fotiadis D, Maeda T, et al. Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice. J Biol Chem 2004;279:48189–48196.

33.Suda K, Filipek S, Palczewski K, Engel A, Fotiadis D. The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes. Mol Membr Biol 2004;21: 435–446.

34.Jastrzebska B, Fotiadis D, Jang GF, Stenkamp RE, Engel A, Palczewski K. Functional and structural characterization of rhodopsin oligomers. J Biol Chem 2006;281:11917–11922.

35.Schlyer S, Horuk R. I want a new drug: G-protein-coupled receptors in drug development. Drug Discovery Today 2006;11:481–493.

36.Nathans J, Hogness DS. Isolation and nucleotide sequence of the gene encoding human rhodopsin. Proc Natl Acad Sci U S A 1984;81:4851–4855.

37.Lem J, Krasnoperova NV, Calvert PD, et al. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci U S A 1999;96:736–741.

38.Eley L, Yates LM, Goodship JA. Cilia and disease. Current Opin Genet Dev 2005;15:308– 314.

39.Ames A 3d, Walseth T, Heyman R, Barad M, Graeff R, Goldberg N. Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors. J Biol Chem 1986;261:13034–13042.

40.Doan T, Mendez A, Detwiler PB, Chen J, Rieke F. Multiple phosphorylation sites confer reproducibility of the rod’s single-photon responses. Science 2006;313:530–533.

41.Hofmann K, Pulvermuller A, Buczylko J, Van Hooser P, Palczewski K. The role of arrestin and retinoids in the regeneration pathway of rhodopsin. J Biol Chem 1992;267:15701– 15706.

42.Palczewski K, Hargrave PA, McDowell JH, Ingebritsen TS. The catalytic subunit of phosphatase 2A dephosphorylates phosphoopsin. Biochemistry 1989;28:415–419.

43.Mathies R, Stryer L. Retinal has a highly dipolar vertically excited singlet state: implications for vision. Proc Natl Acad Sci U S A 1976;73:2169–2173.

44.Jager F, Fahmy K, Sakmar TP, Siebert F. Identification of glutamic acid 113 as the Schiff base proton acceptor in the metarhodopsin II photointermediate of rhodopsin. Biochemistry 1994;33:10878–10882.

45.Yan ECY, Kazmi MA, Ganim Z, et al. Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin. Proc Natl Acad Sci U S A 2003;100:9262–9267.

46.Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 1996;274:768–770.

47.Janz JM, Farrens DL. Rhodopsin activation exposes a key hydrophobic binding site for the transducin {alpha}-subunit C terminus. J Biol Chem 2004;279:29767–29773.

48.Kisselev OG, Downs MA. Rhodopsin controls a conformational switch on the transducin [gamma] subunit. Structure 2003;11:367–373.

49.Palczewski K. G Protein×2013;coupled receptor rhodopsin. Annu Rev Biochem 2006;75:743–767.

50.Stojanovic A, Hwa J. Rhodopsin and retinitis pigmentosa: shedding light on structure and function. Recept Channels 2002;8:33–50.

51.Okada T, Fujiyoshi Y, Silow M, Navarro J, Landau EM, Shichida Y. Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography. Proc Natl Acad Sci U S A 2002;99:5982–5987.

52.Okada T, Sugihara M, Bondar A-N, Elstner M, Entel P, Buss V. The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. J Mol Biol 2004;342:571–583.

53.Yeagle PL, Albert AD. Structure of the G-protein-coupled receptor, rhodopsin: a domain approach. Biochem Soc Trans 1998;26:520–531.

54.Klein-Seetharaman J, Getmanova EV, Loewen MC, Reeves PJ, Khorana HG. NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR. Proc Natl Acad Sci U S A 1999;96:13744–13749.

55.Patel AB, Crocker E, Reeves PJ, et al. Changes in interhelical hydrogen bonding upon rhodopsin activation. J Mol Biol 2005;347:803–812.

56.Smith SO, Palings I, Copie V, et al. Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin. Biochemistry 1987;26:1606–1611.

57.Creemers AF, Kiihne S, Bovee-Geurts PH, DeGrip WJ, Lugtenburg J, de Groot HJ. (1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin. Proc Natl Acad Sci U S A 2002;99:9101–9106.

58.Spooner PJ, Sharples JM, Goodall SC, et al. Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate. Biochemistry 2003;42:13371–13378.

59.Cai K, Klein-Seetharaman J, Altenbach C, Hubbell WL, Khorana HG. Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between cysteine pairs engineered in cytoplasmic loops 1, 3, and 4. Biochemistry 2001;40:12479–12485.

60.Altenbach C, Klein-Seetharaman J, Hwa J, Khorana HG, Hubbell WL. Structural features and light-dependent changes in the sequence 59–75 connecting helices I and II in rhodopsin: a site-directed spin-labeling study. Biochemistry 1999;38:7945–7949.

61.Altenbach C, Yang K, Farrens DL, Farahbakhsh ZT, Khorana HG, Hubbell WL. Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study. Biochemistry 1996;35:12470–12478.

62.Yang K, Farrens DL, Hubbell WL, Khorana HG. Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show positionspecific effects in transducin activation. Biochemistry 1996;35:12464–12469.

63.Altenbach C, Cai K, Khorana HG, Hubbell WL. Structural features and light-dependent changes in the sequence 306–322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study. Biochemistry 1999;38:7931–7937.

64.Langen R, Cai K, Altenbach C, Khorana HG, Hubbell WL. Structural features of the C- terminal domain of bovine rhodopsin: a site-directed spin-labeling study. Biochemistry 1999;38:7918–7924.

65.Yang K, Farrens DL, Hubbell WL, Khorana HG. Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show positionspecific effects in transducin activation. Biochemistry 1996;35:12464–12469.

66.Reeves PJ, Hwa J, Khorana HG. Structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket. Proc Natl Acad Sci U S A 1999;96:1927–1931.

67.Rader AJ, Anderson G, Isin B, Khorana HG, Bahar I, Klein-Seetharaman J. Identification of core amino acids stabilizing rhodopsin. Proc Natl Acad Sci U S A 2004;101:7246–7251.

68.Kim J-M, Hwa J, Garriga P, Reeves PJ, RajBhandary UL, Khorana HG. Light-driven activation of beta2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta2adrenergic receptor cytoplasmic loops. Biochemistry 2005;44:2284–2292.

69.Fotiadis D, Jastrzebska B, Philippsen A, Muller DJ, Palczewski K, Engel A. Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors. Current Opin Struct Biol 2006;16:252–259.

70.Angers S, Salahpour A, Bouvier M. Dimerization: an emerging concept for G protein-cou- pled receptor ontogeny and function. Annu Rev Pharmacol Toxicol 2002;42:409–435.

71.Kota P, Reeves PJ, Rajbhandary UL, Khorana HG. Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface. Proc Natl Acad Sci U S A 2006;103:3054–3059.

72.Mansoor SE, Palczewski K, Farrens DL. Rhodopsin self-associates in asolectin liposomes. Proc Natl Acad Sci U S A 2006;103:3060–3065.

73.Jordan BA, Trapaidze N, Gomes I, Nivarthi R, Devi LA. Oligomerization of opioid receptors with beta 2-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation. Proc Natl Acad Sci U S A 2001;98:343–348.

74.Wilson SJ, Roche AM, Kostetskaia E, Smyth EM. Dimerization of the human receptors for prostacyclin and thromboxane facilitates thromboxane receptor-mediated cAMP generation. J Biol Chem 2004;279:53036–53047.

75.Filipek S, Krzysko KA, Fotiadis D, et al. A concept for G protein activation by G pro- tein-coupled receptor dimers: the transducin/rhodopsin interface. Photochem Photobiol Sci 2004;3:628–638.

76.Lohr HR, Kuntchithapautham K, Sharma AK, Rohrer B. Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death. Exp Eye Res 2006;83:380–389.

77.Huang P, Gaitan A, Hao Y, Petters R, Wong F. Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene. Proc Natl Acad Sci U S A 1993;90:8484–8488.

78.Berson EL. Retinitis pigmentosa: unfolding its mystery. Proc Natl Acad Sci U S A 1996;93:4526–4528.

79.Tao YX. Inactivating mutations of G protein-coupled receptors and diseases: structure-function insights and therapeutic implications. Pharmacol Ther 2006;111:949–973.

80.Barak LS, Oakley RH, Laporte SA, Caron MG. Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus. Proc Natl Acad Sci U S A 2001;98:93–98.

81.Min KC, Zvyaga TA, Cypess AM, Sakmar TP. Characterization of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Mutations on the cytoplasmic surface affect transducin activation. J Biol Chem 1993;268:9400–9404.

82.Chuang J-Z, Vega C, Jun W, Sung C-H. Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes. J Clin Invest 2004;114:131–140.

83.Andres A, Kosoy A, Garriga P, Manyosa J. Mutations at position 125 in transmembrane helix III of rhodopsin affect the structure and signalling of the receptor. Eur J Biochem 2001;268:5696–5704.

84.Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD. Constitutively active mutants of rhodopsin. Neuron 1992;9:719–725.

85.Richards JE, Kuo CY, Boehnke M, Sieving PA. Rhodopsin Thr58Arg mutation in a family with autosomal dominant retinitis pigmentosa. Ophthalmology 1991;98:1797–1805.

86.Li T, Franson W, Gordon J, Berson E, Dryja T. Constitutive activation of phototransduction by K296E opsin is not a cause of photoreceptor degeneration. Proc Natl Acad Sci U S A 1995;92:3551–3555.

87.Rao VR, Cohen GB, Oprian DD. Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. 1994;367:639–642.

88.Najma al-Jandal GJF, Kiang AS, Humphries MM, Bannon N, Findlay JBC, Humphries P, Kenna PF. A novel mutation within the rhodopsin gene (Thr-94-Ile) causing autosomal dominant congenital stationary night blindness. Hum Mutat 1999;13:75–81.

89.Moench SJ, Moreland J, Stewart DH, Dewey TG. Fluorescence studies of the localization and membrane accessibility of the palmitoylation sites of rhodopsin. Biochemistry 1994;33:5791–5796.

90.Moench SJ, Terry CE, Dewey TG. Fluorescence labeling of the palmitoylation sites of rhodopsin. Biochemistry 1994;33:5783–5790.

91.Ernst OP, Meyer CK, Marin EP, et al. Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits. J Biol Chem 2000;275:1937–1943.

92.Tai AW, Chuang J-Z, Bode C, Wolfrum U, Sung C-H. Rhodopsin’s carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. Cell 1999;97:877–887.

93.Garriga P, Liu X, Khorana HG. Structure and function in rhodopsin: Correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125 right-arrow Arg in the transmembrane helix C. Proc Natl Acad Sci U S A 1996;93:4560–4564.

94.Reeves PJ, Thurmond RL, Khorana HG. Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines. Proc Natl Acad Sci U S A 1996;93:11487–11492.

95.Kaushal S, Khorana HG. Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochemistry 1994;33:6121–6128.

96.Doi T, Molday R, Khorana H. Role of the intradiscal domain in rhodopsin assembly and function. Proc Natl Acad Sci U S A 1990;87:4991–4995.

97.Janz JM, Fay JF, Farrens DL. Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2. J Biol Chem 2003;278:16982–16991.

98.Isele J, Sakmar TP, Siebert F. Rhodopsin activation affects the environment of specific neighboring phospholipids: an FTIR spectroscopic study. Biophys J 2000;79:3063–3071.

99.SanGiovanni JP, Chew EY. The role of omega-3 long-chain polyunsaturated fatty acids in health and disease of the retina. Prog Retin Eye Res 2005;24:87–138.

100.Berson EL, Rosner B, Weigel-DiFranco C, Dryja TP, Sandberg MA. Disease progression in patients with dominant retinitis pigmentosa and rhodopsin mutations. Invest Ophthalmol Vis Sci 2002;43:3027–3036.

101.Li T, Sandberg MA, Pawlyk BS, et al. Effect of vitamin A supplementation on rhodopsin mutants threonine-17 right-arrow methionine and proline-347 right-arrow serine in transgenic mice and in cell cultures. Proc Natl Acad Sci U S A 1998;95:11933–11938.

102.Saliba RS, Munro PMG, Luthert PJ, Cheetham ME. The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation. J Cell Sci 2002;115:2907–2918.

103.Noorwez SM, Kuksa V, Imanishi Y, et al. Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J Biol Chem 2003;278:14442–14450.

104.Noorwez SM, Malhotra R, McDowell JH, Smith KA, Krebs MP, Kaushal S. Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H. J Biol Chem 2004;279:16278–16284.

105.Kuksa V, Bartl F, Maeda T, et al. Biochemical and physiological properties of rhodopsin regenerated with 11-cis-6-ring- and 7-ring-retinals. J Biol Chem 2002;277:42315–42324.

106.Vogel R, Siebert F, Ludeke S, Hirshfeld A, Sheves M. Agonists and partial agonists of rhodopsin: retinals with ring modifications. Biochemistry 2005;44:11684–11699.

107.Morrison SA, Russell RM, Carney EA, Oaks EV. Zinc deficiency: a cause of abnormal dark adaptation in cirrhotics. Am J Clin Nutr 1978;31:276–281.

108.Dorea JG, Olson JA. The rate of rhodopsin regeneration in the bleached eyes of zinc-defi- cient rats in the dark. J Nutr 1986;116:121–127.

109.Leure-duPree AE, McClain CJ. The effect of severe zinc deficiency on the morphology of the rat retinal pigment epithelium. Invest Ophthalmol Vis Sci 1982;23:425–434.

110.Kim HJ, Kim YY, Kim SY. Effect of zinc on the visual sensitivity of the bullfrog’s eye. Korean J Ophthalmol 2000;14:53–59.

111.Tam SW, Wilber KE, Wagner FW. Light sensitive zinc content of protein fractions from bovine rod outer segments. Biochem Biophys Res Commun 1976;72:302–309.

112.Shuster TA, Nagy AK, Conly DC, Farber DB. Direct zinc binding to purified rhodopsin and disc membranes. Biochem J 1992;282(Pt 1):123–128.

113.Stojanovic A, Stitham J, Hwa J. Critical role of transmembrane segment zinc binding in the structure and function of rhodopsin. J Biol Chem 2004;279:35932–35941.

114.del Valle LJ, Ramon E, Canavate X, Dias P, Garriga P. Zinc-induced decrease of the thermal stability and regeneration of rhodopsin. J Biol Chem 2003;278:4719–4724.

115.Patel AB, Crocker E, Reeves PJ, et al. Changes in interhelical hydrogen bonding upon rhodopsin activation. J Mol Biol 2005;347:803–812.

116.Swaminath G, T.W. Lee, and B. Kobilka. Identification of an allosteric binding site for Zn2+ on the beta2 adrenergic receptor. J Biol Chem 2003;278:352–356.

117.Swaminath G, et al. Allosteric modulation of beta2-adrenergic receptor by Zn(2+). Mol Pharmacol 2002;61:65–72.

118.Schetz JA, Sibley DR. Zinc allosterically modulates antagonist binding to cloned D1 and D2 dopamine receptors. J Neurochem 1997;68:1990–1997.

119.Holst B, Schwartz TW. Molecular mechanism of agonism and inverse agonism in the melanocortin receptors: Zn(2+) as a structural and functional probe. Ann N Y Acad Sci 2003;994:1–11.

120.Holst B, Elling CE, Schwartz TW. Metal ion-mediated agonism and agonist enhancement in melanocortin MC1 and MC4 receptors. J Biol Chem 2002;277:47662–47670.

121.Wang J, Luthey-Schulten ZA, Suslick KS. Is the olfactory receptor a metalloprotein? Proc Natl Acad Sci U S A 2003;100:3035–3039.

122.Bastek J, Bogden J, Cinotti A, et al. Trace metals in a family with sex-linked retinitis pigmentosa. Adv Exp Med Biol 1977;77:43–50.

123.Mao W, Zeng L, Ma Q, et al. [Primary study of iontophoresis of zinc ion in treatment of retinitis pigmentosa]. Yan Ke Xue Bao 1990;6:88–90.

124.Karcioglu ZA, Stout R, Hahn HJ. Serum zinc levels in retinitis pigmentosa. Curr Eye Res 1984;3:1043–1048.