Download Photoreceptor Membrane Proteins, Phototransduction, and Retinal

Photoreceptor Membrane Proteins,
Phototransduction, and Retinal
Degenerative Diseases
The Friedenwald Lecture
Robert S. Molday
R
od and cone photoreceptor cells are specialized neurons of the vertebrate retina that function in the primary events of vision. Rod cells are highly sensitive to
light and operate under dim lighting conditions. Cones are less
sensitive and function in bright light and in color vision. Both
photoreceptors are elongated cells consisting of several morphologically and functionally distinct regions (Fig. 1). The
photoreceptor outer segment located adjacent to the retinal
pigment epithelial (RPE) cell layer is a specialized compartment 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 renewal
process in which newly synthesized membrane is added at the
base of the outer segment, whereas packets of outer segment
membrane are phagocytized at the distal end by the RPE
cells. '"3 A thin, nonmotile cilium links the outer segment to the
inner segment, a cellular compartment that contains the mitochondria, endoplasmic reticulum, golgi apparatus, and other
subcellular organelles. Adjoining the inner segment is the cell
body containing the nucleus. This region further extends into
the synaptic region where the electrical signal generated in the
photoreceptor cell is transmitted to other neurons of the retina.
Photoreceptor outer segments have been extensively studied at a cellular level/'"8 In the rod cell, the outer segment
appears as a cylindrical structure up to 60 /xm in length and 1.5
to 2 jam in diameter for mammals and 6 to 8 jam in diameter for
amphibians. The cone outer segment is generally shorter and
often is tapered or conical.
The outer segment consists of hundreds of flattened disk
membranes assembled in an ordered axial array (Fig. 1). In rod
cells, the stack of closed disks is surrounded by a separate
plasma membrane over the entire length of the outer segment
except at its base. Here, newly formed disk membranes evaginate from the ciliary membrane to form a folded membrane
system.8 Each rod outer segment (ROS) disk consists of two
closely 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 Research Institute Award, Fort Worth, Texas.
Reprint requests: Robert S. Molday, Department of Biochemistry
and Molecular Biology, 2146 Health Sciences Mall, Vancouver, BC,
Canada V6T 1Z3. E-mail: [email protected].
Investigative Ophthalmology & Visual Science, December 1998, Vol. 39, No. 13
Copyright © Association for Research in Vision and Ophthalmology
loop called the rim region. The continuous disk membrane
separates an intradiskal space or lumen from the cytoplasm.
The perimeter of the rod disk is interrupted by one or more
incisures that penetrate toward the center of the disk. Cone
outer segments also consist of a stack of disk membranes, but
unlike ROS disks, cone disks remain open and do not contain
incisures.9'10 Fibrous elements extend from the rim regions of
disks to adjacent disks and to the plasma membrane.""13 This
cytoskeletal-like system serves to maintain the precise distances between adjacent disk and plasma membranes and stabilize the outer segment structure.
ROS
Isolation and Characterization
Although ultrastructural features of ROS had been described in
considerable detail by the early 1970s, relatively few studies
dealt directly with the molecular composition of this lightsensitive organelle. This was due largely to the lack of a pure,
well-characterized ROS preparation and to technical difficulties
inherent in the molecular analysis of biological membranes,
and in particular membrane proteins. A significant advancement came in 1974 when David Papermaster, working as a
postdoctoral fellow in William Dreyer's laboratory at California
Institute of Technology in Pasadena, developed a highly pure
preparation of bovine ROS.14 This procedure, based in part on
earlier protocols,1516 takes advantage of the fragile nature of
the connecting cilium, the stability of intact ROS in sucrose
solutions, and the low density of ROS relative to other subcellular organelles. Briefly, retina tissue immersed in sucrose solution is gently agitated to detach the ROS. Differential velocity
centrifugation and sucrose density gradient sedimentation is
then used to isolate ROS, largely free of contaminating subcellular organelles. Minor modifications to this procedure have
been introduced over the years, but in general these preparations exhibit similar protein and lipid compositions. A typical
profile of ROS proteins fractionated on a sodium dodecyl sulfate (SDS) polyacrylamide gel is shown in Figure 2 (lane a).
Rhodopsin represents the major stained band, accounting for
more than 70% of the total ROS protein. Numerous less abundant proteins of various molecular weights are also observed.
Isolated ROS in sucrose solutions retain their soluble and
weakly associated membrane proteins. Herman Kiihn, also a
former postdoctoral fellow at Cal-Tech and later a research
scientist at the Neurobiology Institute in Jiilich, Germany, developed several procedures to selectively extract soluble and
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JOVS, December 1998, Vol. 39, No. 13
Incisure
Disks
Rod outer segment
plasma membrane
Plasma Membrane-
\
Rod outer segment
Connecting Cilium
Golgi complex
Rod inner segment
Endoplasmic reticulum
> Cell body
Synaptic terminal
Synaptic vesicles
1. Left: schematic diagram of a rod photoreceptor cell. Right: diagram of a rod outer
segment. Adapted with permission from Young RW. Visual cells. Set Am. 1970;223:81-91-
FIGURE
weakly associated membrane proteins for biochemical analysis. 1718 One such procedure involves hypotonic lysis of darkadapted ROS followed by centrifugation to separate the supernatant fraction from the membrane pellet. 1719 As shown in
Figure 2 (lanes b, c), a distinct protein profile is observed for
each fraction. The supernatant contains numerous proteins
ranging in molecular mass from approximately 10 kDa to more
than 100 kDa. Many of these proteins have been characterized
by various laboratories and shown to function as key enzymes
and regulatory proteins in phototransduction and metabolism
(Table 1). The membrane fraction of ROS is dominated by
rhodopsin. In addition, numerous less abundant membrane
proteins are observed in the range of 30 kDa to 240 kDa (see
Table 2).
Separation of ROS Disk and Plasma Membranes
My laboratory has focused primarily on the molecular analysis
of integral membrane proteins of ROS and elucidation of their
role in phototransduction, outer segment structure and metabolism, and retinal degenerative diseases. As a first step, it was
important to analyze the protein composition of the ROS disk
and plasma membrane. One view at the time held that the two
membranes are identical in composition. This was supported
by electron microscopic studies showing that the disk and
plasma membranes arise from the same newly formed membranes at the base of the outer segment8 and immunocytochemical studies revealing the presence of high levels of rhodopsin in both membranes.20"22 Furthermore, several
laboratories reported that both the disk and plasma membrane
contain cGMP-gated channel activity and Na/Ca exchange activity.23"25 In contrast, biochemical labeling studies indicated
that several proteins present in the ROS plasma membrane are
absent in disks, suggesting that the two membranes may differ
in protein composition.26'27
To resolve this issue, we developed a ricin-gold affinity
density perturbation method that can effectively separate the
plasma membrane, constituting ~5% of the ROS membrane,
from the more abundant disk membranes.19'28 In this procedure, isolated ROS are first treated with neuraminidase to
remove terminal sialic acid residues on surface sialoglycopro-
The Friedenwald Lecture
IOVS, December 1998, Vol. 39, No. 13
kDa
205
-
97
-
68
-
45
-
-
PDE(a,p)
.^
Rhodopsin
Kinase
\ ^
Arrestin
_
Rhodopsin I
_- Transducin
i
a
FIGURE 2. Sodium dodecyl sulfate (SDS) gel electrophoresis of
bovine rod outer segment (ROS) proteins. Dark adapted, purified ROS (lane a) were lysed in hypotonic buffer, and the
membrane fraction (lane b) was separated from the soluble
fraction (lane c) by high-speed centrifugation. Fractions were
run on a 9% SDS polyacrylamide gel and stained with Coomassie blue. Bands corresponding to rhodopsin, phosphodiesterase sub-units (PDEa,|3), rhodopsin kinase, arrestin, and transducin (a- and j3-subunits) are indicated.19
teins.27 Exposed galactose residues are then specifically labeled with the plant lectin ricin conjugated to gold particles.
The small uniform gold particles (~10 nm diameter) are physically dense and therefore, increase the density of the plasma
membrane to which they bind. They are also electron dense
and serve as excellent visual markers for electron microscopy
(EM). The gold-labeled ROS are lysed in hypotonic buffer to
produce fragments consisting of unlabeled disks radiating from
the cytoplasmic surface of the gold-labeled plasma membrane.
Trypsin is then used to detach the disks from the plasma
membrane, and sucrose gradient centrifugation is used to separate the unlabeled disks from the labeled plasma membrane.
The fractionation procedure, as elegantly carried out by Laurie
Molday, is documented in the electron micrographs in Figures
3A, 3B, 3C, and 3D.
SDS gel electrophoresis and functional assays indicate that
disk and plasma membranes differ in protein composition1928
(see Fig. 5A), Although both membranes contain rhodopsin as
the major membrane protein, many proteins present in the
plasma membrane are absent in disk membranes (Table 2).
Furthermore, the plasma membrane exhibits cGMP-gated channel activity29 and Na/Ca-K exchange activity,30 whereas disk
membranes do not. Differences in lipid composition between
the disk and plasma membrane have also been reported.31-32
MONOCLONAL ANTIBODIES TO ROS
MEMBRANE PROTEINS
To begin to identify, characterize, and localize photoreceptor
membrane proteins, highly sensitive and specific reagents are
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required. Monoclonal antibodies are particularly valuable since
they typically bind to a well-defined site or epitope consisting
of four to eight amino acids, can be produced in unlimited
quantities, and have broad applications with highly sensitive
immunochemical techniques. For example, monoclonal antibodies can be used to (1) detect and identify proteins by
western blot analysis; (2) isolate proteins and peptides by
immunoaffinity chromatography; (3) map the cellular and subcellular distribution of proteins by light and EM; (4) screen
cDNA expression libraries for cloning and sequence analysis;
(5) analyze membrane protein topology; (6) resolve proteinprotein interactions; (7) identify specific cells for developmental and transplantation studies; and (8) detect protein expression in gene therapy protocols.
Monoclonal Antibodies to Rhodopsin
Our studies on the generation and characterization of monoclonal antibodies began in the early 1980s. Using ROS as an
immunogen, we were able to generate a panel of antibodies to
rhodopsin and other ROS membrane proteins.33 Our initial
studies focused on the analysis of anti-rhodopsin antibodies.33"39 In collaboration with Paul Hargrave at the University
of Florida and Bob Hodges at the University of Alberta, we
were able to precisely map the epitopes for a number of
anti-rhodopsin antibodies.37"39 Four antibodies have been
widely used in studies carried out in my laboratory and in other
research laboratories throughout the world. These include the
rho4D2 and rho3A6 antibodies generated by David Hicks, a
former postdoctoral fellow in my laboratory,21 and rholD4 and
rho4B4 antibodies produced by Don MacKenzie, a former
graduate student.35"37
The rho4D2 antibody, directed against a highly conserved
epitope near the N terminus of rhodopsin, cross-reacts with
rhodopsin from a broad range of vertebrate species.21'33'39 It
has been widely used to study the cellular and subcellular
distribution of rhodopsin in rod cells and as a rod cell-specific
marker in retinal developmental and transplantation studi e s 2],33,<fo,<n ^ n e rnolrj>4 antibody reacts with the conserved
eight amino acid C terminus of rhodopsin.57'38 This antibody
has been routinely used to purify rhodopsin from ROS and
heterologous cells for structure-function analysis.'""43 The
rho4B4 antibody specifically binds to an internal loop connecting helices 4 and 5 and has been used to confirm the organization of rhodopsin in disk membranes.33'34'30" Finally, the
rho3A6 antibody shows more limited cross-reactivity, binding
to bovine and human rhodopsin, but not mouse rhodopsin.36'38 This property has been used effectively to monitor the
expression and distribution of human rhodopsin in transgenic
mice that serve as models for retinitis pigmentosa and other
retinal
Monoclonal Antibodies to Other ROS Proteins
As part of our studies, we have generated a library of monoclonal antibodies against other ROS disk and plasma membrane
proteins including peripherin/rrfs, rom-1, the cGMP-gated
channel, guanylate cyclase, the Na/Ca-K exchanger, glyceraldehyde-3-phosphate dehydrogenase, ABCR/RIM protein, and
others (see Table 2). Applications of some of these antibodies
in the identification, characterization, and localization of specific photoreceptor proteins are described below.
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TABLE
Molday
IOVS, December 1998, Vol. 39, No. 13
1. Partial List of Soluble and Membrane-Associated ROS Proteins
Protein
Approximate Mr
Phosphodiesterase
Function
Phototransduction
a-subunit
jS-subunit
y-subunit
8-subunit
Rhodopsin kinase
Arrestin
p44 arrestin variant
Transducin
a-subunit
j3-subunit
y-subunit
RGS protein
Phosducin
G lyceraldeliyde-3-phosphate dehydrogenase
Other glycolytic enzymes
Creatine kinase
Phosphatase 2A
Guanylate kinase
Nucleotide diphosphokinase
Hexose monophosphate enzymes
88,000
84,000
11,000
17,000
68,000
48,000
44,000
f-GARP
t-GARP
Recoverin
65,000
32,000
26,000
Calmodulin
20,000
GCAP1 and GCAP2
24,000
Pyrophosphatase
Actin
36,000
45,000
Tubulin
Myosin
Protein kinase C
Others
205,000
85,000
39,000
37,000
8,000
57,000
33,000
38,000
43,000
38,000
54,000
Rhodopsin phosphorylation
Rhodopsin inactivation
Rhodopsin inactivation
Phototransduction
Activator of transducin GTPase activity
Interaction with transducin subunits
Glucose metabolism and energy production
Glucose metabolism and energy production
ATP regeneration
Regulation
Nucleotide metabolism
Nucleotide metabolism
Glucose metabolism and regeneration of
NADPH
Unknown
Unknown
Calcium-dependent modulator of rhodopsin
kinase
Calcium-dependent modulator of the cGMPgated channel
Calcium-dependent modulator of guanylate
cyclase
Pyrophosphate hydrolysis
Cytoskeletal protein localized at the base of
the outer segment
Component of microtubules
Connecting cilium
Protein regulation
Mr, molecular weight; GARP, glutamic acid-rich protein.
PHOTOTRANSDUCTION AND THE CYCLIC GMPGATED CHANNEL OF ROD PHOTORECEPTORS
The cGMP-gated channel plays a central role in phototransduction by controlling the flow of Na+ and Ca2+ ions into the
outer segment in response to light induced changes in intracellular cGMP concentrations (Fig. 4). 46 -' 18 Briefly, in darkadapted rod cells, Na+ and Ca2+ ions flow into the ROS
through cGMP-gated channels maintained in their open state
by a relatively high concentration of cGMP. K+ ions flow out of
the inner segment through voltage-gated K+ channels, thereby
completing a dark current loop. Na+ and K+ gradients are
maintained by an active Na,K ATPase localized in the plasma
membrane of the inner segment, and the Ca2+ concentration
in the outer segment is kept at ~400 nM by the balanced efflux
of Ca2+ through the Na/Ca-K exchanger in the ROS plasma
membrane. Under these conditions, the rod cell is in its depolarized state (—40 mV), and there is a constant release of the
glutamate transmitter from the synaptic region of the photoreceptor cell.49
Photoexcitation is initiated when a photon converts the
ll-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 transducin 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 in
intracellular cGMP causes the cGMP-gated channels to close
and the rod cell to become hyperpolarized. Under this condition, glutamate release at the synaptic region of the rod cell is
inhibited. The closure of cGMP-gated channels also causes
Ca2+ levels in the outer segment to decrease since the Na/Ca-K
exchanger continues to extrude Ca2+ from the outer segment.
After photoexcitation, the photoreceptor cell returns to
its dark state by the shutdown of the visual cascade system and
resynthesis of cGMP.51'54 Rhodopsin is inactivated by ATPdependent phosphorylation at its C terminus and the subsequent binding of arrestin. Transducin and PDE are inactivated
by the hydrolysis of GTP to GDP on Ta by its intrinsic GTPase
activity. Guanylate cyclase, the enzyme responsible for the
The Friedenwald Lecture
IOVS, December 1998, Vol. 39, No. 13
TABLE
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2. Major ROS Plasma Membrane and Disk Membrane Proteins
Protein
Plasma membrane proteins
Rhodopsin
cGMP-channel
a-subunit
Approximate
Quantity (%)
Mr
36,000 (38,000)
60
63,000 (79,600)
7
Function
Phototransduction
Monoclonal
Antibodies
RholD4
Rho4D2
Phototransduction
PMclDl
PMc6E7
/3-subunit
240,000(155,000)
7
Ca 2+ homeostatis
50,000
4
Glucose transport
36,000 (38,000)
85
Phototransduction
Guanylate cyclase (RetGCl)
112,000
1
Phototransduction
Peripherin/rds
35,000 (39,000)
4
Outer segment structure
Rom-1
37,000 (37,000)
Na/Ca-K exchanger
Glucose transporter
230,000 (130,000)
Others
Disk Membrane Proteins
Rhodopsin
ABCR/RIM
Retinol dehydrogenaset
Others
220,000 257,000
33,500
Outer segment structure
3
Transporter?
Retinal reduction
PMs5El 1
PMblC9
PMe2D9
PMelB3
Polyclonal
antibody
RholD4
Rho4D2
GC16G7
GC12H6
Per2B6
Per3B6
RomlD5
RomlC5
Rim3F4
mAbA..
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 in
intracellular Ca 2+ after photoexcitation, a process that is mediated by GCAP proteins.55"57 As the cGMP concentration
increases, the cGMP-gated channels reopen, and the photoreceptor cell is returned to its depolarized state. A corresponding
increase in intracellular Ca 2+ restores guanylate cyclase to its
basal level of activity. Calcium feedback is also thought to
facilitate photorecovery through the regulation of rhodopsin
phosphorylation by recoverin58 and modulation of the channel
sensitivity for cGMP by calmodulin.59
Since the initial patch clamp studies of Fesenko et al.,60
many laboratories have studied the physiological properties of
the cGMP-gated channel of rod photoreceptors (for review, see
Refs. 46 and 47). Generally, the rod channel is cooperatively
activated by cGMP with a K l/2 of 10 to 50 /xM and a Hill
coefficient of 1.7 to 35 and permeable to a wide range of
monovalent and divalent cations including Na + , K+, Li+, Cs + ,
Rb"1", Ca 2+ , Mg 2+ , Mn 2+ , and Ba2+. The pharmacological
agent, l-cis diltiazem is an effective inhibitor of channel activity
and divalent cations, such as Ca 2+ and Mg2+ are known to
significantly decrease the conductance of the channel.
Molecular Characterization and Subcellular
Distribution of the cGMP-gated Channel
Molecular characterization of the rod cGMP-gated channel began in the late 1980s when Neil Cook and Benjamin Kaupp
isolated a 63-kDa protein from bovine ROS that exhibited
cGMP-dependent channel activity when reconstituted into
lipid 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 reported
to be the rod channel. At the same time, Delyth Reid, a
postdoctoral fellow in my laboratory, had generated a monoclonal antibody, designated PMclDl, which reacted with a
63-kDa protein in ROS plasma membranes. In collaboration
with Benjamin Kaupp's group in Germany, we were able to
show that this antibody recognizes the 63-kDa channel protein
in both ROS and purified channel preparations.66 Furthermore,
the PMclDl antibody immobilized on Sepharose quantitatively
immunoprecipitated the 63-kDa protein and cGMP-dependent
channel activity.66'67 These studies provided compelling evidence that the 63-kDa protein, and not the other candidate
proteins, is a component of the rod cGMP-gated channel. Using
isolated disk and plasma membrane preparations, we further
showed by western blot analysis and activity measurements
that the 63-kDa channel protein is present in the ROS plasma
membrane, but absent in disks (Fig. 5A, 5B).66 Immunohistochemical techniques have confirmed that the cGMP-gated
channel and the Na/Ca-K exchanger are targeted to the ROS
plasma membrane and are not present in detectable quantities
in 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 a
subunit of the rod channel and, in addition, yielded information about its primary structure. The cloned protein having a
molecular mass of —79.6 kDa is considerably larger than the
63-kDa channel protein of ROS. This is attributed to the absence of the N-terminal 92 amino acids in die ROS channel,
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IOVS, December 1998, Vol. 39, No. 13
V
CV i DV
FIGURE 3.
Electron micrographs showing the separation of rod outer segment (ROS) disk and
plasma membranes by the ricin gold affinity density perturbation method.2** (A) Isolated
neuraminidase-treated ROS densely labeled with ricin gold particles (10 nm diameter). (B)
Membrane fragments obtained after hypotonic lysis of ROS. Unlabeled disks are seen radiating
from the cytoplasmic surface of the gold-labeled plasma membrane. (C) Disk membrane
fraction obtained after sucrose density centrifugation. (D) Ricin gold-labeled plasma membrane fraction obtained after sucrose density centrifugation.
presumably due to photoreceptor-specific posttranslational
proteolysis.6* When expressed in Xenopus oocytes or HEK
293 cells, the protein assembles into a functional channel that
is activated by cGMP.7172 These results led to the early view
that the rod cGMP-gated channel is an oligomeric complex
consisting of identical a-subunits.
The first indication that the rod channel contains a second
subunit was obtained from the cloning studies of King Wai Yau
and coworkers.73 They isolated a cDNA encoding a 102-kDa
polypeptide that is 30% identical in sequence to a-subunit and
possesses similar structural features. Although this subunit,
referred to as subunit 2, does not assemble into a functional
channel when expressed by itself, coexpression with the rod
a-subunit produces a functional channel that exhibits physio-
logical properties characteristic of the ROS channel. These
include rapid opening and closing orflickeringbehavior, inhibition by l-cis diltiazem and modulation by Ca-calmodulin.73'74
Questions regarding the true nature of the second subunit
arose, however, since a 102-kDa polypeptide is not observed in
isolated channel preparations from bovine or human ROS.6774
Instead, a larger polypeptide is observed that migrates on SDS
polyacrylamide gels with an apparent molecular mass of 240
kDa (Fig. 6). To begin to characterize this channel-associated
protein, we isolated a number of proteolytic peptides derived
from the 240-kDa protein. The sequence of several peptides
matched sequences present in subunit 2, suggesting this subunit is part of the 240-kDa protein.74 However, some peptide
sequences were not found in subunit 2: but instead were
The Friedenwald Lecture
IOVS, December 1998, Vol. 39, No. 13
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Exchanger
4
RGS
jr
GDPT\ --*-?••—..
Ca 2 *t
Transducin y
Light
^>
N
* Channel
GD
Rhodopsin^
Disk Membrane
Plasma Membrane
FIGURE 4.
Diagram depicting the principal reactions in phototransduction. Light initiates the
isomerization of 11-cis retinal to all-trans retinal resulting in the activated (Meta II) state of
rhodopsin. Meta II rhodopsin catalyzes the exchange of GDP for GTP on transducin and the
dissociation of the a-subunit (Tot) from the j37-subunits (T/3y). To: interacts with phosphodiesterase (PDE) to release the inhibitory constraint on the enzyme. Activated PDE catalyzes the
hydrolysis of cGMP to 5'GMP. The decrease in intracellular cGMP concentration causes the
channel to close and the rod cell to hyperpolarized. Intracellular Ca2+ levels decrease as the
Na/Ca-K exchanger continues to extrude Ca2+ from the outer segment. Photorecovery is
initiated 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 by
rhodopsin kinase (RK) and the subsequent binding of arrestin; (2) hydrolysis of GTP to GDP
on a-subunit of transducin, a reaction activated by RGS protein. PDE also returns to its inactive
state as a result of this reaction; and (3) reassociation of the a-subunit of transducin with its
jBy-subunits to form the inactivated transducin heterotrimer. Low intracellular Ca2+ concentrations lead to (1) the activation of guanylate cyclase, a process that is mediated by the
calcium-binding protein GCAP; and (2) an increase in the sensitivity of the channel to cGMP
as a result of the dissociation of calmodulin from the channel. As cGMP concentration
increases, the channels reopen and the cell is returned to its depolarized state. The increase
in Ca2+ also converts guanylate cyclase to its inactive or basal level of activity. The solid
arrows show the photoexcitation process; dashed arrows show the photorecovery process.
identical with sequences present in a previously cloned retinal
glutamic acid-rich protein called GARP.75'76 The relationship
between subunit 2 and GARP was resolved when, in collaboration with Benjamin Kaupp's laboratory, we were able to
show that the 240-kDa polypeptide represents the full length
j3-subunit of the cGMP-gated channel and contains both subunit 2 and GARP.75
Molecular Structure and Regulation of the cGMPGated Channel
Topological models for the a- and /3-subunits of the rod channel as developed from sequence analysis and immunochemical
labeling studies are shown in Figures 7A and 7B/ 5877 - 80 The
core structural unit consists of six membrane-spanning segments (S1-S6) followed by a cGMP binding domain. A voltage
sensor-like motif comprising the S4 segment and a pore region
of approximately 20 to 30 amino acids located between the S5
and S6 transmembrane segments are also evident,81 A negatively charged glutamate residue in the pore region of the
a-subunit has been shown to be responsible for external diva-
lent cation blockage.82"3 Since the S4 segment, the pore region, and the folding pattern of the cGMP-gated channel subunits are characteristic features of voltage-gated cation
channels, it has been suggested that cyclic nucleotide-gated
channels and voltage-gated channels are members of a superfamily of cation channels that have evolved from a common
primordial 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 800
amino acids contains the core structural unit of the channel
required for activity. The N terminus contains the GARP part,
a region having a high content of glutamic acid and proline
residues. Two shorter spliced variants of GARP, called f-GARP
and t-GARP, are also present in ROS.84'85 The functions of the
various GARP variants are currently under investigation.
The channel is a heterotetrameric complex,86 most likely
consisting of two a- and two |3-subunits (Fig. 7C). The pore
regions of the individual subunits are oriented toward the
central cavity of the channel where they serve as the ion
selectivity filter and gate.87 Recent high-resolution x-ray analy-
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IOVS, December 1998, Vol. 39, No. 13
Coomassie
Blue
PMC1D1
205976845
29-
r
a
b
63 kDa
c
a
b c
B
0,02-
cGMP
Plasma
Membrane
AA
0.01-
Disk
Membrane
50
100
sis of a related K + selective channel from Streptomyces lividans has provided a detailed structure of the pore region of
this channel and insight into how such pore regions function in
the translocation of ions through these channels. 88
As part of our studies on the rod channel, we investigated
factors that regulate the activity of the rod cGMP-gated channel. Yi-Te Hsu, a former graduate student, first showed that
calmodulin modulates the apparent affinity of the channel for
cGMP in a calcium dependent manner. 5 9 8 9 Using both native
and reconstituted channels, he showed that Ca-calmodulin
shifts the dose-response curve to higher cGMP concentrations
(Fig. 8) : an effect that has been reproduced in various experimental systems. 74 ' 7590 He also showed that calmodulin-Sepharose can be used to purify the channel and that the binding site
for calmodulin is localized on the £-subunit. 59 ' 88 More recently, this site has been mapped near the N terminus of the
core structural unit of the j3-subunit.9192
Modulation of the channel by calmodulin operates under
physiological Ca 2+ concentrations 59 ' 89 This has led us to suggest that Ca-calmodulin regulation of the channel may play a
role in facilitating photorecovery after bleaching by increasing
the sensitivity of the channel to cGMP. 5979 The finding that
calmodulin modulates the rod channel has led to the important
discovery by King Wai Yau's group that the olfactory cyclic
nucleotide-gated channel is also strongly modulated by calmodulin, a mechanism that is important in olfactory adaptation.
kDa
205-
- 240 kDa
(p-subunit)
97-
6845FIGURE 5- Localization of die cGMP-gated channel to the rod
outer segment (ROS) plasma membrane. (A) Sodium dodecyl
sulfate polyacrylamide gel stained with Coomassie blue (left) and
the corresponding western blot analysis labeled with monoclonal
antibody PMclDl against the 63-kDa osubunit of the channel.66
Lane a: rod outer segment (ROS) membranes; lane b: isolated
disk membrane fraction; lane c: isolated plasma membrane fraction. (B) cGMP dependent channel activity. Purified disk and
plasma membrane fractions solubilized in CHAPS detergent were
reconstituted into Ca2+-containing lipid vesicles. The release of
Ca 2+ was initiated with 150 fjM cGMP and detected by a change
in absorbance (AA) using Arsenazo in as an indicator. Only the
plasma membrane exhibits channel activity.66 (C) Electron micrograph of bovine ROS membranes labeled with antibodies
against the cGMP-gated channel. ROS membrane fragments were
labeled with the PMc6E7 anti-channel antibody followed by immunogold particles (10 nm diameter). The cytoplasmic surface of
the inside out plasma membrane vesicles (arrowheads) is
densely labeled. The disk membranes radiating from the plasma
membrane vesicles are not labeled.68
63 kDa
(ct-subunit)
29-
FIGURE 6. Immunoaffinity purification of the channel from
rod outer segment (ROS). ROS membranes (lane a) were
solubilized in CHAPS buffer and passed through a PMc6E7Sepharose affinity column. After the column was washed to
remove unbound proteins, the channel (lane b) was eluted
with a peptide corresponding to the epitope for the PMc6E7
monoclonal antibody. The samples were run on a 9% sodium
dodecyl sulfate polyacrylamide gel and stained with Coomassie
blue. The purified channel contains two prominent polypeptides: the 63-kDa a-subunit and the 240-kDa 0-subunit.
The Friedenwald Lecture 2501
IOVS, December 1998, Vol. 39, No. 13
Pore Region
Pore Region
S1 S2 S3 S4S5I S6
\ \
I / /
| I
RinHinn ^ite
Binding Site
6 g o
Garp Part
CHO
(V-Part
f-Garp
t-Garp
Pore Region
Pore Region
COOH
p-Part
a - Subunit
Garp Part
- Subunit
a
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) is
missing in the channel present in rod outer segment (ROS). (B) Linear representation (top) and
topological model (bottom) of the rod j3-subunit. In addition to many structural features found
in the a-subunit, the j3-subunit contains a calmodulin (CaM) binding site and an extended
N-terminal region called the GARP part. Two additional spliced variants of GARP, called f-GARP
and t-GARP, are also present in ROS.84 Unlike the a-subunit, the /3-subunit does not contain an
N-ltnked glycosylation site or a glutamate residue in the pore region. (C) Model for the
cGMP-gated channel complex. Two cc-subunits and two /3-subunits assemble into a tetrameric
complex. The S6 and S5 segments are visualized to line the central cavity of the channel. The
pore regions joining the S5 and S6 segments near the extracellular surface of the membrane
extend toward the center of the cavity where they can function as a gate and ion selectivity
filter. The glutamate residue (0) in the pore region of the a-subunit is responsible for external
divalent cation blockage of the channel. Although the channel is shown with identical subunits
across from each other, it is possible that the two identical subunits may be adjacent.
FIGURE
IOVS, December 1998, Vol. 39, No. 13
amounts of ATP are produced by anaerobic glycolysis to sustain cGMP levels in dark-adapted ROS.102 Glucose is also used
by the hexose monophosphate pathway in ROS to regenerate
NADPH for retinal reduction after the photobleaching of rhodopsin and glutathione reduction for the protection of outer
segments from oxidative stress.102 A phosphocreatine shuttle
system most likely serves as an additional source of energy.
Biochemical pathways involved in the production of energy
and reducing equivalents in ROS are depicted in Figure 10. The
importance of anaerobic glycolysis in retinal function has been
addressed in considerable detail in the physiological and biochemical studies of Barry Winkler and coworkers.104'105
RETINAL DEGENERATIVE DISEASES
60
cGMP Concentration
FIGURE 8. Modulation of the rod cGMP-gated channel by Cacalmodulin. Rod outer segment (ROS) membrane vesicles
loaded with Arsenazo III were placed in a calcium-containing
buffer in the absence (•) or presence (•) of calmodulin, and
calcium uptake was initiated by the addition of cGMP. The
relative initial velocity (VJVm:ix) is plotted against cGMP concentration. The solid line is the sigmoidal binding curve calculated using a Michaelis constant (Km~) of 19 /xM and a Hill
coefficient 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 calmodulin.59
In 1990, Ted Dryja and coworkers first reported that a P23H
mutation in rhodopsin is responsible for a form of autosomal
dominant retinitis pigmentosa (ADRP).106 Since this time,
more than 70 different rhodopsin mutations have been linked
to autosomal dominant and recessive forms of retinitis pigmentosa, and congenital stationary nightblindness (CSNB).107'109
GLYCOLYTIC ENZYMES AND GLUCOSE
METABOLISM IN ROS AND CONE OUTER
SEGMENTS
Phototransduction and related outer segment processes require considerable amounts of energy and reducing equivalents in the form of ATP, GTP, and NADPH. Over the years,
questions arose as to whether these cofactors are generated in
the outer segments or produced in the inner segment and
shuttled to the outer segment. In early studies, glycolytic and
hexose monophosphate enzyme activities were detected in
outer segment preparations,95"98 but the activities were low in
comparison to other retinal cell layers. As a result, it has been
unclear whether these enzyme activities come from endogenous outer segment enzymes or alternatively, from contaminants of the preparations. A report indicating that outer segments contain considerable amounts of creatine kinase also led
to the possibility that energy in the form of ATP can be
transferred from the inner segment to the outer segment by a
phosphocreatine shuttle pathway.99
Our interest in glucose metabolism arose when Shu-Chan
Hsu, a former graduate student, identified an abundant 38-kDa
ROS plasma membrane-associated protein as glyceraldehyde3-phosphate dehydrogenase, a key enzyme in anaerobic glycolysis. 10° She further showed by immunofluorescence, western
blot analysis and enzyme activity measurements that other
glycolytic enzymes and a GLUT-1 glucose transporter are also
present in ROS and cone outer segments.101 Moreover, quantitative measurements indicated that isolated ROS preparations
have the capacity to take up glucose and convert glucose to
lactate by anaerobic glycolysis (Figs. 9A, 9B).101"103 Sufficient
0
50
100
150
Time (seconds)
200
B
to
O
ac 400 CD
200 -
5
10
Time (min)
FIGURE 9-
15
A Uptake of glucose into isolated bovine rod outer
segment (ROS). The uptake of 45 /xM external 3-O-[l4C]methylglucose (3-O-MG), a nonmetabolizable analogue of glucose,
was measured in the absence (•) and presence (O) of 0.05 mM
cytochalasin B, an inhibitor of glucose transport.101 (B) Glycolytic flux in isolated bovine ROS. Time course of lactate production by isolated bovine ROS in the presence (•) and absence (V) of 5 mM glucose.102
The Friedenwald Lecture
IOVS, December 1998, Vol. 39, No. 13
GP
_
GSSG
Glutathione
redox cycle
GSH
OS
PHOTOTRANSDUCTION
IS
FIGURE 10. Diagram showing the role of glycolysis, the hexose monophosphate pathway, and the phosphocreatine shuttle
in ATP, GTP, and NADPH production. In photoreceptor outer
segments (OS), glucose is transported across the plasma membrane by a GLUT-1-type glucose transporter (GT). Glucose is
metabolized to lactate by anaerobic glycolysis resulting in the
production of ATP and GTP for phototransdviction and other
energy requiring processes. Glucose can also be used by the
hexose monophosphate pathway (HMP) for the regeneration
of NADPH. The latter is required for the reduction of all-trans
retinal to all-trans retinol by retinol dehydrogenase (RDH) after
the bleaching of rhodopsin and for glutathione reduction by
glutathione reductase (GR) for protection of the outer segment
against oxidative damage. Additional energy is obtained from
the inner segment (IS). High-energy phosphocreatine (PCr) is
transferred to the outer segment by the PCr and used to
generate ATP. This reaction is catalyzed by a brain-type creatine kinase (Ckb).102
In addition, positional cloning and candidate gene approaches
have led to the identification of large number of genes associated with retinitis pigmentosa and other retinal degenerative
diseases (Table 3).108-109 Many genes code for photoreceptorspecific proteins that play key roles in phototransduction and
outer segment morphogenesis. Our interest in this area has
focused on the peripherin/rds-rom-1 complex and the ABCR/
RIM protein, two disk membrane proteins implicated in multiple retinal degenerative diseases.
PERIPHERIN/RDS—ROM-1 COMPLEX
Molecular Structure and Subcellular Distribution
Peripherin/rds and rom-1 are homologous subunits of an oligomeric membrane protein found in disks. Peripherin/TYfe,
2503
formerly called peripherin, was first detected as a 33- to 35-kDa
protein in bovine disk membranes with monoclonal antibodies110 and subsequently cloned from a retinal expression library
by Greg Connell, a former graduate student in my laboratory. 1 " Several years later, Rod Mclnnes's laboratory in Toronto
cloned rom-1, a protein that is 30% identical in sequence to
peripherin/rds."2 Immunochemical labeling studies and sequence analysis has led to a topological model for these proteins (Fig. II). 1 1 1 "" 4 Characteristic structural features include
four membrane- spanning segments (M1-M4), an extended
carboxyl terminal domain exposed on the cytoplasmic side of
disks and a large intradiskal loop (L3-L4) containing seven
conserved cysteine residues.
Peripherin/rds and rom-1 associate to form a tetrameric
complex as determined by immunoprecipitation studies and
hydrodynamic measurements."2"1'6 Since dimers of peripherin/rds and rom-1 are routinely observed by SDS gel electrophoresis under nonreducing conditions," 0 "" 3 it was initially
thought that a disulfide-linked homodimer of peripherin/rcls
interact noncovalently with a disulfide-linked homodimer of
rom-1 to form a heterotetramer. " 4 " " 6 More recently, however, Chris Loewen, a graduate student in my laboratory, has
shown that the peripherin/rd.s-rom-1 tetramer does not contain
intermolecular disulfide bonds.117 Instead, intermolecular disulfide bonds, mediated by Cys 150 within the large intradiskal
loop,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 subcellular distribution. Preembedding and postembedding immunogold labeling studies have localized peripherin/rds to the rim
region of ROS and cone outer segment disks (Figs. 12A,
1 2 B ) iio,iu,u<f, 119 i mt i a i studies suggested that rom-1 is only
COOH
Cytoplasmic
side
Intradiscal
side
L3-4
FIGURE 11. Topological model for peripherin/rcfe and rom-1
based on sequence analysis and immunochemical studies. Both
proteins are shown to contain four transmembrane segments
(M1-M4), cytoplasmic N and C termini, and a large intradiskal
loop (L3-4). Perpherin/rds contains an N-linked oligosaccharide chain (hexagons') that is not present in rom-1. Stretches of
conserved amino acids and conserved cysteine residues are
indicated.1' 1~113
2504
TABLE
Molday
IOVS, December 1998, Vol. 39, No. 13
3- Proteins Associated with Various Retinal Degenerative Diseases
Protein
Localization
Disease
Rhodopsin
Phosphodiesterase O,/3)
cGMP-gated channel (a)
Guanylate cyclase (Ret GC1)
GCAP1
Arresttn
Rliodopsin kinase
Transducin (a)
Peripherin/r^/s
Rod cells
Rod cells
Rod cells
Cone and rod cells
Cone and rod cells
Rod cells
Rod cells
Rod cells
Rod and cone cells
ADRp'CKS.107 , m
Rom-1
ABCR/RIM
RPGR
CRALBP
CRX (transcriptional factor)
RPE65
TIMP3
XLRS1
Myosin VIIA
Bestrophin
Rod and cone cells
Rod cells
Rod cells
RPE and Miiller cells
Rod and cone cells
RPE cells
Bruch's membrane
Extracellular (photoreceptors)
Photoreceptors/RPE
RPE
Digenic ADRP 137 ' 138
Stargardt's MD, 149 ' 152 ARRP,15'' CRD,' 55 and AMD?153
X-linked RP (RP3) 172 ' 182
ARRP15y
LCA and CRD 1 5 7 1 5 8
LCA and ARRP160'161
Sorby's macular dystrophy 162
X-linked retinoschisis 170
Usher syndrome (USH1)171
Best's MD(VMDd2) 163164
ARRP 168169 and CSNB177
ARRP166
LCA167 and CRD1*0
Cone dystrophy 165
CSNB (Oguchi disease) 173
CSNB (Oguchi disease) 174
CSNB (Nougaret disease) 175
ADRP, 126 - 128 ' 130 ' 131 MD, 1 3 1 1 3 4 1 3 6 pattern
dystrophy, 132133 and digenic ADRP 137 ' 138
ADR]*, autosomal dominant retinitis pigmentosa; ARRP, autosomal recessive retinitis pigmentosa; CSNB, congenital stationary nightblindness;
X-linked RP, X-linked retinitis pigmentosa; MD, macular dystrophy; AMD, age-related macular dystrophy; CRD, cone-rod dystrophy; LCA, Leber's
congenital amaurosis.
present in ROS disks. 112 However, Orson Moritz, a former
graduate student in my laboratory, has clearly shown that
rom-1 is present along the rim region of cone and rod disk
membranes as shown in Figure 12C. 113
B
Role of Peripherin/rrfs in Outer Segment
Morphogenesis and Retinal Degeneration in the
RDS Mouse
The importance of peripherin/r^ in outer segment morphogenesis originated from the cellular and molecular analysis of
the retinal degeneration slow or rds mouse. Sanyal and coworkers 120 ' 121 showed that mice homozygous for the rds mutation fail to develop outer segments and the photoreceptor
cells undergo slow degeneration such that few cells remain 1
year after birth. Heterozygous rds mice exhibit short, highly
disorganized outer segments that often appear as whorls of
membrane. 122 In the late 1980s, Grabriel Travis and colleagues 123 used subtractive hybridization techniques to identify a mutation in a photoreceptor cell-specific gene that is
responsible for the rds phenotype. We subsequently showed
that this gene codes for peripherin/refe.124 Finally, the research
groups of Gabriel Travis and Dean Bok have reported that
introduction of the normal peripherin/rds gene into a homozy-
ROS
COS
CIS
FIGURE 12. Localization of peripherin/rrfs and rom-1 to the
rim region of rod outer segment (ROS) and cone outer segment
(COS) disks. (A, B) Electron micrographs of an isolated bovine
ROS disk and a group of disks labeled with Per 3B6 antiperiphcrin/rds monoclonal antibody and immunogold particles (10
nra diameter). The gold particles are localized to the rim region
of the disks. (C) Electron micrograph of an ROS, a COS, and a
cone inner segment (CIS) labeled for rom-1. Bovine retina was
embedded in LR White resin. Sections were then labeled with
the anti-rom-1 antibody (Rom L4) and immunogold particles.
Gold particles are distributed along the peripheral region of
both the ROS and COS and the incisures of ROS (arrow-
head)^
The Friedenwald Lecture
IOVS, December 1998, Vol. 39, No. 13
330.
2505
346
Cytoplasmic
side
OOH
Trp25FS
Intradiskal
side
Lysl 53Arg
Lysl 53Del
Pro2l6Leu
Pro2l6Ser
*Cys2l4Ser
Ser212Gly
Pro210Arg
Pro210Ser
Lysl93Del
13- Location of peripherin/rds mutations associated with retinal degenerative diseases. Unframed mutations have been linked to monogenic autosomal dominant retinitis
pigmentosa (ADRP). Mutations framed in black have been implicated macular and pattern
dystrophies. The mutation framed in gray is linked to digenic ADRP. Insert/RDS is the site of
an insertion mutation in the rds mouse; FS indicates a frameshift resulting in a change in
reading frame and premature stop.
FIGURE
gous rds mouse results in outer segment formation and suppression of photoreceptor degeneration.125 Taken together,
these studies indicate that peripherin/rds is plays an essential
role in ROS and cone outer segment morphogenesis, and the
absence or reduced amounts of this membrane protein result
in photoreceptor degeneration.
Role of Peripherin/rds in ADRP and Macular
Degeneration
The finding that a mutation in the peripherin/rds gene causes
photoreceptor degeneration in the rds mouse prompted many
laboratories to determine whether mutations in the human
gene are responsible for inherited retinal diseases. This has
proved to be the case and to date more than 30 mutations in
the peripherin/rds gene have been implicated in various retinal degenerative diseases (Fig. 13).126"130 Interestingly,
whereas some peripherin/rds mutations cause ADRP, others
result in macular degeneration and pattern dystrophies that go
under such names as, butterfly-shaped pigment dystrophy,
fundus flavimaculatus, cone-rod dystrophy and Bull's Eye
maculopathy. l31~136 Similar efforts to link mutations in rom-1
with retinal degenerative diseases have been less successful.
However, several mutations in the rom-1 gene have been
linked to a novel form of digenic ADRP.137"138
To begin to understand the mechanism by which mutations in peripherin/rds cause specific disease phenotypes,
Andy Goldberg, a former postdoctoral fellow in my laboratory, developed a heterologous cell expression system to
study the molecular properties and subunit interactions of
peripherin/rds and rom-1. 115 He showed that coexpression
of peripherin/rds and rom-1 results in a heterotetrameric
complex having properties similar to those of the complex
derived from ROS. However, many mutations in the large
intradiskal segment, including the C214S peripherin/rds mutant-linked ADRP,130 result in abnormal protein folding and
the inability of peripherin/rds to associate with rom-1." 8
From these studies, we have concluded that segments of the
large intradiskal loop are important in subunit-subunit interactions, a property important in normal outer segment
formation.
We have also examined the R172W peripherin/rds mutant
linked to a mild form of macular degeneration.131136 Interestingly, this mutant folds normally and assembles with rom-1 into
a nativelike tetrameric complex.139 Apparently, subtle differ-
2506
Molday
ences in the structure and/or stability of this complex result in
preferential cone degeneration.136
Recently, a novel digenic form of ADRP linked to a
L185P mutation in peripherin/nfc and a null mutation in
rom-1 has been described in several families. 137138 Individuals who coinherit both mutations (double heterozygotes)
exhibit an ADRP disease phenotype, whereas family members who inherit only one of these mutations (single heterozygotes) are essentially normal. We postulated that this
disease pattern might arise from defective peripherin/rdsrom-1 subunit interactions. To investigate the role of subunit
assembly in this form of digenic RP, Andy Goldberg analyzed
the hydrodynamic properties of the complexes formed
when either wild-type peripherin/rds or the L185P mutant is
expressed in COS-1 cells in the presence and absence of
rom-1. l39 The results of this study are diagramed in Figure
14. Coexpression of the L185P peripherin/rds mutant with
rom-1 (simulating an individual who inherits only the peripherin/n/s mutation) results in a heterotetrameric complex similar to the wild-type complex. Wild-type peripherin/
rds in the absence of rom-1 (simulating an individual who
inherits a rom-1 null mutation) self-assembles into a homotetrameric complex. In contrast, L185P peripherm/rds in
the absence of rom-1 (simulating individuals who inherit
both mutations) fails to self-assemble into a tetramer, but
instead remains in a dissociated state. From these studies we
have concluded that peripherin/rds-containing tetramers
are crucial in outer segment formation and stability. Reduced levels of this complex can lead to abnormal and
unstable outer segments and progressive photoreceptor degeneration.139
Implicit in this model is the hypothesis that peripherin/rc/s
homotetramers can effectively substitute for peripherin/rdsrom-1 heterotetramers and promote outer segment disk morphogenesis. Recently, we have had the opportunity to test this
prediction in a Rom-1 knockout mouse produced by Geoff
Clark and Rod Mclnnes.l4° Biochemical and ultrastructural
analyses of these mice, indeed, indicate that peripherin/rds
self-assembles into a homotetramer in the absence of rom-1,
and this complex supports ROS and cone outer segment formation (Goldberg A, Molday L, Molday R, Clark G, Mclnnes R,
unpublished results, 1997). Comparative analysis of the rom-1
knockout mouse and the rds mouse leads to the conclusion
that peripherin//Y/s is the dominant subunit required for disk
morphogenesis. Rom-1 is relegated to a more minor role, perhaps enhancing the stability of the outer segment and/or fine
tuning the structure of disks.
THE ABCR/RIM PROTEIN AND STARGARDT'S
MACULAR DEGENERATION
More recent studies in my laboratory have focused on the
identification and characterization of the abundant high-molecular-weight rim protein first identified by David Papermaster
and colleagues in frog photoreceptors"""' 2 and subsequently
detected in mammalian ROS.1''314'1 Michelle Illing, as part of
her graduate research in my laboratory, purified and cloned the
bovine 220-kDa rim protein and showed that this protein is a
member of the superfamily of ABC (ATP binding cassette)
transporters.145 This extensive family of proteins include the
cysticfibrosistransmembrane regulator (CFTR) linked to cystic
IOVS, December 1998, Vol. 39, No. 13
Molecular Complex
B
D
Phenotype
WT Per/rds
WT Rom-1
Normal
Ll 85P Per/rds
WT Rom-1
"Normal"
Wt Per/rds
Null Rom-1
"Normal"
L185PPer
Null Rom-1
Digenic
ADRP
14. Subunit assembly model for digenic autosomal
dominant retinitis pigmentosa (ADRP) linked to a L185P mutation in peripherin/rds and a null allele in Rom-1. Left: molecular complexes of peripherin/rds (dark) and rom-1 (light)
determined by velocity sedimentation measurements.l39 Right:
corresponding phenotypes for individuals who inherit one or
both mutations. (A) Wild-type (WT) peripherinAv5fo-rom-l
complex characteristic of normal individuals. (B) L185P peripherin/r^5-rom-l complex predicted to exist in individuals
who inherit only the L185P mutation. This tetrameric complexes is suggested to support outer segment formation resulting in a borderline "Normal" phenotype. (C) Peripherin/rrfs
homotetrameric complex predicted to exist in individuals who
inherit a null mutation in rom-1. This complex can take the
place of the peripherin/rc/s-rom-1 heterotetramer and support
outer segment morphogenesis and structure. A borderline
"Normal" phenotype can result from a small net reduction in
the amount of peripherin/rcfc containing tetramers. (D) Dissociated L185P peripherin/r<3?s mutant in the absence of rom-1.
This species is not expected to support outer segment morphogenesis or structure. A significant reduction in the level of
peripherin/rc/.s containing tetramer will result in disorganized,
unstable outer segments, a condition which underlies photoreceptor degeneration and a ADRP phenotype.
FIGURE
fibrosis, P-glycoprotein involved in multidrug resistance in cancer, TAP1 and TAP2 proteins that serve as peptide transporters
in lymphocytes, prokaryotic permeases, and others.146 Like
CFTR and P-glycoprotein, the rod ABC protein consists of two
structurally related halves, each of which contains a multiple
membrane-spanning domain followed by a cytoplasmic ATP
binding cassette. On the basis of sequence analysis and biochemical studies, we have developed a working model for the
topological organization of this glycoprotein in disk membranes as shown in Figure 15.
The Friedenwald Lecture
10VS, December 1998, Vol. 39, No. 13
2507
N1614FS
G818E
Del WAIC
INTRADISKAL
SPACE
R2106C
CYTOPLASMIC
SPACE
L2027F I
R2038W
\R2077W
V2050L
E1087K
L962FS
V1072A
N965S
A1028V
S1071FS
E1036K
FIGURE 15- Topological model for ABCR/RIM based on sequence analysis, location of N-linked
oligosaccharides, and analogy with other ABC transporters. The model shows 12 transmembrane segments, 2 ATP binding cassettes (ABC) on the cytoplasmic side of the membrane, and
2 N-linked oligosaccharide chains (hexagons') on the intradiskal side. 145 Several mutations
linked to Stargardt's macular dystrophy are indicated. 149152
The cellular and subcellular distribution of this ABC protein has been investigated using both immunochemical and
molecular biology techniques. The protein is abundantly expressed in the outer segments of rod photoreceptors, but is
absent in cone cells. 1 4 5 1 4 7 ' 1 '' 8 This is in agreement with in situ
hybridization and northern blot analysis, indicating that mRNA
expression is restricted to rod cells. 149 At a higher resolution,
postembedding, immunogold labeling studies carried out by
Laurie Molday have shown that the bovine rod ABC transporter, like its frog counterpart, is localized to the rim region of
rod disk membranes (Fig. l 6 ) . 1 4 1 1 4 5
During the course of our studies, Allkimets and coworkers
elegantly showed that mutations in the ABCR gene coding for
a retinal rod-specific ABC protein are responsible for Stargardt's disease, an autosomal recessive macular dystrophy with
a juvenile onset. 149 This disease, characterized by progressive
loss in central vision, bilateral atrophy of the macular region of
the retina and RPE layer, and the appearance of orange-yellow
flecks, accounts for up to 7% of the human retinal degenerative
diseases. 150151 Analysis of the amino acid sequences of human,
bovine, and mouse proteins indicate that the ABCR protein and
the rim protein are one in the same. 1 4 5 ' 1 4 7 ' 1 4 8 ' 1 5 2 The location
of several reported Stargardt's mutations l 4 y 1 5 2 in context to
our working model of rod ABC protein, now referred to as the
ABCR/RIM protein, is shown in Figure 15.
More recent studies suggest that mutations in the ABCR
gene can also cause other disease phenotypes. Allkimets et
al. 153 have reported that some mutations in the ABCR gene are
associated age-related macular degeneration, a result that has
generated considerable controversy. Homozygous and compound heterozygous mutations in the ABCR gene resulting in
null alleles have also been implicated in autosomal recessive RP
and cone-rod dystrophy. 154 ' 155
The function of the ABCR/RIM protein is not currently
known. Other members of this superfamily are known to
mediate the active transport of a wide range of compounds
including drugs, metabolites, peptides, and lipids. l46 A related
protein, ABC1, has also been implicated in the engulfment of
cell 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 the
rim and incisures of rod outer segment (ROS) disks. Electron
microgniphs of bovine rod photoreceptor cells embedded in
LR White resin, sectioned, and labeled with an anti-ABCR/RIM
antibody and immunogold particles. Labeling is restricted to
the peripheral region and incisures (arrowhead) of the ROS, a
pattern that is characteristic of disk rim labeling. The rod inner
segment (RIS) is not labeled. 145
2508
Molday
IOVS, December 1998, Vol. 39, No. 13
Retina) Derivative
Peptide
Antioxidant
Phospholipid
Other
A DP + Pi
ABCR/RIM
Peripherin/rds-Rom-1
Complex
FIGURE 17. Diagram showing possible transport functions of the ABCR/RIM protein. Retinal
derivatives, peptides, antioxidants, phospholipids, or other agents may be actively transported
into or out of the disk membrane or translocated from one side of the disk lipid bilayer to the
other using ATP hydrolysis as a source of energy. Pi, inorganic phosphate.
Glutathtone Cycle
Hexose
Moitophosphate
Pathway
Retinal Reduclioi
Glucose
Transporter
Glucose
Rhodopsm
Kmase
CSMB
Arnwtin
CSNB
cGMP-gated
Channel
Na/Ca-K
Exchanger
4Na
dopsin
Guanylate
Cyclase
LCAI
•
"
Disk
Lamellar
ADRP
MD
ABCR/RIM
XLRS1
Slgdl MD
O
'
Disk
Rim
Plasma
Membrane
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 retinoschists 1; XLRP3, X-linked retinitis pigmentosa 3.
The Friedenwald Lecture
JOVS, December 1998, Vol. 39, No. 13
is involved in the transport of retinal derivatives, phospholipids, peptides or other endogenous substrates across the disk
membrane CFig- 17)- Molecular and biochemical approaches
are now being used to define in more detail the structure and
function of this unique disk protein and to understand how
specific mutations cause various disease phenotypes.
SUMMARY AND FUTURE DIRECTIONS
Over the past two decades, remarkable progress has been
made on the cellular and molecular characterization of ROS
and cone outer segments. Many membrane and soluble proteins have been identified and localized within the outer segment, and their structural and functional properties have been
studied in considerable detail. This has led to a comprehensive
understanding of phototransduction and supporting metabolic
pathways and a glimpse into mechanisms underlying outer
segment structure, morphogenesis, and renewal. Recent studies have also indicated that mutations in many photoreceptor
outer segment-specific proteins cause a multitude of inherited
retinal degenerative diseases (Fig. 18). Molecular studies are
now beginning to provide insight into how specific mutations
in these proteins lead to various retinal diseases.
Although we have learned a great deal about the molecular composition and function of ROS and cone outer segments,
much remains to be learned. The staicture and function of
many proteins associated with retinal degenerative diseases,
such as the ABCR/RJM associated with Stargardt's disease,
XLRS1 implicated in X-linked juvenile retinoschisis, and RPGR
associated with X-linked RP3 remain to be determined. Regulatory mechanisms underlying phototransduction need to be
understood in a more detailed, quantitative manner. Molecular
mechanisms and protein-protein interactions that form and
stabilize the unique ROS and cone outer segment structure and
target proteins to the outer segment and more specifically to
the outer segment disk or plasma membrane have to be understood at a molecular level.
Although the future challenges are numerous, one only
can be optimistic that the rapid advances in basic and clinical
research will lead to a complete understanding of photoreceptor structure and function and detailed insight into the molecular basis for retinal diseases. From this knowledge, it should
be 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 and
present research colleagues who have contributed greatly to the research accomplishments embodied in this award and extend special
thanks to Laurie Molday who carried out many of the electron microscopic and biochemical studies described in this article. Other postdoctoral fellows, graduate students, and research assistants who have
made significant contributions include Jinhi Ahn, Steve Clark, Carole
Colville, Greg Connell, Andrea Dose, Andrew Goldberg, David Hicks,
Theresa Hii, Shu-Chan Hsu, Yi-Te Hsu, Michelle Tiling, Tom Kim, Dale
Laird, Chris Loewen, Don MacKenzie, Orson Moritz, Igor Nassonkin,
Delyth Reid, and Simon Wong. I also thank my friends and collaborators, King Wai Yau, Benjamin Kaupp, Barry Winkler and Bernhard
Weber, and other prominent vision investigators with whom I have
had an opportunity to collaborate and discuss research of mutual
interest. These include Gobind Khorana, David Papermaster, Dan
Oprian, Paul Hargrave, Ted Dryja, David Williams, Rod Mclnnes, Neal
2509
Cook, Heinz Wassle, Kris Palczewski, Karl Koch, Paul Bauer, Steve
Fisher, and Jeremy Nathans. Finally, I gratefully acknowledge the longstanding grant support from the National Eye Institute, IIP Foundation
of Canada, and the Medical Research Council.
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The Friedenwald Lecture
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