Photoreceptor Membrane Proteins,Phototransduction, and RetinalDegenerative DiseasesThe Friedenwald Lecture
Robert S. Molday
Rod and cone photoreceptor cells are specialized neu-rons of the vertebrate retina that function in the pri-mary events of vision. Rod cells are highly sensitive to
light and operate under dim lighting conditions. Cones are lesssensitive and function in bright light and in color vision. Bothphotoreceptors are elongated cells consisting of several mor-phologically and functionally distinct regions (Fig. 1). Thephotoreceptor outer segment located adjacent to the retinalpigment epithelial (RPE) cell layer is a specialized compart-ment uniquely designed to carry out phototransduction, i.e.,the capture of light and its conversion to an electrical signal.This membrane-rich organelle undergoes a continual renewalprocess in which newly synthesized membrane is added at thebase of the outer segment, whereas packets of outer segmentmembrane are phagocytized at the distal end by the RPEcells. '"3 A thin, nonmotile cilium links the outer segment to theinner segment, a cellular compartment that contains the mito-chondria, endoplasmic reticulum, golgi apparatus, and othersubcellular organelles. Adjoining the inner segment is the cellbody containing the nucleus. This region further extends intothe synaptic region where the electrical signal generated in thephotoreceptor cell is transmitted to other neurons of the ret-ina.
Photoreceptor outer segments have been extensively stud-ied at a cellular level/'"8 In the rod cell, the outer segmentappears as a cylindrical structure up to 60 /xm in length and 1.5to 2 jam in diameter for mammals and 6 to 8 jam in diameter foramphibians. The cone outer segment is generally shorter andoften is tapered or conical.
The outer segment consists of hundreds of flattened diskmembranes assembled in an ordered axial array (Fig. 1). In rodcells, the stack of closed disks is surrounded by a separateplasma membrane over the entire length of the outer segmentexcept at its base. Here, newly formed disk membranes evagi-nate from the ciliary membrane to form a folded membranesystem.8 Each rod outer segment (ROS) disk consists of twoclosely spaced lamellar membranes circumscribed by a hairpin
From the Department of Biochemistry and Molecular Biology,University of British Columbia, Vancouver, BC, Canada.
Supported by Grant EY02422 from the National Eye Institute,Bethesda, Maryland; The RP Research Foundation-Fighting Blindness,Canada; the Medical Research Council of Canada; and an Alcon Re-search Institute Award, Fort Worth, Texas.
Reprint requests: Robert S. Molday, Department of Biochemistryand Molecular Biology, 2146 Health Sciences Mall, Vancouver, BC,Canada V6T 1Z3. E-mail: [email protected].
loop called the rim region. The continuous disk membraneseparates an intradiskal space or lumen from the cytoplasm.The perimeter of the rod disk is interrupted by one or moreincisures that penetrate toward the center of the disk. Coneouter segments also consist of a stack of disk membranes, butunlike ROS disks, cone disks remain open and do not containincisures.9'10 Fibrous elements extend from the rim regions ofdisks to adjacent disks and to the plasma membrane.""13 Thiscytoskeletal-like system serves to maintain the precise dis-tances between adjacent disk and plasma membranes and sta-bilize the outer segment structure.
ROS
Isolation and Characterization
Although ultrastructural features of ROS had been described inconsiderable detail by the early 1970s, relatively few studiesdealt directly with the molecular composition of this light-sensitive organelle. This was due largely to the lack of a pure,well-characterized ROS preparation and to technical difficultiesinherent in the molecular analysis of biological membranes,and in particular membrane proteins. A significant advance-ment came in 1974 when David Papermaster, working as apostdoctoral fellow in William Dreyer's laboratory at CaliforniaInstitute of Technology in Pasadena, developed a highly purepreparation of bovine ROS.14 This procedure, based in part onearlier protocols,1516 takes advantage of the fragile nature ofthe connecting cilium, the stability of intact ROS in sucrosesolutions, and the low density of ROS relative to other subcel-lular organelles. Briefly, retina tissue immersed in sucrose so-lution is gently agitated to detach the ROS. Differential velocitycentrifugation and sucrose density gradient sedimentation isthen used to isolate ROS, largely free of contaminating subcel-lular organelles. Minor modifications to this procedure havebeen introduced over the years, but in general these prepara-tions exhibit similar protein and lipid compositions. A typicalprofile of ROS proteins fractionated on a sodium dodecyl sul-fate (SDS) polyacrylamide gel is shown in Figure 2 (lane a).Rhodopsin represents the major stained band, accounting formore than 70% of the total ROS protein. Numerous less abun-dant proteins of various molecular weights are also observed.
Isolated ROS in sucrose solutions retain their soluble andweakly associated membrane proteins. Herman Kiihn, also aformer postdoctoral fellow at Cal-Tech and later a researchscientist at the Neurobiology Institute in Jiilich, Germany, de-veloped several procedures to selectively extract soluble and
FIGURE 1. Left: schematic diagram of a rod photoreceptor cell. Right: diagram of a rod outersegment. Adapted with permission from Young RW. Visual cells. Set Am. 1970;223:81-91-
weakly associated membrane proteins for biochemical analy-sis.1718 One such procedure involves hypotonic lysis of dark-adapted ROS followed by centrifugation to separate the super-natant fraction from the membrane pellet.1719 As shown inFigure 2 (lanes b, c), a distinct protein profile is observed foreach fraction. The supernatant contains numerous proteinsranging in molecular mass from approximately 10 kDa to morethan 100 kDa. Many of these proteins have been characterizedby various laboratories and shown to function as key enzymesand regulatory proteins in phototransduction and metabolism(Table 1). The membrane fraction of ROS is dominated byrhodopsin. In addition, numerous less abundant membraneproteins are observed in the range of 30 kDa to 240 kDa (seeTable 2).
Separation of ROS Disk and Plasma Membranes
My laboratory has focused primarily on the molecular analysisof integral membrane proteins of ROS and elucidation of theirrole in phototransduction, outer segment structure and metab-olism, and retinal degenerative diseases. As a first step, it was
important to analyze the protein composition of the ROS diskand plasma membrane. One view at the time held that the twomembranes are identical in composition. This was supportedby electron microscopic studies showing that the disk andplasma membranes arise from the same newly formed mem-branes at the base of the outer segment8 and immunocyto-chemical studies revealing the presence of high levels of rho-dopsin in both membranes.20"22 Furthermore, severallaboratories reported that both the disk and plasma membranecontain cGMP-gated channel activity and Na/Ca exchange ac-tivity.23"25 In contrast, biochemical labeling studies indicatedthat several proteins present in the ROS plasma membrane areabsent in disks, suggesting that the two membranes may differin protein composition.26'27
To resolve this issue, we developed a ricin-gold affinitydensity perturbation method that can effectively separate theplasma membrane, constituting ~5% of the ROS membrane,from the more abundant disk membranes.19'28 In this proce-dure, isolated ROS are first treated with neuraminidase toremove terminal sialic acid residues on surface sialoglycopro-
IOVS, December 1998, Vol. 39, No. 13
kDa
205 -
97 -
68 -
45 -
Rhodopsin -
- PDE(a,p)
.^ Rhodopsin\ ^ Kinase
Arrestin
_ _- Transducin
Ia
i
FIGURE 2. Sodium dodecyl sulfate (SDS) gel electrophoresis ofbovine rod outer segment (ROS) proteins. Dark adapted, puri-fied ROS (lane a) were lysed in hypotonic buffer, and themembrane fraction (lane b) was separated from the solublefraction (lane c) by high-speed centrifugation. Fractions wererun on a 9% SDS polyacrylamide gel and stained with Coomas-sie blue. Bands corresponding to rhodopsin, phosphodiester-ase sub-units (PDEa,|3), rhodopsin kinase, arrestin, and trans-ducin (a- and j3-subunits) are indicated.19
teins.27 Exposed galactose residues are then specifically la-beled with the plant lectin ricin conjugated to gold particles.The small uniform gold particles (~10 nm diameter) are phys-ically dense and therefore, increase the density of the plasmamembrane to which they bind. They are also electron denseand serve as excellent visual markers for electron microscopy(EM). The gold-labeled ROS are lysed in hypotonic buffer toproduce fragments consisting of unlabeled disks radiating fromthe cytoplasmic surface of the gold-labeled plasma membrane.Trypsin is then used to detach the disks from the plasmamembrane, and sucrose gradient centrifugation is used to sep-arate the unlabeled disks from the labeled plasma membrane.The fractionation procedure, as elegantly carried out by LaurieMolday, is documented in the electron micrographs in Figures3A, 3B, 3C, and 3D.
SDS gel electrophoresis and functional assays indicate thatdisk and plasma membranes differ in protein composition1928
(see Fig. 5A), Although both membranes contain rhodopsin asthe major membrane protein, many proteins present in theplasma membrane are absent in disk membranes (Table 2).Furthermore, the plasma membrane exhibits cGMP-gated chan-nel activity29 and Na/Ca-K exchange activity,30 whereas diskmembranes do not. Differences in lipid composition betweenthe disk and plasma membrane have also been reported.31-32
MONOCLONAL ANTIBODIES TO ROS
MEMBRANE PROTEINS
To begin to identify, characterize, and localize photoreceptormembrane proteins, highly sensitive and specific reagents are
The Friedenwald Lecture 2495
required. Monoclonal antibodies are particularly valuable sincethey typically bind to a well-defined site or epitope consistingof four to eight amino acids, can be produced in unlimitedquantities, and have broad applications with highly sensitiveimmunochemical techniques. For example, monoclonal anti-bodies can be used to (1) detect and identify proteins bywestern blot analysis; (2) isolate proteins and peptides byimmunoaffinity chromatography; (3) map the cellular and sub-cellular distribution of proteins by light and EM; (4) screencDNA expression libraries for cloning and sequence analysis;(5) analyze membrane protein topology; (6) resolve protein-protein interactions; (7) identify specific cells for developmen-tal and transplantation studies; and (8) detect protein expres-sion in gene therapy protocols.
Monoclonal Antibodies to Rhodopsin
Our studies on the generation and characterization of mono-clonal antibodies began in the early 1980s. Using ROS as animmunogen, we were able to generate a panel of antibodies torhodopsin and other ROS membrane proteins.33 Our initialstudies focused on the analysis of anti-rhodopsin antibod-ies.33"39 In collaboration with Paul Hargrave at the Universityof Florida and Bob Hodges at the University of Alberta, wewere able to precisely map the epitopes for a number ofanti-rhodopsin antibodies.37"39 Four antibodies have beenwidely used in studies carried out in my laboratory and in otherresearch laboratories throughout the world. These include therho4D2 and rho3A6 antibodies generated by David Hicks, aformer postdoctoral fellow in my laboratory,21 and rholD4 andrho4B4 antibodies produced by Don MacKenzie, a formergraduate student.35"37
The rho4D2 antibody, directed against a highly conservedepitope near the N terminus of rhodopsin, cross-reacts withrhodopsin from a broad range of vertebrate species.21'33'39 Ithas been widely used to study the cellular and subcellulardistribution of rhodopsin in rod cells and as a rod cell-specificmarker in retinal developmental and transplantation stud-i e s 2],33,<fo,<n ^ n e rnolrj>4 antibody reacts with the conservedeight amino acid C terminus of rhodopsin.57'38 This antibodyhas been routinely used to purify rhodopsin from ROS andheterologous cells for structure-function analysis.'""43 Therho4B4 antibody specifically binds to an internal loop connect-ing helices 4 and 5 and has been used to confirm the organi-zation of rhodopsin in disk membranes.33'34'30" Finally, therho3A6 antibody shows more limited cross-reactivity, bindingto bovine and human rhodopsin, but not mouse rhodop-sin.36'38 This property has been used effectively to monitor theexpression and distribution of human rhodopsin in transgenicmice that serve as models for retinitis pigmentosa and otherretinal
Monoclonal Antibodies to Other ROS Proteins
As part of our studies, we have generated a library of mono-clonal antibodies against other ROS disk and plasma membraneproteins including peripherin/rrfs, rom-1, the cGMP-gatedchannel, guanylate cyclase, the Na/Ca-K exchanger, glyceral-dehyde-3-phosphate dehydrogenase, ABCR/RIM protein, andothers (see Table 2). Applications of some of these antibodiesin the identification, characterization, and localization of spe-cific photoreceptor proteins are described below.
2496 Molday IOVS, December 1998, Vol. 39, No. 13
TABLE 1. Partial List of Soluble and Membrane-Associated ROS Proteins
Activator of transducin GTPase activityInteraction with transducin subunitsGlucose metabolism and energy productionGlucose metabolism and energy productionATP regenerationRegulationNucleotide metabolismNucleotide metabolismGlucose metabolism and regeneration of
NADPHUnknownUnknownCalcium-dependent modulator of rhodopsin
kinaseCalcium-dependent modulator of the cGMP-
gated channelCalcium-dependent modulator of guanylate
cyclasePyrophosphate hydrolysisCytoskeletal protein localized at the base of
the outer segmentComponent of microtubulesConnecting ciliumProtein regulation
PHOTOTRANSDUCTION AND THE CYCLIC GMP-GATED CHANNEL OF ROD PHOTORECEPTORS
The cGMP-gated channel plays a central role in phototransduc-tion by controlling the flow of Na+ and Ca2+ ions into theouter segment in response to light induced changes in intra-cellular cGMP concentrations (Fig. 4).46-'18 Briefly, in dark-adapted rod cells, Na+ and Ca2+ ions flow into the ROSthrough cGMP-gated channels maintained in their open stateby a relatively high concentration of cGMP. K+ ions flow out ofthe inner segment through voltage-gated K+ channels, therebycompleting a dark current loop. Na+ and K+ gradients aremaintained by an active Na,K ATPase localized in the plasmamembrane of the inner segment, and the Ca2+ concentrationin the outer segment is kept at ~400 nM by the balanced effluxof Ca2+ through the Na/Ca-K exchanger in the ROS plasmamembrane. Under these conditions, the rod cell is in its depo-larized state (—40 mV), and there is a constant release of theglutamate transmitter from the synaptic region of the photore-ceptor cell.49
Photoexcitation is initiated when a photon converts thell-cis retinal chromophore of rhodopsin to its all-trans isomer.This reaction leads to the formation of Meta II rhodopsin or R*and activation of the visual cascade.30"53 R* catalyzes transdu-cin activation via the exchange of GDP for GTP on its a-subunit(Ta). This in turn leads to the activation of phosphodiesterase(PDE) and the hydrolysis of cGMP to 5'-GMP. The decrease inintracellular cGMP causes the cGMP-gated channels to closeand the rod cell to become hyperpolarized. Under this condi-tion, glutamate release at the synaptic region of the rod cell isinhibited. The closure of cGMP-gated channels also causesCa2+ levels in the outer segment to decrease since the Na/Ca-Kexchanger continues to extrude Ca2+ from the outer segment.
After photoexcitation, the photoreceptor cell returns toits dark state by the shutdown of the visual cascade system andresynthesis of cGMP.51'54 Rhodopsin is inactivated by ATP-dependent phosphorylation at its C terminus and the subse-quent binding of arrestin. Transducin and PDE are inactivatedby the hydrolysis of GTP to GDP on Ta by its intrinsic GTPaseactivity. Guanylate cyclase, the enzyme responsible for the
IOVS, December 1998, Vol. 39, No. 13
TABLE 2. Major ROS Plasma Membrane and Disk Membrane Proteins
ROS, rod outer segment; Mr, molecular weight estimated by sodium dodecyl sulfate gel electrophoresis.* Values in parentheses are determined from sequence.t Only the cone retinol clehydrogenase (retSDRl) has been cloned183; localization in ROS has not yet been determined.
synthesis of cGMP from GTP, is activated by the decrease inintracellular Ca2+ after photoexcitation, a process that is me-diated by GCAP proteins.55"57 As the cGMP concentrationincreases, the cGMP-gated channels reopen, and the photore-ceptor cell is returned to its depolarized state. A correspondingincrease in intracellular Ca2+ restores guanylate cyclase to itsbasal level of activity. Calcium feedback is also thought tofacilitate photorecovery through the regulation of rhodopsinphosphorylation by recoverin58 and modulation of the channelsensitivity for cGMP by calmodulin.59
Since the initial patch clamp studies of Fesenko et al.,60
many laboratories have studied the physiological properties ofthe cGMP-gated channel of rod photoreceptors (for review, seeRefs. 46 and 47). Generally, the rod channel is cooperativelyactivated by cGMP with a Kl/2 of 10 to 50 /xM and a Hillcoefficient of 1.7 to 35 and permeable to a wide range ofmonovalent and divalent cations including Na+, K+, Li+, Cs+,Rb"1", Ca2+, Mg2+, Mn2+, and Ba2+. The pharmacologicalagent, l-cis diltiazem is an effective inhibitor of channel activityand divalent cations, such as Ca2+ and Mg2+ are known tosignificantly decrease the conductance of the channel.
Molecular Characterization and SubcellularDistribution of the cGMP-gated ChannelMolecular characterization of the rod cGMP-gated channel be-gan in the late 1980s when Neil Cook and Benjamin Kauppisolated a 63-kDa protein from bovine ROS that exhibitedcGMP-dependent channel activity when reconstituted intolipid vesicles or planar bilayers.61'62 These studies, however,
were not without considerable controversy since rhodopsin,63
a 39-kDa protein64 and a 250-kDa protein65 were also reportedto be the rod channel. At the same time, Delyth Reid, apostdoctoral fellow in my laboratory, had generated a mono-clonal antibody, designated PMclDl, which reacted with a63-kDa protein in ROS plasma membranes. In collaborationwith Benjamin Kaupp's group in Germany, we were able toshow that this antibody recognizes the 63-kDa channel proteinin both ROS and purified channel preparations.66 Furthermore,the PMclDl antibody immobilized on Sepharose quantitativelyimmunoprecipitated the 63-kDa protein and cGMP-dependentchannel activity.66'67 These studies provided compelling evi-dence that the 63-kDa protein, and not the other candidateproteins, is a component of the rod cGMP-gated channel. Usingisolated disk and plasma membrane preparations, we furthershowed by western blot analysis and activity measurementsthat the 63-kDa channel protein is present in the ROS plasmamembrane, but absent in disks (Fig. 5A, 5B).66 Immunohisto-chemical techniques have confirmed that the cGMP-gatedchannel and the Na/Ca-K exchanger are targeted to the ROSplasma membrane and are not present in detectable quantitiesin disk membranes or in other retinal neurons (Fig. 5C) 3O(56-70
Molecular cloning and expression studies of Kaupp et al.7'provided the most direct evidence that the 63-kDa protein is asubunit of the rod channel and, in addition, yielded informa-tion about its primary structure. The cloned protein having amolecular mass of —79.6 kDa is considerably larger than the63-kDa channel protein of ROS. This is attributed to the ab-sence of the N-terminal 92 amino acids in die ROS channel,
2498 Molday IOVS, December 1998, Vol. 39, No. 13
VCV i -
D V
FIGURE 3. Electron micrographs showing the separation of rod outer segment (ROS) disk andplasma membranes by the ricin gold affinity density perturbation method.2** (A) Isolatedneuraminidase-treated ROS densely labeled with ricin gold particles (10 nm diameter). (B)Membrane fragments obtained after hypotonic lysis of ROS. Unlabeled disks are seen radiatingfrom the cytoplasmic surface of the gold-labeled plasma membrane. (C) Disk membranefraction obtained after sucrose density centrifugation. (D) Ricin gold-labeled plasma mem-brane fraction obtained after sucrose density centrifugation.
presumably due to photoreceptor-specific posttranslationalproteolysis.6* When expressed in Xenopus oocytes or HEK293 cells, the protein assembles into a functional channel thatis activated by cGMP.7172 These results led to the early viewthat the rod cGMP-gated channel is an oligomeric complexconsisting of identical a-subunits.
The first indication that the rod channel contains a secondsubunit was obtained from the cloning studies of King Wai Yauand coworkers.73 They isolated a cDNA encoding a 102-kDapolypeptide that is 30% identical in sequence to a-subunit andpossesses similar structural features. Although this subunit,referred to as subunit 2, does not assemble into a functionalchannel when expressed by itself, coexpression with the roda-subunit produces a functional channel that exhibits physio-
logical properties characteristic of the ROS channel. Theseinclude rapid opening and closing or flickering behavior, inhi-bition by l-cis diltiazem and modulation by Ca-calmodulin.73'74
Questions regarding the true nature of the second subunitarose, however, since a 102-kDa polypeptide is not observed inisolated channel preparations from bovine or human ROS.6774
Instead, a larger polypeptide is observed that migrates on SDSpolyacrylamide gels with an apparent molecular mass of 240kDa (Fig. 6). To begin to characterize this channel-associatedprotein, we isolated a number of proteolytic peptides derivedfrom the 240-kDa protein. The sequence of several peptidesmatched sequences present in subunit 2, suggesting this sub-unit is part of the 240-kDa protein.74 However, some peptidesequences were not found in subunit 2: but instead were
IOVS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2499
4 RGS jrExchanger
GDPT\ --*-?••—..
LightTransducin y
GD
R h o d o p s i n ^
Ca2*t ^> N* Channel
Disk Membrane Plasma Membrane
FIGURE 4. Diagram depicting the principal reactions in phototransduction. Light initiates theisomerization of 11-cis retinal to all-trans retinal resulting in the activated (Meta II) state ofrhodopsin. Meta II rhodopsin catalyzes the exchange of GDP for GTP on transducin and thedissociation of the a-subunit (Tot) from the j37-subunits (T/3y). To: interacts with phosphodi-esterase (PDE) to release the inhibitory constraint on the enzyme. Activated PDE catalyzes thehydrolysis of cGMP to 5'GMP. The decrease in intracellular cGMP concentration causes thechannel to close and the rod cell to hyperpolarized. Intracellular Ca2+ levels decrease as theNa/Ca-K exchanger continues to extrude Ca2+ from the outer segment. Photorecovery isinitiated by the shutoff of the visual cascade and the calcium mediated feedback mechanism.The visual cascade system is inactivated through (1) the phosphorylation of rhodopsin byrhodopsin kinase (RK) and the subsequent binding of arrestin; (2) hydrolysis of GTP to GDPon a-subunit of transducin, a reaction activated by RGS protein. PDE also returns to its inactivestate as a result of this reaction; and (3) reassociation of the a-subunit of transducin with itsjBy-subunits to form the inactivated transducin heterotrimer. Low intracellular Ca2+ concen-trations lead to (1) the activation of guanylate cyclase, a process that is mediated by thecalcium-binding protein GCAP; and (2) an increase in the sensitivity of the channel to cGMPas a result of the dissociation of calmodulin from the channel. As cGMP concentrationincreases, the channels reopen and the cell is returned to its depolarized state. The increasein Ca2+ also converts guanylate cyclase to its inactive or basal level of activity. The solidarrows show the photoexcitation process; dashed arrows show the photorecovery process.
identical with sequences present in a previously cloned retinalglutamic acid-rich protein called GARP.75'76 The relationshipbetween subunit 2 and GARP was resolved when, in collabo-ration with Benjamin Kaupp's laboratory, we were able toshow that the 240-kDa polypeptide represents the full lengthj3-subunit of the cGMP-gated channel and contains both sub-unit 2 and GARP.75
Molecular Structure and Regulation of the cGMP-Gated Channel
Topological models for the a- and /3-subunits of the rod chan-nel as developed from sequence analysis and immunochemicallabeling studies are shown in Figures 7A and 7B/5877-80 Thecore structural unit consists of six membrane-spanning seg-ments (S1-S6) followed by a cGMP binding domain. A voltagesensor-like motif comprising the S4 segment and a pore regionof approximately 20 to 30 amino acids located between the S5and S6 transmembrane segments are also evident,81 A nega-tively charged glutamate residue in the pore region of thea-subunit has been shown to be responsible for external diva-
lent cation blockage.82"3 Since the S4 segment, the pore re-gion, and the folding pattern of the cGMP-gated channel sub-units are characteristic features of voltage-gated cationchannels, it has been suggested that cyclic nucleotide-gatedchannels and voltage-gated channels are members of a super-family of cation channels that have evolved from a commonprimordial channel.81
The |3-subunit contains an unusual bipartite structure (Fig.yB-j 75,8'f T h e c-terminal region or 0' part of approximately 800amino acids contains the core structural unit of the channelrequired for activity. The N terminus contains the GARP part,a region having a high content of glutamic acid and prolineresidues. Two shorter spliced variants of GARP, called f-GARPand t-GARP, are also present in ROS.84'85 The functions of thevarious GARP variants are currently under investigation.
The channel is a heterotetrameric complex,86 most likelyconsisting of two a- and two |3-subunits (Fig. 7C). The poreregions of the individual subunits are oriented toward thecentral cavity of the channel where they serve as the ionselectivity filter and gate.87 Recent high-resolution x-ray analy-
2500 Molday IOVS, December 1998, Vol. 39, No. 13
2 0 5 -
9 7 -
6 8 -
45
2 9 -
CoomassieBlue
ra b c
PMC1D1
63 kDa
a b c
B
0 , 0 2 -
AA
0 . 0 1 -
cGMP PlasmaMembrane
DiskMembrane
50 100
FIGURE 5- Localization of die cGMP-gated channel to the rodouter segment (ROS) plasma membrane. (A) Sodium dodecylsulfate polyacrylamide gel stained with Coomassie blue (left) andthe corresponding western blot analysis labeled with monoclonalantibody PMclDl against the 63-kDa osubunit of the channel.66
Lane a: rod outer segment (ROS) membranes; lane b: isolateddisk membrane fraction; lane c: isolated plasma membrane frac-tion. (B) cGMP dependent channel activity. Purified disk andplasma membrane fractions solubilized in CHAPS detergent werereconstituted into Ca2+-containing lipid vesicles. The release ofCa2+ was initiated with 150 fjM cGMP and detected by a changein absorbance (AA) using Arsenazo in as an indicator. Only theplasma membrane exhibits channel activity.66 (C) Electron mi-crograph of bovine ROS membranes labeled with antibodiesagainst the cGMP-gated channel. ROS membrane fragments werelabeled with the PMc6E7 anti-channel antibody followed by im-munogold particles (10 nm diameter). The cytoplasmic surface ofthe inside out plasma membrane vesicles (arrowheads) isdensely labeled. The disk membranes radiating from the plasmamembrane vesicles are not labeled.68
sis of a related K+ selective channel from Streptomyces livi-dans has provided a detailed structure of the pore region ofthis channel and insight into how such pore regions function inthe translocation of ions through these channels.88
As part of our studies on the rod channel, we investigatedfactors that regulate the activity of the rod cGMP-gated chan-nel. Yi-Te Hsu, a former graduate student, first showed thatcalmodulin modulates the apparent affinity of the channel forcGMP in a calcium dependent manner.5989 Using both nativeand reconstituted channels, he showed that Ca-calmodulinshifts the dose-response curve to higher cGMP concentrations(Fig. 8): an effect that has been reproduced in various experi-mental systems.74'7590 He also showed that calmodulin-Sepha-rose can be used to purify the channel and that the binding sitefor calmodulin is localized on the £-subunit.59'88 More re-cently, this site has been mapped near the N terminus of thecore structural unit of the j3-subunit.9192
Modulation of the channel by calmodulin operates underphysiological Ca2+ concentrations59'89 This has led us to sug-gest that Ca-calmodulin regulation of the channel may play arole in facilitating photorecovery after bleaching by increasingthe sensitivity of the channel to cGMP.5979 The finding thatcalmodulin modulates the rod channel has led to the importantdiscovery by King Wai Yau's group that the olfactory cyclicnucleotide-gated channel is also strongly modulated by cal-modulin, a mechanism that is important in olfactory adapta-tion.
kDa
205-
97-
68-
4 5 -
29-
- 240 kDa(p-subunit)
63 kDa(ct-subunit)
FIGURE 6. Immunoaffinity purification of the channel fromrod outer segment (ROS). ROS membranes (lane a) weresolubilized in CHAPS buffer and passed through a PMc6E7-Sepharose affinity column. After the column was washed toremove unbound proteins, the channel (lane b) was elutedwith a peptide corresponding to the epitope for the PMc6E7monoclonal antibody. The samples were run on a 9% sodiumdodecyl sulfate polyacrylamide gel and stained with Coomassieblue. The purified channel contains two prominent polypep-tides: the 63-kDa a-subunit and the 240-kDa 0-subunit.
IOVS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2501
Pore Region
S1 S2 S3 S4S5I S6 RinHinn ^ite\ \ I / / | I Binding Site 6 g o
Pore Region
CHO
Garp Part
f-Garp
t-Garp
(V-Part
Pore Region Pore Region
COOH
a - Subunitp-Part
Garp Part
- Subunit
aFIGURE 7. Schematic models of the rod cGMP-gated channel. (A) Linear representation (top)and topological model (bottom) of the rod a-subunit. The membrane spanning segments(S1-S6), the pore region containing a glutamate residue (0), the N-linked glycosylation site(CHO), and the cGMP binding site are shown. The N-terminal 92 amino acids (dashed line) ismissing in the channel present in rod outer segment (ROS). (B) Linear representation (top) andtopological model (bottom) of the rod j3-subunit. In addition to many structural features foundin the a-subunit, the j3-subunit contains a calmodulin (CaM) binding site and an extendedN-terminal region called the GARP part. Two additional spliced variants of GARP, called f-GARPand t-GARP, are also present in ROS.84 Unlike the a-subunit, the /3-subunit does not contain anN-ltnked glycosylation site or a glutamate residue in the pore region. (C) Model for thecGMP-gated channel complex. Two cc-subunits and two /3-subunits assemble into a tetramericcomplex. The S6 and S5 segments are visualized to line the central cavity of the channel. Thepore regions joining the S5 and S6 segments near the extracellular surface of the membraneextend toward the center of the cavity where they can function as a gate and ion selectivityfilter. The glutamate residue (0) in the pore region of the a-subunit is responsible for externaldivalent cation blockage of the channel. Although the channel is shown with identical subunitsacross from each other, it is possible that the two identical subunits may be adjacent.
60
cGMP Concentration
FIGURE 8. Modulation of the rod cGMP-gated channel by Ca-calmodulin. Rod outer segment (ROS) membrane vesiclesloaded with Arsenazo III were placed in a calcium-containingbuffer in the absence (•) or presence (•) of calmodulin, andcalcium uptake was initiated by the addition of cGMP. Therelative initial velocity (VJVm:ix) is plotted against cGMP con-centration. The solid line is the sigmoidal binding curve calcu-lated using a Michaelis constant (Km~) of 19 /xM and a Hillcoefficient of 3-7 in the absence of calmodulin and a Km of 33/xM and a Hill coefficient of 3-5 in the presence of calmodu-lin.59
GLYCOLYTIC ENZYMES AND GLUCOSE
METABOLISM IN ROS AND CONE OUTER
SEGMENTS
Phototransduction and related outer segment processes re-quire considerable amounts of energy and reducing equiva-lents in the form of ATP, GTP, and NADPH. Over the years,questions arose as to whether these cofactors are generated inthe outer segments or produced in the inner segment andshuttled to the outer segment. In early studies, glycolytic andhexose monophosphate enzyme activities were detected inouter segment preparations,95"98 but the activities were low incomparison to other retinal cell layers. As a result, it has beenunclear whether these enzyme activities come from endoge-nous outer segment enzymes or alternatively, from contami-nants of the preparations. A report indicating that outer seg-ments contain considerable amounts of creatine kinase also ledto the possibility that energy in the form of ATP can betransferred from the inner segment to the outer segment by aphosphocreatine shuttle pathway.99
Our interest in glucose metabolism arose when Shu-ChanHsu, a former graduate student, identified an abundant 38-kDaROS plasma membrane-associated protein as glyceraldehyde-3-phosphate dehydrogenase, a key enzyme in anaerobic glyco-lysis. 10° She further showed by immunofluorescence, westernblot analysis and enzyme activity measurements that otherglycolytic enzymes and a GLUT-1 glucose transporter are alsopresent in ROS and cone outer segments.101 Moreover, quan-titative measurements indicated that isolated ROS preparationshave the capacity to take up glucose and convert glucose tolactate by anaerobic glycolysis (Figs. 9A, 9B).101"103 Sufficient
IOVS, December 1998, Vol. 39, No. 13
amounts of ATP are produced by anaerobic glycolysis to sus-tain cGMP levels in dark-adapted ROS.102 Glucose is also usedby the hexose monophosphate pathway in ROS to regenerateNADPH for retinal reduction after the photobleaching of rho-dopsin and glutathione reduction for the protection of outersegments from oxidative stress.102 A phosphocreatine shuttlesystem most likely serves as an additional source of energy.Biochemical pathways involved in the production of energyand reducing equivalents in ROS are depicted in Figure 10. Theimportance of anaerobic glycolysis in retinal function has beenaddressed in considerable detail in the physiological and bio-chemical studies of Barry Winkler and coworkers.104'105
RETINAL DEGENERATIVE DISEASES
In 1990, Ted Dryja and coworkers first reported that a P23Hmutation in rhodopsin is responsible for a form of autosomaldominant retinitis pigmentosa (ADRP).106 Since this time,more than 70 different rhodopsin mutations have been linkedto autosomal dominant and recessive forms of retinitis pigmen-tosa, and congenital stationary nightblindness (CSNB).107'109
BtoOacCD
0 50 100 150Time (seconds)
400 -
200
200 -
5 10Time (min)
15
FIGURE 9- A Uptake of glucose into isolated bovine rod outersegment (ROS). The uptake of 45 /xM external 3-O-[l4C]meth-ylglucose (3-O-MG), a nonmetabolizable analogue of glucose,was measured in the absence (•) and presence (O) of 0.05 mMcytochalasin B, an inhibitor of glucose transport.101 (B) Glyco-lytic flux in isolated bovine ROS. Time course of lactate pro-duction by isolated bovine ROS in the presence (•) and ab-sence (V) of 5 mM glucose.102
IOVS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2503
GP
_ GlutathioneG S S G redox cycle G S H
PHOTO-TRANSDUCTION
OS
IS
FIGURE 10. Diagram showing the role of glycolysis, the hex-ose monophosphate pathway, and the phosphocreatine shuttlein ATP, GTP, and NADPH production. In photoreceptor outersegments (OS), glucose is transported across the plasma mem-brane by a GLUT-1-type glucose transporter (GT). Glucose ismetabolized to lactate by anaerobic glycolysis resulting in theproduction of ATP and GTP for phototransdviction and otherenergy requiring processes. Glucose can also be used by thehexose monophosphate pathway (HMP) for the regenerationof NADPH. The latter is required for the reduction of all-transretinal to all-trans retinol by retinol dehydrogenase (RDH) afterthe bleaching of rhodopsin and for glutathione reduction byglutathione reductase (GR) for protection of the outer segmentagainst oxidative damage. Additional energy is obtained fromthe inner segment (IS). High-energy phosphocreatine (PCr) istransferred to the outer segment by the PCr and used togenerate ATP. This reaction is catalyzed by a brain-type creat-ine kinase (Ckb).
102
In addition, positional cloning and candidate gene approacheshave led to the identification of large number of genes associ-ated with retinitis pigmentosa and other retinal degenerativediseases (Table 3).108-109 Many genes code for photoreceptor-specific proteins that play key roles in phototransduction andouter segment morphogenesis. Our interest in this area hasfocused on the peripherin/rds-rom-1 complex and the ABCR/RIM protein, two disk membrane proteins implicated in mul-tiple retinal degenerative diseases.
PERIPHERIN/RDS—ROM-1 COMPLEX
Molecular Structure and Subcellular DistributionPeripherin/rds and rom-1 are homologous subunits of an oli-gomeric membrane protein found in disks. Peripherin/TYfe,
formerly called peripherin, was first detected as a 33- to 35-kDaprotein in bovine disk membranes with monoclonal antibod-ies110 and subsequently cloned from a retinal expression libraryby Greg Connell, a former graduate student in my laborato-ry.1" Several years later, Rod Mclnnes's laboratory in Torontocloned rom-1, a protein that is 30% identical in sequence toperipherin/rds."2 Immunochemical labeling studies and se-quence analysis has led to a topological model for these pro-teins (Fig. II).111""4 Characteristic structural features includefour membrane- spanning segments (M1-M4), an extendedcarboxyl terminal domain exposed on the cytoplasmic side ofdisks and a large intradiskal loop (L3-L4) containing sevenconserved cysteine residues.
Peripherin/rds and rom-1 associate to form a tetramericcomplex as determined by immunoprecipitation studies andhydrodynamic measurements."2"1'6 Since dimers of periph-erin/rds and rom-1 are routinely observed by SDS gel electro-phoresis under nonreducing conditions,"0""3 it was initiallythought that a disulfide-linked homodimer of peripherin/rclsinteract noncovalently with a disulfide-linked homodimer ofrom-1 to form a heterotetramer. " 4 " " 6 More recently, how-ever, Chris Loewen, a graduate student in my laboratory, hasshown that the peripherin/rd.s-rom-1 tetramer does not containintermolecular disulfide bonds.117 Instead, intermolecular di-sulfide bonds, mediated by Cys 150 within the large intradiskalloop,118 link individvial tetramers into higher order oligomers,a mechanism that may underlie disk morphogenesis.117
The peripherin/rds-rom-1 complex has a unique subcel-lular distribution. Preembedding and postembedding immuno-gold labeling studies have localized peripherin/rds to the rimregion of ROS and cone outer segment disks (Figs. 12A,12B) iio,iu,u<f, 119 imtiai studies suggested that rom-1 is only
Cytoplasmicside
COOH
Intradiscalside
L3-4
FIGURE 11. Topological model for peripherin/rcfe and rom-1based on sequence analysis and immunochemical studies. Bothproteins are shown to contain four transmembrane segments(M1-M4), cytoplasmic N and C termini, and a large intradiskalloop (L3-4). Perpherin/rds contains an N-linked oligosaccha-ride chain (hexagons') that is not present in rom-1. Stretches ofconserved amino acids and conserved cysteine residues areindicated.1' 1~113
2504 Molday IOVS, December 1998, Vol. 39, No. 13
TABLE 3- Proteins Associated with Various Retinal Degenerative Diseases
Rod cellsRod cellsRod cellsCone and rod cellsCone and rod cellsRod cellsRod cellsRod cellsRod and cone cells
Rod and cone cellsRod cellsRod cellsRPE and Miiller cellsRod and cone cellsRPE cellsBruch's membraneExtracellular (photoreceptors)Photoreceptors/RPERPE
present in ROS disks.112 However, Orson Moritz, a formergraduate student in my laboratory, has clearly shown thatrom-1 is present along the rim region of cone and rod diskmembranes as shown in Figure 12C.113
B
ROS
COS
CIS
Role of Peripherin/rrfs in Outer SegmentMorphogenesis and Retinal Degeneration in theRDS MouseThe importance of peripherin/r^ in outer segment morpho-genesis originated from the cellular and molecular analysis ofthe retinal degeneration slow or rds mouse. Sanyal and co-workers120'121 showed that mice homozygous for the rds mu-tation fail to develop outer segments and the photoreceptorcells undergo slow degeneration such that few cells remain 1year after birth. Heterozygous rds mice exhibit short, highlydisorganized outer segments that often appear as whorls ofmembrane.122 In the late 1980s, Grabriel Travis and col-leagues123 used subtractive hybridization techniques to iden-tify a mutation in a photoreceptor cell-specific gene that isresponsible for the rds phenotype. We subsequently showedthat this gene codes for peripherin/refe.124 Finally, the researchgroups of Gabriel Travis and Dean Bok have reported thatintroduction of the normal peripherin/rds gene into a homozy-
FIGURE 12. Localization of peripherin/rrfs and rom-1 to therim region of rod outer segment (ROS) and cone outer segment(COS) disks. (A, B) Electron micrographs of an isolated bovineROS disk and a group of disks labeled with Per 3B6 antiperiph-crin/rds monoclonal antibody and immunogold particles (10nra diameter). The gold particles are localized to the rim regionof the disks. (C) Electron micrograph of an ROS, a COS, and acone inner segment (CIS) labeled for rom-1. Bovine retina wasembedded in LR White resin. Sections were then labeled withthe anti-rom-1 antibody (Rom L4) and immunogold particles.Gold particles are distributed along the peripheral region ofboth the ROS and COS and the incisures of ROS (arrow-head)^
IOVS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2505
330.
Cytoplasmicside
346OOH
Trp25FS
Intradiskalside
Lysl 53ArgLysl 53Del
Pro2l6LeuPro2l6Ser
*Cys2l4Ser
Ser212Gly
Pro210ArgPro210Ser
Lysl93Del
FIGURE 13- Location of peripherin/rds mutations associated with retinal degenerative dis-eases. Unframed mutations have been linked to monogenic autosomal dominant retinitispigmentosa (ADRP). Mutations framed in black have been implicated macular and patterndystrophies. The mutation framed in gray is linked to digenic ADRP. Insert/RDS is the site ofan insertion mutation in the rds mouse; FS indicates a frameshift resulting in a change inreading frame and premature stop.
gous rds mouse results in outer segment formation and sup-pression of photoreceptor degeneration.125 Taken together,these studies indicate that peripherin/rds is plays an essentialrole in ROS and cone outer segment morphogenesis, and theabsence or reduced amounts of this membrane protein resultin photoreceptor degeneration.
Role of Peripherin/rds in ADRP and MacularDegeneration
The finding that a mutation in the peripherin/rds gene causesphotoreceptor degeneration in the rds mouse prompted manylaboratories to determine whether mutations in the humangene are responsible for inherited retinal diseases. This hasproved to be the case and to date more than 30 mutations inthe peripherin/rds gene have been implicated in various reti-nal degenerative diseases (Fig. 13).126"130 Interestingly,whereas some peripherin/rds mutations cause ADRP, othersresult in macular degeneration and pattern dystrophies that gounder such names as, butterfly-shaped pigment dystrophy,fundus flavimaculatus, cone-rod dystrophy and Bull's Eyemaculopathy. l31~136 Similar efforts to link mutations in rom-1with retinal degenerative diseases have been less successful.
However, several mutations in the rom-1 gene have beenlinked to a novel form of digenic ADRP.137"138
To begin to understand the mechanism by which mu-tations in peripherin/rds cause specific disease phenotypes,Andy Goldberg, a former postdoctoral fellow in my labora-tory, developed a heterologous cell expression system tostudy the molecular properties and subunit interactions ofperipherin/rds and rom-1.115 He showed that coexpressionof peripherin/rds and rom-1 results in a heterotetramericcomplex having properties similar to those of the complexderived from ROS. However, many mutations in the largeintradiskal segment, including the C214S peripherin/rds mu-tant-linked ADRP,130 result in abnormal protein folding andthe inability of peripherin/rds to associate with rom-1."8
From these studies, we have concluded that segments of thelarge intradiskal loop are important in subunit-subunit in-teractions, a property important in normal outer segmentformation.
We have also examined the R172W peripherin/rds mutantlinked to a mild form of macular degeneration.131136 Interest-ingly, this mutant folds normally and assembles with rom-1 intoa nativelike tetrameric complex.139 Apparently, subtle differ-
2506 Molday
ences in the structure and/or stability of this complex result inpreferential cone degeneration.136
Recently, a novel digenic form of ADRP linked to aL185P mutation in peripherin/nfc and a null mutation inrom-1 has been described in several families.137138 Individ-uals who coinherit both mutations (double heterozygotes)exhibit an ADRP disease phenotype, whereas family mem-bers who inherit only one of these mutations (single het-erozygotes) are essentially normal. We postulated that thisdisease pattern might arise from defective peripherin/rds-rom-1 subunit interactions. To investigate the role of subunitassembly in this form of digenic RP, Andy Goldberg analyzedthe hydrodynamic properties of the complexes formedwhen either wild-type peripherin/rds or the L185P mutant isexpressed in COS-1 cells in the presence and absence ofrom-1.l39 The results of this study are diagramed in Figure14. Coexpression of the L185P peripherin/rds mutant withrom-1 (simulating an individual who inherits only the pe-ripherin/n/s mutation) results in a heterotetrameric com-plex similar to the wild-type complex. Wild-type peripherin/rds in the absence of rom-1 (simulating an individual whoinherits a rom-1 null mutation) self-assembles into a ho-motetrameric complex. In contrast, L185P peripherm/rds inthe absence of rom-1 (simulating individuals who inheritboth mutations) fails to self-assemble into a tetramer, butinstead remains in a dissociated state. From these studies wehave concluded that peripherin/rds-containing tetramersare crucial in outer segment formation and stability. Re-duced levels of this complex can lead to abnormal andunstable outer segments and progressive photoreceptor de-generation.139
Implicit in this model is the hypothesis that peripherin/rc/shomotetramers can effectively substitute for peripherin/rds-rom-1 heterotetramers and promote outer segment disk mor-phogenesis. Recently, we have had the opportunity to test thisprediction in a Rom-1 knockout mouse produced by GeoffClark and Rod Mclnnes.l4° Biochemical and ultrastructuralanalyses of these mice, indeed, indicate that peripherin/rdsself-assembles into a homotetramer in the absence of rom-1,and this complex supports ROS and cone outer segment for-mation (Goldberg A, Molday L, Molday R, Clark G, Mclnnes R,unpublished results, 1997). Comparative analysis of the rom-1knockout mouse and the rds mouse leads to the conclusionthat peripherin//Y/s is the dominant subunit required for diskmorphogenesis. Rom-1 is relegated to a more minor role, per-haps enhancing the stability of the outer segment and/or finetuning the structure of disks.
THE ABCR/RIM PROTEIN AND STARGARDT'S
MACULAR DEGENERATION
More recent studies in my laboratory have focused on theidentification and characterization of the abundant high-molec-ular-weight rim protein first identified by David Papermasterand colleagues in frog photoreceptors"""'2 and subsequentlydetected in mammalian ROS.1''314'1 Michelle Illing, as part ofher graduate research in my laboratory, purified and cloned thebovine 220-kDa rim protein and showed that this protein is amember of the superfamily of ABC (ATP binding cassette)transporters.145 This extensive family of proteins include thecystic fibrosis transmembrane regulator (CFTR) linked to cystic
IOVS, December 1998, Vol. 39, No. 13
Molecular Complex Phenotype
WT Per/rdsWT Rom-1 Normal
B Ll 85P Per/rdsWT Rom-1
"Normal"
Wt Per/rdsNull Rom-1
"Normal"
DL185PPerNull Rom-1
DigenicADRP
FIGURE 14. Subunit assembly model for digenic autosomaldominant retinitis pigmentosa (ADRP) linked to a L185P mu-tation in peripherin/rds and a null allele in Rom-1. Left: molec-ular complexes of peripherin/rds (dark) and rom-1 (light)determined by velocity sedimentation measurements.l39 Right:corresponding phenotypes for individuals who inherit one orboth mutations. (A) Wild-type (WT) peripherinAv5fo-rom-lcomplex characteristic of normal individuals. (B) L185P pe-ripherin/r^5-rom-l complex predicted to exist in individualswho inherit only the L185P mutation. This tetrameric com-plexes is suggested to support outer segment formation result-ing in a borderline "Normal" phenotype. (C) Peripherin/rrfshomotetrameric complex predicted to exist in individuals whoinherit a null mutation in rom-1. This complex can take theplace of the peripherin/rc/s-rom-1 heterotetramer and supportouter segment morphogenesis and structure. A borderline"Normal" phenotype can result from a small net reduction inthe amount of peripherin/rcfc containing tetramers. (D) Disso-ciated L185P peripherin/r<3?s mutant in the absence of rom-1.This species is not expected to support outer segment mor-phogenesis or structure. A significant reduction in the level ofperipherin/rc/.s containing tetramer will result in disorganized,unstable outer segments, a condition which underlies photo-receptor degeneration and a ADRP phenotype.
fibrosis, P-glycoprotein involved in multidrug resistance in can-cer, TAP1 and TAP2 proteins that serve as peptide transportersin lymphocytes, prokaryotic permeases, and others.146 LikeCFTR and P-glycoprotein, the rod ABC protein consists of twostructurally related halves, each of which contains a multiplemembrane-spanning domain followed by a cytoplasmic ATPbinding cassette. On the basis of sequence analysis and bio-chemical studies, we have developed a working model for thetopological organization of this glycoprotein in disk mem-branes as shown in Figure 15.
10VS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2507
G818E N1614FS
Del WAIC
CYTOPLASMICSPACE
INTRADISKALSPACE
R2106C
L962FS
N965S
A1028V E1036K
L2027F I \R2077W
R2038W V2050L
E1087K
V1072A
S1071FS
FIGURE 15- Topological model for ABCR/RIM based on sequence analysis, location of N-linkedoligosaccharides, and analogy with other ABC transporters. The model shows 12 transmem-brane segments, 2 ATP binding cassettes (ABC) on the cytoplasmic side of the membrane, and2 N-linked oligosaccharide chains (hexagons') on the intradiskal side.145 Several mutationslinked to Stargardt's macular dystrophy are indicated.149152
The cellular and subcellular distribution of this ABC pro-tein has been investigated using both immunochemical andmolecular biology techniques. The protein is abundantly ex-pressed in the outer segments of rod photoreceptors, but isabsent in cone cells.145147'1 ' '8 This is in agreement with in situhybridization and northern blot analysis, indicating that mRNAexpression is restricted to rod cells.149 At a higher resolution,postembedding, immunogold labeling studies carried out byLaurie Molday have shown that the bovine rod ABC trans-porter, like its frog counterpart, is localized to the rim region ofrod disk membranes (Fig. l6 ) . 1 4 1 1 4 5
During the course of our studies, Allkimets and coworkerselegantly showed that mutations in the ABCR gene coding fora retinal rod-specific ABC protein are responsible for Star-gardt's disease, an autosomal recessive macular dystrophy witha juvenile onset.149 This disease, characterized by progressiveloss in central vision, bilateral atrophy of the macular region ofthe retina and RPE layer, and the appearance of orange-yellowflecks, accounts for up to 7% of the human retinal degenerativediseases.150151 Analysis of the amino acid sequences of human,bovine, and mouse proteins indicate that the ABCR protein andthe rim protein are one in the same.145'147'148'152 The locationof several reported Stargardt's mutations l 4 y 1 5 2 in context toour working model of rod ABC protein, now referred to as theABCR/RIM protein, is shown in Figure 15.
More recent studies suggest that mutations in the ABCRgene can also cause other disease phenotypes. Allkimets etal.153 have reported that some mutations in the ABCR gene areassociated age-related macular degeneration, a result that hasgenerated considerable controversy. Homozygous and com-pound heterozygous mutations in the ABCR gene resulting innull alleles have also been implicated in autosomal recessive RPand cone-rod dystrophy.154'155
The function of the ABCR/RIM protein is not currentlyknown. Other members of this superfamily are known tomediate the active transport of a wide range of compoundsincluding drugs, metabolites, peptides, and lipids.l46 A relatedprotein, ABC1, has also been implicated in the engulfment ofcell corpses after apoptosis.l5<5 It is possible that the ABCR/RIM
ROS
ROS
RIS
8
FIGURE 16. (A, B) Localization of ABCR/RIM protein to therim and incisures of rod outer segment (ROS) disks. Electronmicrogniphs of bovine rod photoreceptor cells embedded inLR White resin, sectioned, and labeled with an anti-ABCR/RIMantibody and immunogold particles. Labeling is restricted tothe peripheral region and incisures (arrowhead) of the ROS, apattern that is characteristic of disk rim labeling. The rod innersegment (RIS) is not labeled.145
FIGURE 17. Diagram showing possible transport functions of the ABCR/RIM protein. Retinalderivatives, peptides, antioxidants, phospholipids, or other agents may be actively transportedinto or out of the disk membrane or translocated from one side of the disk lipid bilayer to theother using ATP hydrolysis as a source of energy. Pi, inorganic phosphate.
Glutathtone Cycle
Retinal Reduclioi
HexoseMoitophosphate
Pathway
RhodopsmKmase Arnwtin
CSMB CSNB
GlucoseTransporter
Glucose
cGMP-gatedChannel
Na/Ca-KExchanger
4Na
dopsin
Guanylate ABCR/RIMCyclase
LCAI• " '
ADRPMD
Slgdl MD
XLRS1
ODisk
LamellarDiskRim
PlasmaMembrane
FIGURE 18. Diagram showing the distribution of various proteins in the rod outer segment(ROS). Retinal diseases linked to mutations in these proteins are also indicated. ADRP,autosomal dominant retinitis pigmentosa; ARRP, autosomal recessive retinitis pigmentosa;CSNB, congenital stationary night blindness; CD, cone dystrophy; CRD, cone-rod dystrophy;MD, macular dystrophy; Stgdt MD, Stargardt's macular dystrophy; XLRS1, X-linked retinoschi-sts 1; XLRP3, X-linked retinitis pigmentosa 3.
JOVS, December 1998, Vol. 39, No. 13 The Friedenwald Lecture 2509
is involved in the transport of retinal derivatives, phospholip-ids, peptides or other endogenous substrates across the diskmembrane CFig- 17)- Molecular and biochemical approachesare now being used to define in more detail the structure andfunction of this unique disk protein and to understand howspecific mutations cause various disease phenotypes.
SUMMARY AND FUTURE DIRECTIONS
Over the past two decades, remarkable progress has beenmade on the cellular and molecular characterization of ROSand cone outer segments. Many membrane and soluble pro-teins have been identified and localized within the outer seg-ment, and their structural and functional properties have beenstudied in considerable detail. This has led to a comprehensiveunderstanding of phototransduction and supporting metabolicpathways and a glimpse into mechanisms underlying outersegment structure, morphogenesis, and renewal. Recent stud-ies have also indicated that mutations in many photoreceptorouter segment-specific proteins cause a multitude of inheritedretinal degenerative diseases (Fig. 18). Molecular studies arenow beginning to provide insight into how specific mutationsin these proteins lead to various retinal diseases.
Although we have learned a great deal about the molecu-lar composition and function of ROS and cone outer segments,much remains to be learned. The staicture and function ofmany proteins associated with retinal degenerative diseases,such as the ABCR/RJM associated with Stargardt's disease,XLRS1 implicated in X-linked juvenile retinoschisis, and RPGRassociated with X-linked RP3 remain to be determined. Regu-latory mechanisms underlying phototransduction need to beunderstood in a more detailed, quantitative manner. Molecularmechanisms and protein-protein interactions that form andstabilize the unique ROS and cone outer segment structure andtarget proteins to the outer segment and more specifically tothe outer segment disk or plasma membrane have to be under-stood at a molecular level.
Although the future challenges are numerous, one onlycan be optimistic that the rapid advances in basic and clinicalresearch will lead to a complete understanding of photorecep-tor structure and function and detailed insight into the molec-ular basis for retinal diseases. From this knowledge, it shouldbe possible to develop novel and effective treatments for most,if not all, the diseases that lead to the loss in vision.
Acknowledgments
I want to convey my sincere gratitude to the numerous past andpresent research colleagues who have contributed greatly to the re-search accomplishments embodied in this award and extend specialthanks to Laurie Molday who carried out many of the electron micro-scopic and biochemical studies described in this article. Other post-doctoral fellows, graduate students, and research assistants who havemade significant contributions include Jinhi Ahn, Steve Clark, CaroleColville, Greg Connell, Andrea Dose, Andrew Goldberg, David Hicks,Theresa Hii, Shu-Chan Hsu, Yi-Te Hsu, Michelle Tiling, Tom Kim, DaleLaird, Chris Loewen, Don MacKenzie, Orson Moritz, Igor Nassonkin,Delyth Reid, and Simon Wong. I also thank my friends and collabora-tors, King Wai Yau, Benjamin Kaupp, Barry Winkler and BernhardWeber, and other prominent vision investigators with whom I havehad an opportunity to collaborate and discuss research of mutualinterest. These include Gobind Khorana, David Papermaster, DanOprian, Paul Hargrave, Ted Dryja, David Williams, Rod Mclnnes, Neal
Cook, Heinz Wassle, Kris Palczewski, Karl Koch, Paul Bauer, SteveFisher, and Jeremy Nathans. Finally, I gratefully acknowledge the long-standing grant support from the National Eye Institute, IIP Foundationof Canada, and the Medical Research Council.
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