visual transduction and non-visual light perception || rhodopsin structure, function, and...

8Rhodopsin Structure, Function, and Involvement

in Retinitis Pigmentosa

Scott Gleim and John Hwa

CONTENTS

IntroductionHistorical PerspectiveRhodopsin as the Prototypical G Protein-Coupled ReceptorRhodopsin, Localization, and SignalingDark State and ActivationStructural AnalysisRetinitis PigmentosaImplications of Receptor MisfoldingNongenetic Contributions to RPConclusionReferences

INTRODUCTION

Rhodopsin is the dim-light sensitive photoreceptor, densely packed in the rod cells of the retina. Organisms from bacteria to humans have evolved highly specialized systems for the detection of light, driven by survival-based interests, ranging from energy capture to visual sensing. Phylogenic analysis suggests that photopic vision arose first as cone receptors, which diverged into four groups, with one of these groups further diverging to enable scotopic vision via rhodopsin [1]. Such an evolutionary scheme would suggest that highly sensitive dim-light photoreception developed through optimized specializa-tion, that is, mutations surrounding the chromophore to refine photoactivation in terms of wavelength selectivity and, more importantly, sensitivity. The remarkable sensitivity of rhodopsin, activated by single photons, enables scotopic and peripheral vision. The 200-fs photoisomerization of rhodopsin remains among the fastest and most efficient biological photochemical reactions known. This capture of light energy and the corre-sponding visual response has mesmerized philosophers and scientists alike.

From: Ophthalmology Research: Visual Transduction and Non-Visual Light PerceptionEdited by: J. Tombran-Tink and C. J. Barnstable © Humana Press, Totowa, NJ

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172 Gleim and Hwa

Rhodopsin and Retinitis Pigmentosa 173

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174 Gleim and Hwa

HISTORICAL PERSPECTIVE

Systematic investigation into photoreception can be traced back to the years following the scientific revolution (Fig. 1A) when geographer, microscope enthusiast, and pioneer of micro-biology Anton van Leeuwenhoek first observed retinal rod and cone cells in 1722, providing the first suggestion that light reception may occur somewhere other than at the lens. Thomas Young, famed for the double-slit experiment leading to the wave theory of light, subsequently proposed that color perception depends on three different color-sensitive nerve fibers, later defined by Hermann von Helmholtz to be blue, green, and red, a theory so advanced it was not proven until a century later. Heinrich Mueller suggested that retina rod and cone cells were involved in photoreception, further stimulating investigations into the retina. Franciscus Donders, around 1857, coined the term retinitis pigmentosa in a letter to Helmholz describing spicules of pigmentation he found throughout a patient’s degenerated retina.

The discovery of rhodopsin is owed to the combined efforts of Franz Boll and Willy Kühne through an interesting series of exchanges reviewed elsewhere [2]. Bloch linked night blindness to malnutrition in 1917, from which Blegvad subsequently identified the deficient agent to be vitamin A. Involvement of vitamin A deficiency in night blindness provided sup-plemental evidence in the seminal identification of the active combination of vitamin A and opsin in 1935 by Wald (Fig. 1B), whose accomplishments have enabled incalculable benefits in vision research [3]. Over the past three decades, astounding progress has been made since the discovery of the opsin gene by Nathans [4]; these include the discovery of naturally occurring mutations leading to retinitis pigmentosa [5], deciphering the signaling pathway through transducin [6–12], determination of critical structural features such as the Schiff’s base counterion [13] and disulfide bond [14, 15], electron cryomicroscopy structure [16, 17], conformational movements required for activation [18, 19], and the crystal structure of rhodopsin [20] (Fig. 1C). Further details of activation are being intensely investigated at the molecular and biophysical levels [8, 21, 22]. Insights into the nature of misfolded rhodopsin [23–29] and dimeric packing of rhodopsin [30–34] have also been major achievements. This exponential rate of discovery will likely unfold many further details on the intriguing structure and function of the opsin protein and disease associations.

RHODOPSIN AS THE PROTOTYPICAL G PROTEIN-COUPLED RECEPTOR

Rhodopsin receives considerable research interest, particularly in structural and functional studies, owing in part to its reputation as the prototypical member of the seven-transmembrane-spanning, guanine nucleotide-binding protein (G protein)-coupled receptor superfamily, which accounts for approximately 5% of genes in the human genome. The G protein-coupled receptors (GPCRs) are arguably among the most medi-cally important protein families as over 30% of available pharmaceuticals target proteins within this family [35]. Furthermore, rhodopsin remains the only crystallographic struc-ture available to represent the GPCR superfamily. GPCRs are generally responsible for transmitting extracellular information into the cellular environment with ligand stimuli covering the range of biochemical diversity: large macromolecules, peptides, amino acids, nucleic acids, lipids, ions, and even, as with rhodopsin, a single photon of light.


Much of what is known about rhodopsin, and thus GPCRs in general, is due in large part to studies performed on bovine rhodopsin. The bovine eye provides an ample source from which substantial amounts of this protein can be purified or studied directly. Sequencing of the bovine rhodopsin gene [4] gave an arrangement of five exons, subsequently identified (Fig. 1C) also to represent human rhodopsin gene configuration [36]. The 6.4-kb gene consists of a 96-bp 5' untranslated region; a 1,044-bp coding region; and a sur-prisingly long, approximately 1,400-bp 3' untranslated region and are divided into five exons by four introns that interrupt the coding region [4]. The human gene (Gene ID 6010) is located on chromosome 3 (3q21–q24). The resulting proteins are 93.4% homol-ogous with completely conserved cytoplasmic loops.

RHODOPSIN, LOCALIZATION, AND SIGNALING

Expression of rhodopsin is required for normal cell morphology, as the rod outer seg-ment (ROS) does not form in rhodopsin knockout mice (−/−) [37]. Interestingly, ROS formation takes on typical morphology in rhodopsin heterozygotes (+/−) [32], but with about 50–60% of typical ROS volume, decreased rhodopsin concentration, decreased 11-cis retinal concentration, and impaired light sensitivity [32, 37]. Rhodopsin is densely packed (most probably as dimers; [30]) into stacked disks within the ROS, constituting more than 90% of membrane protein in the lipid bilayer. Opsin is synthesized, folded, and transported through a nonmotile ciliary connection [38] between the cell body and the outer segment, where it functions as a G protein-coupled photon receptor. Disks are shed regularly, with the outermost ROS segments endocytosed by retinal pigment epithelia (RPE) and resulting vesicles trafficked to the RPE proteasomal compartment for opsin degradation.

Isomerization of the retinal moiety, by light excitation, extends the twisted spring-like carbon chain, forcing away nearby residues (Fig. 2). This conformational change exposes the hydrophobic binding site for a heterotrimeric G protein, transducin (G

t), to

associate and become catalytically active, exchanging guanosine diphosphate (GDP) for guanosine triphosphate (GTP). In the case of rhodopsin, the GDP/GTP exchange disso-ciates G

t from opsin to bind phosphodiesterase, removing the inhibitory γ-subunits. This

generates active cyclic guanosine monophosphate (cGMP)-phosphodiesterase, which in turn hydrolyses cGMP at a rate of 103 per second, rapidly closing cGMP-gated Na+ channels and hyperpolarizing the rod cell. Hyperpolarization stops neurotransmitter release, predominantly glutamate, to neighboring ganglia.

A single GPCR activates multiple G proteins, which in turn activate multiple down-stream signaling factors, resulting in a highly amplified signal of at least 10,000 hydrolyzed cGMP molecules per photon under dim-light conditions [39]. The signal is rapidly quenched through rhodopsin kinase (RK) phosphorylation of multiple serine and threo-nine residues along the C-terminal tail, allowing arrestin to bind and preventing further G

t

interactions. These multiple phosphorylation sites appear to be critical to the remarkable reproducibility of the rhodopsin signal [40]. Arrestin binding promotes the hydrolysis and release of all-trans retinal, allowing for association of a new 11-cis retinal molecule, dependent on release of arrestin [41], thereby promoting dephosphorylation by protein phosphatase A (PPA) [42], regenerating a light-sensitive receptor.

176 Gleim and Hwa


DARK STATE AND ACTIVATION

Generally, opsins are large integral membrane proteins of approximately 360 amino acid residues, half comprising the GPCR characteristic seven-membrane-traversing regions (Fig. 3A). Despite countless similarities and conserved regions, rhodopsin is unique among this superfamily in a number of other ways. For instance, rhodopsin uniquely functions as a holoprotein, a working assembly of the precursor opsin apoprotein and a prosthetic inverse agonist, 11-cis retinal. This vitamin A derivative attaches covalently to lysine 296 through Schiff base formation, stabilizing opsin in a completely inactive conformation. This inverse agonist serves as the chromophore, finely tuning the absorption wavelength of the receptor in conjunction with surrounding residues. Such precision is required to prevent visual noise and allow for optimal visual sensitivity. In the absence of the 11-cis retinal, a significant degree of transducin coupling and thus signaling can occur.

The spectrophotometric absorption profile of rhodopsin is defined by binding pocket interactions with the chromophore. This fortuitously allows tracking of struc-tural changes by measuring the shift in the local absorption maxima (λ

max) of the spectra.

Dark-state rhodopsin maintains a characteristic absorption peak (λmax

) at 498 nm, in which the bound ligand is maintained in a state reminiscent of a twisted spring. A photon of light energy strikes. On excitation, the positive charge, once localized to the Schiff base, redistributes along the π-electron system [43]. Charge transfer to an alternative counterion accompanies isomerization of the retinal molecule into all-trans retinal, sterically pushing apart transmembrane segments three (TM3) and six (TM6) [18].

Surrounding features of the holoprotein respond to the excitation in rapid succession, through a series of excited states, before final energy decay into a form relaxed enough to release all-trans retinal and activate the waiting effector, G

t. Bathorhodopsin (529 nm)

develops equilibrium with a blue-shift intermediate (BSI) state (477 nm), which decays into a counterion transition state, lumirhodopsin (492 nm). Lumirhodopsin represents a transient state in which proton transfer from the E113 dark-state counterion [44] in TM3 across S186 and through an integral water molecule to protonate alternative coun-terion E181 [45] in the second intradiskal loop results in formation of meta I rhodopsin (478 nm). By the MI intermediate state, the ligand spring has untwisted, isomerized into all-trans retinylidene, still covalently attached to the opsin, and still incapable of activating transducin. Receptor activation, or conversion of meta I into meta II (MII or R*) (380 nm), requires deprotonation of the Schiff base, releasing the isomerized ligand.

Fig. 2. A Cartoon of the Rhodopsin Activation Cycle. A simplified rhodopsin activation scheme highlighting important events in the rhodopsin lifecycle. This schematic shows 11-cis-retinal in (A) dark-state rhodopsin, with the protonated Schiff base stabilized by a counter-ion at Glu113. Photon energy catalyzes isomerization of the ligand to (B) all-trans-retinal, followed by counter-ion transfer across Ser186, during (C) lumi-rhodopsin, to Glu181, with a distancing of transmembrane helices 3 and 6 forming (D) active meta-II rhodopsin. Transducin (G

t) acti-

vation continues until (E) deactivating phosphorylation of the carboxy-tail, which promotes arrestin association and dissociation of all-trans-retinal. (F) Free opsin combines with new or re-formed 11-cis-retinal to reinitiate the cycling.

178 Gleim and Hwa

Fig. 3. Structural representations of rhodopsin. (A) The primary sequence of rhodopsin, written as a schematic of the secondary structure, shows the organization of transmembrane helices. Auto-somal dominant retinitis pigmentosa (RP) mutations are shown demonstrating the diverse range of affected residues and their locations. (B) A representation of rhodopsin, based on the 1LH9 2.6-Å crystal structure, highlights important structural features relevant to RP. Specifically, the amino (N-) and car-boxy (C-) termini, the disulfide bridge, the retinal-binding site, and the highly conserved glutamate, arginine, tyrosine motif (ERY) activation-related sequence are associated with certain RP mutants.


Accompanied rigid-body movements of TM3 and TM6 [46] expose a hydrophobic cleft between TM5 and TM6 [47]. Exposure of this hydrophobic site draws a nearby pheny-lalanine (F64) of the G

tγ, an orientation otherwise unfavored in an aqueous environment,

resulting in amphipathic helix formation of the Gtγ-C-terminus, stabilized by activated

rhodopsin, allowing allosteric regulation of nucleotide exchange [48].

STRUCTURAL ANALYSIS

The current state of structural and functional knowledge regarding rhodopsin has been recently reviewed [49, 50]. Rhodopsin is ellipsoid in shape with dimensions of approxi-mately 75 × 48 × 35 Å. The 348 amino acids of bovine rhodopsin are posttranslationally modified with N-terminus acetylation, N-terminus dual glycosylations (at N2 and N15), an intradiskal disulfide bond (C110–C187), dual palmitoylations at the C-terminus (C322 and C323), and multiple C-terminus light-activated phosphorylations.

Electron Cryomicroscopy and Crystal Structure

Most structural information on rhodopsin, and thereby on GPCRs, is based on an inactive, inverse-agonist-bound, dark state. This is because the most definitive structural data available are from x-ray crystallography. Electron cryomicroscopy leveraged two-dimensional crystal formations to provide the earliest, albeit low-resolution, pictures of rhodopsin structure [16]. Originally solved to 2.8 Å [20], the crystal structure of rhodopsin demonstrated a number of important principles in rhodopsin function, the structure of GPCRs, and general aspects of large integral membrane proteins. Impor-tantly, as mentioned, the helical arrangement was shown to be significantly different from, and more organized than, bacterial rhodopsin (another intensely studied seven-transmembrane-spanning protein that serves as a proton pump from halophilic archae-bacteria). Identification of proline-induced bending of transmembrane helices (e.g., highly conserved P267) showed significant distortion from an ideal helix, facilitating interhelical interactions and allowing for chromophore accommodation. Of particular interest was identification of a water-mediated interhelical interaction network centered around Asp83 on TM2, connecting this helix to TM3 and TM7 through interactions with Gly120 and Asn302, respectively. Also of mechanistic importance was the structural suggestion that β-ionone movement toward TM3 may result in helical displacement.

Additional crystal analyses improved structural detail by adding missing residues (protein databank (http://rscb.org) structure identification numbers (pdf:1HZX), increasing resolution to 2.6 Å (1L9H) [51] (Fig. 3B), further improving resolution to 2.2 Å (1U19) [52], and refining earlier structures in different crystal space groups (1GZM). Increased resolving power provided confirmation of the structural importance of water molecules and their likely participation in spectral tuning and proton transfer [51]. Further details provided definition of the cytoplasmic region and chromophore, demonstrating a 6 s-cis conformation of the ionone ring and an unusual twisted and extended π-system with a delocalized charge–carboxylate interaction [52]. Also clarified was the hydrogen-bond-ing network connecting E113 to E181 through a required water molecule, a network later confirmed to transmit the counterion shift important in rhodopsin activation [45].

http://rscb.org

180 Gleim and Hwa

Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) resolution of rhodopsin domain peptides have also been used to study the structure of rhodopsin [53]. Nuclear magnetic resonance has also extended crystallographic interpretations toward the elucidation of structural changes involved in opsin activation [54, 55]. Solid-state NMR, in particular, providing advantages for dealing with integral membrane proteins, is uniquely adept for follow-ing particular interactions, with studies utilizing isotopically labeled retinal providing direct chemical observation of modifications to ligand–protein interactions following light activation [55, 56]. The complete 1H and 13C assignments of the chromophore in the bound state showed interactions between 11-cis retinylidene’s H16/H17 and Phe 208, Phe 212, and H18 with Trp265 [57]. NMR provides considerable advantages in the context of activation as light-activated rhodopsin remains elusive to crystallization, and pho-toactivated intermediates, likely due to their transient nature, prove similarly difficult to crystallize [58]. The side chain of Glu122 and backbone of His211 were shown to be disrupted in meta II [55].

Cysteine Mutagenesis and Electron Paramagnetic Resonance

Site-directed cysteine mutagenesis and sulfhydryl modification chemistry provide remarkable resources for structural studies facilitating spin labeling, disulfide construc-tion [18, 59], and metal-binding site engineering [19]. Paramagnetic spin labeling adds unique topological information, measuring solvent accessibility of the modified resi-due, overall mobility of particular residues, and even demonstrating residue interactions. A series of spin-labeling studies revealed structural details of cytoplasmic loop 1 connect-ing helices 1 and 2 [60], loop 2 between helices 3 and 4 [12], loop 3 between helices 5 and 6 [61, 62], loop 4 leading from helix 7 to the palmitoylation site [63], and the C-terminal tail [64]. Together, these results map a range of light-initiated structural changes. Evaluating light-activated changes in mobility of cytoplasmic loop 3, the transducin-interacting domain connecting transmembrane helices 5 and 6, demonstrated a dramatic loss of mobility for residue V227, with a smaller decrease in mobility for K231. The loss of mobility corresponds to formation of tertiary contacts, whereas an increased mobility, as observed with V250, T251, M253, and Q244, indicates that tertiary contacts are lost in conversion to MII. This has considerable mechanistic importance as Q244 has been identified as a required residue for G

t activation [65]. As such, it can be seen that residues

in cytoplasmic loop 3 are exposed to allow transducin interaction with nearby residues, forming new contacts to maintain structural integrity of the receptor.

Other Approaches

Breaking the protein into subsections for structural analysis of the components attempts to alleviate some of these concerns; however, less-direct biophysical measure-ments of intact protein have allowed structural inferences to fill gaps left by direct meas-urements. Fourier-transform infrared (FTIR) spectroscopy, for example, resolved proton movements involved in activation-induced counterion shift [44], and ultraviolet-visible (UV/Vis) spectrophotometry is routinely used to evaluate rhodopsin purity, structural stability, regeneration rate, and activation state [66]. Such techniques prove quite powerful


at leveraging structural information provided by crystallographic data, particularly when combined with complementary tools such as site-directed mutagenesis and molecular modeling. In fact, molecular modeling has been pivotal in the study of rhodopsin, as it is a critical refining step in processing crystallographic and NMR data, and facilitates mutagenic approaches and biophysical data interpretation. Not surprisingly, as modeling tech-niques continue to mature, they become utilized as an experimental approach in their own right, with energy-induced decay of the protein structure revealing a core set of stabiliz-ing interactions providing a folding scaffold for the overall rhodopsin structure [67].

Genetic manipulation techniques have proven useful in structural investigations of rhodopsin. From deletion of segments and chimeric recombination of protein to muta-genesis of individual residues and chemical modification of localized functional groups, each distinct application provides creative insight into structural features of this remark-able protein. Supporting a common GPCR activation principal is construction of a chimeric rhodopsin spliced with the cytoplasmic regions of the β-adrenergic receptor, resulting in a light-activated GPCR that elicits a β-adrenergic Gα

s stimulation of cyclic

adenosine monophosphate (cAMP) [68].Investigations into rhodopsin structure and function parallel many major unanswered

questions facing general protein biochemistry. Structural motions involved in translating binding of a ligand or allosteric modulator across the membrane bilayer to activate intrac-ellular signals are of general interest, particularly if findings extrapolate to a wider range of GPCRs. More structurally accessible than most GPCRs, rhodopsin continues to provide a uniquely suited prototype for studying general GPCR features. One interesting feature to develop over recent years is the concept of GPCR dimerization, carrying uncertain poten-tial impact [69, 70]. Rhodopsin has demonstrated potential to form dimers, as well as higher-order oligomers, in disk membranes [33], expression systems [71], liposomes [72] and in solubilizing detergents [34]. However, demonstration of native receptor dimeriza-tion, along with a functional requirement for dimerization, remains elusive. Perhaps the most compelling evidence in favor of rhodopsin dimer formation was demonstrated using atomic force microscopy [30]. A large battery of additional techniques, including electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, proteolysis, and cross-linking support the idea that rhodopsin is capable of dimer formation in both isolated disk membranes and when solubilized in detergent [33]. Site-directed mutagenesis, combined with fluorescence resonance energy transfer (FRET) and cysteine cross-linking, suggests a hydrophobic interaction between W175 in the sec-ond intradiskal loop, and Y206 in TM5 participates in rhodopsin dimer formation [71]. Molecular modeling has provided additional details for the putative interface [31].

Implications of native dimer formation for GPCRs range from transport considera-tions [73] to activation responses [74]. An interesting approach using solubilized rho-dopsin in detergent micelles of increasing size accommodating mixtures of differing oligomeric sizes suggested that larger dimeric organizations might be structurally preferred, reflected by increased receptor stability [34]. Although these organizations may not be required for transducin activation, increasing levels of oligomerization corresponded to dramatically increased rates of G

t activation, without modifying MII decay, consistent

with putatively improved Gt binding by dimeric rhodopsin [75].

182 Gleim and Hwa

RETINITIS PIGMENTOSA

Retinitis pigmentosa (RP) is a collection of inherited neurodegenerative disorders in which rod cell apoptosis spreads throughout the retina, resulting in progressive vision loss and the characteristic pigmented retinal appearance. RP genetics demonstrate extreme heterogeneity in severity, progression rate, and mode of inheritance, covering autosomal dominant, autosomal recessive, X-linked, and sporadic mutations. Over 30 different genes have been identified to produce RP, with additional loci also implicated. Of at least 15 autosomal dominant RP (adRP) inherited disease genes, some have obvious correlations to RP biochemistry, while others have uncertain or indeterminate relation-ship to the disease. By definition, all RP mutants result in photoreceptor degeneration. Degenerative effects trigger multiple cell-death pathways, including caspase-dependent apoptosis, complement activation, and autophagy [76], with phase profiles reflecting the initial cause of cell death, be it calcium overload, structural defects, or oxidative damage. Uniform retinal degeneration was demonstrated to be independent of cellular genotype in chimeric retinas [77], indicating that transcellular factors or interactions are responsible for the final global retinal degeneration.

Rhodopsin mutations account for an estimated 40% of autosomal dominant gain-of-function mutations, with over 100 distinct mutations within the receptor (Fig. 3A). Initial association of RP with single-point mutations in rhodopsin stemmed from identification of the predominant rhodopsin RP mutant, P23H [5]. Clinical visual parameters of P23H RP suggest increased ROS shedding and impaired ROS renewal [78], findings consistent with the notion of misfolded opsin impairing the disk integrity. With an astounding number of different single-point mutations in rhodopsin leading to autosomal dominant RP [79], it becomes useful to categorize the resulting effects when evaluating disease mechanism and therapeutic approaches. Although the final pathology may appear to be similar, rhodopsin mutations are a heterogeneous group at the molecular level, with differing structural perturbations. Potential for pharmacological and molecular therapy may ultimately depend on the location of the mutation and amino acid change.

Classification of RP mutants in terms of structural loci and mechanistic impact can be reduced to three critical structural regions [50]. Most severely, modifications in the cyto-plasmic tail interfere with rhodopsin trafficking to the ROS. Mutants interfering with the normal disulfide bond formation, between C187 on the second intradiskal loop and C110 at the end of the first intradiskal loop, lead to receptor misfolding. Finally, muta-tions among the transmembrane- and chromaphore-binding regions can effect protein folding and receptor activation. Associating cytoplasmic, intradiskal, and transmem-brane mutations with trafficking, misfolding, and activation defects provides a useful means to discuss major routes of rhodopsin-RP development, but also provides drastic overgeneralization of a highly heterogeneous problem.

An extended classification scheme is based on inactivating mutations of GPCRs in general [79]: class I, defective biosynthesis; class II, defective trafficking; class III, defective ligand binding; class IV, defective activation; and class V, unknown defects. Defective biosynthesis predominantly occurs through premature termination, often through frameshift, but may also entail accelerated degradation. Defective surface traf-ficking, in this scheme, covers the majority of GPCR mutations, where normally produced receptors demonstrate intracellular retention. Receptors with ligand-binding defects,


despite proper production and surface expression, are incompetent for ligand binding. Activation-defective receptors, it follows, demonstrate proper production, localization, and ligand binding, failing only to carry out the final activation step, resulting in reduced maximal response or sensitivity (manifest as an increased half maximal response, EC

50).

The final category of unknown defects refers to disease-associated mutants with no apparent mechanistic deficiency, functioning normally in model systems and suggesting a case for which, despite association, the mutant may not be the cause of disease.

While categorizing mutation effects may facilitate discussion of general mechanisms, the differences in location and physicochemical properties of replacement residues likely result in a broad range of subtle changes among each major class. Mutation of the arginine in the conserved [E/D]RY sequence associated with G protein signaling results in impaired activation in the melanocortin MC1R receptor (R142H) through G protein decoupling. The corresponding mutation in the vasopressin V2R receptor, R147H, also causes constitutive internalization and desensitization [80], resulting in nephrogenic diabetes insipidus. Similar RP mutants have been found, R135L and R135W, that are unable to activate transducin despite normal folding and ligand binding [81], a phenotype that could fall into the class IV activation-impaired mutant group. However, further research into these mutants has identified the defect to be caused by constitutive phosphorylation and arrestin binding, leading to constitutive internalization [82]. One could also argue that this may fall into the category of defective trafficking. However, the cellular out-come of this defect remains distinctly different from that of traditional mistrafficking.

Transmembrane RP Rhodopsin Mutants

The transmembrane region of rhodopsin, consisting of half of the protein, is of obvi-ous structural importance. This region also forms the retinal-binding pocket and coordi-nates G protein activation through conformational movements of the transmembrane (TM) domains. As such, rhodopsin mutations within the transmembrane region can be further subdivided by those in proximity of the retinal β-ionone ring or carbon chain and those found in remaining regions of the transmembrane domains (Fig. 4). Evaluation of RP mutants across the transmembrane domains (G51A,V in TM1, G89D in TM2, L125R,A,F in TM3, A164V in TM4, H211P in TM5, P267L,R in TM6, and T297R in TM7) demonstrated that mutations in each TM segment can lead to abnormal bleaching and MII photointermedi-ates [25], indicators of protein misfolding. These mutations appear to result in nonnative packing of the transmembrane helices, which relay misfolding to the intradiskal domain, where they may cause abnormal disulfide formation.

The L125 residue is in TM3, within the ligand-binding pocket, close to the retinal β-ionone ring. A comprehensive list of mutations at this site (G, N, I, H, P, T, D, E, Y, and W) decreased 11-cis retinal binding, causing a red-shift of λ

max, increased solvent

exposure, and decreased thermostability [24, 83]. These findings support the importance of ligand binding in maintaining the structural integrity of rhodopsin, highlighting an interaction between L125 and the β-ionone ring as critical in maintaining the struc-ture of the chromaphore-binding pocket. Further use of mutagenesis in evaluating the structural role of L125 demonstrated the role of this residue in maintaining additional interhelical interactions [29]. Rescue of the L125R RP mutant through compensatory mutations of W126E,D or E122L eliminated steric hindrance caused by L125R, restoring

184 Gleim and Hwa

Fig. 4. (continued)


the TM3–TM5 interaction formed by the salt bridge between E122 and H211, by recon-structing the hydrogen bond between W126 and E122. Similarly, RP mutant A164 in TM4 interfered with residues L119 and I123 in TM3, disrupting the same salt bridge.

Congenital stationary night blindness (CSNB; nyctalopia) is a condition characterized by inability to see in conditions of low illumination. The most common cause of nyctalopia

Fig. 4. Regional clusters of autosomal dominant retinitis pigmentosa (adRP) rhodopsin mutations. Organization of adRP mutations according to structural features demonstrates clusters in relation to (A) the retinal-binding site, (B) the transmembrane scaffold, (C) the cytoplasmic region, and (D) the intradiskal region.

186 Gleim and Hwa

is rod photoreceptor degeneration from RP, often caused by rhodopsin mutations. Non-RP mutations generating constitutive receptor activation cause CSNB. Rhodopsin is constrained in an inactive conformation through the salt-bridge interaction between lysine-296 and glutamate-113 [84]; mutation of either component can generate con-stitutive activity, causing CSNB. Interestingly, one of the earliest symptoms of RP is night blindness [85], demonstrating a fascinating relationship between rhodopsin folding and activity. RP mutation K296E, however, has been shown to cause photo-receptor degeneration through a process independent of constitutive activity [86]. Proximity of glycine-90 to this counterion permits G90D interference of the K296-E113 salt bridge by substituting for E113 in salt-bridge formation, leading to constitutive activation and CSNB [87]. The T94I autosomal dominant CSNB mutation is similarly near the G90 region and also results in constitutive transducin activation, most likely through hydrophobic interference or steric hindrance [88].

Cytoplasmic RP Rhodopsin Mutants

The cytoplasmic region of rhodopsin extends from the surface of disks into the cellu-lar milieu, where transducin (G

t) binding, and hence signaling, occurs. This intracellular

region consists of the first cytoplasmic loop connecting helices 1 and 2, the second cyto-plasmic loop connecting helices 3 and 4, the third cytoplasmic loop, and the C-terminal tail. The C-terminus is anchored to the membrane surface by palmitoylation of residues Cys322 and Cys323, creating what is putatively referred to as the fourth cytoplasmic loop [89, 90]. The proximal portion of this loop forms a part of the binding site for the C-terminal section of the transducin α-subunit [91]. RP mutations within the cytoplas-mic region of rhodopsin are found throughout the region, with the exception of the third cytoplasmic loop (Fig. 4C). The C-terminus directly interacts with a cargo-binding subunit of dynein, providing transport of rhodopsin to the outer segment [92], an interaction abolished by severe RP mutants P347L, P347S, V345M, and Q344ter. C-terminal tail also contains multiple phosphorylation sites, phosphorylated by RK following transducin activation. Phosphorylation of the C-terminal tail regulates interaction of rhodopsin with arrestin, deactivating the receptor. These multiple phosphorylation sites result in uniquely consistent control of the amplitude and duration of the activated rhodopsin signal [40].

Intradiskal RP Rhodopsin Mutants

Structural investigations of the RP mutants and their consequences on the intradiskal region were best highlighted in a series of investigations by Khorana et al. [23, 25, 26, 93–95]. Investigations by this group established a number of techniques critical for furthering rhodopsin research, while demonstrating important general principles of rhodopsin structure and the misfolding properties of numerous RP mutants. Together, their findings suggest that packing of the transmembrane helices, binding of the chro-maphore, and intradiskal structural integrity are physically coupled properties critical for proper folding of rhodopsin. Intradiskal RP mutations are numerous and diverse in their impact on misfolding (Fig. 4D).

Deletion studies of the intradiskal region provided suggestions for the structural importance of this region [96]. N-terminal deletions resulted in partial chromophore


regeneration, whereas deletions in the first and second intradiskal loops caused regeneration-incompetent 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 140 RP 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 vari-ability 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 pro-gression 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. Com-plementary 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.

188 Gleim and Hwa

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 sig-nificant 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 recep-tor 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 E134-mediated 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 supple-mentation. 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 muta-tions 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 classifi-cations, 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 folding-impaired 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 degen-eration 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 coordina -tion 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 chemi-cal shifts are consistent with the presence of Zn2+ within that region [115]. Direct inter-actions between divalent cations and GPCRs are not unprecedented as a diverse list of receptors, including β

2-adrenergic [116, 117], dopaminergic [118], melanocortin MC

1

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. Improve-ments 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.

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