PHYSIOLOGICAL REVIEWS
Vol. 81, No. 4, October 2001
Printed in U.S.A.
Rhodopsin: Structural Basis of Molecular Physiology
SANTOSH T. MENON, MAY HAN, AND THOMAS P. SAKMAR
Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry,
The Rockefeller University, New York, New York
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I. Introduction to Rhodopsin: a Prototypical G Protein-Coupled Receptor
II. Molecular Structure of Rhodopsin
A. Overview of rhodopsin structure
B. Extracellular surface domain of rhodopsin
C. Membrane-embedded domain of rhodopsin
D. Chromophore-binding pocket
E. Cytoplasmic surface domain of rhodopsin
III. Mechanism of the Opsin Shift and Spectral Tuning in Visual Pigments
IV. Molecular Mechanism of Receptor Activation
V. The Rhodopsin Photocycle and the Chromophore Regeneration Pathway
VI. Interaction of R* With Molecules Involved in Visual Transduction
VII. Rhodopsin Mutations as a Cause of Human Disease
VIII. Conclusion
Menon, Santosh T., May Han, and Thomas P. Sakmar. Rhodopsin: Structural Basis of Molecular Physiology.
Physiol Rev 81: 1659 –1688, 2001.—The crystal structure of rod cell visual pigment rhodopsin was recently solved at
2.8-Å resolution. A critical evaluation of a decade of structure-function studies is now possible. It is also possible to
begin to explain the structural basis for several unique physiological properties of the vertebrate visual system,
including extremely low dark noise levels as well as high gain and color detection. The ligand-binding pocket of
rhodopsin is remarkably compact, and several apparent chromophore-protein interactions were not predicted from
extensive mutagenesis or spectroscopic studies. The transmembrane helices are interrupted or kinked at multiple
sites. An extensive network of interhelical interactions stabilizes the ground state of the receptor. The helix
movement model of receptor activation, which might apply to all G protein-coupled receptors (GPCRs) of the
rhodopsin family, is supported by several structural elements that suggest how light-induced conformational
changes in the ligand-binding pocket are transmitted to the cytoplasmic surface. The cytoplasmic domain of the
receptor is remarkable for a carboxy-terminal helical domain extending from the seventh transmembrane segment
parallel to the bilayer surface. Thus the cytoplasmic surface appears to be approximately the right size to bind to the
transducin heterotrimer in a one-to-one complex. Future high-resolution structural studies of rhodopsin and other
GPCRs will form a basis to elucidate the detailed molecular mechanism of GPCR-mediated signal transduction.
I. INTRODUCTION TO RHODOPSIN:
A PROTOTYPICAL G
PROTEIN-COUPLED RECEPTOR
Rhodopsin (Rho) is a highly specialized G proteincoupled receptor (GPCR) that detects photons in the rod
photoreceptor cell. Within the superfamily of GPCRs that
couple to heterotrimeric G proteins, Rho defines the socalled class A GPCRs, which share primary structural
homology (221, 232). Bovine Rho has been the most extensively studied GPCR. A large amount of pigment (0.5–
1.0 mg) can be obtained from a single bovine retina by
sucrose density gradient centrifugation preparation of the
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rod outer segment disk membranes (199). The pigment is
stable enough in the dark that it can be further purified by
various chromatographic procedures, and it remains stable in solubilized form in a variety of detergents (187).
Bovine Rho was the first GPCR to be sequenced by amino
acid sequencing (93, 193), the first to be cloned (174, 175),
the first to be crystallized (186), and the first to yield a
crystal structure (194).
Visual pigments share a number of structural features
(222). Like all GPCRs, they consist of seven transmembrane
segments (H1 to H7). A Lys residue that acts as the linkage
site for the chromophore is conserved within H7 in all
pigments, and a carboxylic acid residue that serves as the
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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features not found in other GPCRs. In particular, visual
pigments are made up of opsin apoprotein plus chromophore. The chromophore is not a ligand in the classical
sense, since it is linked covalently via a protonated Schiff
base bond to a specific Lys residue in the membraneembedded domain of the protein (Fig. 1). The chromophore in all Rhos is derived from the aldehyde of
vitamin A1, 11-cis-retinal (RET). Some fishes, amphibians,
reptiles, and aquatic mammals may also employ the aldehyde of vitamin A2, 11-cis-3,4-didehydroretinal, which
contains an additional carbon-carbon double bond. All
pigments with a vitamin A2-derived chromophore are
called porphyropsins. An important structural feature of
the RET chromophore in Rho, in addition to its Schiff
base linkage, is its extended polyene structure, which
accounts for its visible absorption properties and allows
for resonance structures (206).
FIG. 1. A secondary structure diagram of bovine Rho. Amino acid residues are depicted in single-letter code. The
amino-terminal tail and extracellular domain is toward the top, and the carboxy-terminal tail and cytoplasmic domain is
toward the bottom. Transmembrane ␣-helical segments (H1 to H7) and the cationic amphipathic helix H8 are shown in
colored cylinders. An essential disulfide bond links Cys-110 and Cys-187. Cys-322 and Cys-323 are palmitoylated. Inset:
structure of the 11-cis-retinylidene (RET) chromophore. Carbon atoms are numbered 1 through 20.
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counterion to the protonated, positively charged Schiff base
is conserved within H3. The position analogous to the Schiff
base counterion is one helix turn away from the position of
an Asp residue conserved in biogenic amine receptors that
serves as the counterion to the cationic amine ligands. A pair
of highly conserved Cys residues is found on the extracelluar surface of the receptor and forms a disulfide bond. A
Glu(Asp)/Arg/Tyr(Trp) tripeptide sequence is found at the
cytoplasmic border of H3. This sequence is conserved in
class A GPCRs and has been shown to be involved in G
protein interaction (68, 69). Sites of light-dependent phosphorylation at Ser and Thr residues are found at the carboxy-terminal tail of most visual pigments. These sites are
analogous to phosphorylation sites found on the carboxyterminal tails of other GPCRs (20).
Although it shares many similarities with other GPCR
types, as a visual pigment Rho displays many specialized
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dark noise in Rho and a significant degree of biochemical
amplification. Thermal isomerization in a single Rho molecule at physiological temperature has been estimated to
occur about once in 470 years (16). The possibility of
single pheromone molecule detection by insect olfactory
systems notwithstanding, the visual system is unique
among sensory signal transduction systems in that it can
detect single events.
The gain and kinetics of activation in the vertebrate
visual transduction cascade of the rod cell are of particular interest at the interface of biochemistry and physiology. Activation of a single Rho molecule by a single
photon has been estimated to prevent the entry of as
many as 107 cations into the rod cell. Recent studies have
estimated that at room temperature each R* triggers activation of cGMP PDE at rates of 1,000 –2,000 molecules/s
(134). During the past 5 years, transgenic animal models
have been employed to study the molecular basis of rod
cell physiology in vivo (139). In addition, animal models
have also begun to elucidate the molecular pathophysiology of human diseases that result from defects in visual
transduction or in the biosynthesis of molecules in the
signal transduction pathway.
This review attempts to outline the structural basis of
the molecular physiology of vertebrate Rho. It will focus
on what has been learned in this respect from the recently
published crystal structure of Rho (194). What insight
does the structure provide about the mechanism of the
“opsin shift” and spectral tuning? What is the structural
basis for the incredible stability of Rho in the rod cell disk
membrane in the dark? How does Rho achieve high photochemical specificity and high quantum yield? How does
a single R* catalyze guanine nucleotide exchange by hundreds of Gt molecules? What does the Rho structure tell
us, if anything, about structure-activity relationships in
other GPCRs? Finally, when possible, attempts are made
to reconcile previous key findings from biochemical studies and the analysis of site-directed mutant pigments with
the Rho crystal structure.
II. MOLECULAR STRUCTURE OF RHODOPSIN
A. Overview of Rhodopsin Structure
FIG. 2. A ultraviolet-visible absorption spectrum of purified recombinant COS cell Rho in detergent solution (dark) shows a characteristic
broad visible absorbance with a maximum wavelength (␭max) value of
500 nm. The 280-nm peak represents the protein component. After
exposure to light, the pigment is converted to metarhodopsin II (meta-II)
with a ␭max value of 380 nm characteristic of an unprotonated Schiff
base imine. Meta-II is the active form of the receptor that interacts with
the G protein transducin (Gt). Inset: photobleaching difference spectrum obtained by simply subtracting the light spectrum from the dark
spectrum. Identical results can be obtained with Rho from bovine retinas purified by concanavalin A lectin affinity chromatography.
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Rho is the first GPCR for which a crystal structure
has been reported (194). To obtain crystals, bovine Rho
was purified from rod outer segment membranes and
crystallized from a detergent solution, nonyl-thiol-glucoside supplemented with the small amphiphile heptane
1,2,3-triol (186, 187). The resolution of the crystallographic data is ⬃2.8 Å, but small segments of the cytoplasmic surface domain are not resolved. The structure
represents the inactive form of Rho with its bound RET
chromophore intact. A ribbon diagram of the Rho peptide
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Rho displays a broad visible absorption maximum
(␭max) at ⬃500 nm (Fig. 2). Photon capture leading to
photoisomerization of the 11-cis- to all-trans-form of the
RET chromophore is the primary event in visual signal
transduction, and it is the only light-dependent step (252).
After photoisomerization, the pigment decays thermally
to metarhodopsin II (meta-II) with a ␭max value of 380 nm.
The meta-II intermediate is characterized by a deprotonated Schiff base chromophore linkage. Meta-II is the
active form of the receptor (R*), which catalyzes guanine
nucleotide exchange by the rod cell heterotrimeric G
protein transducin (Gt). In contrast to vertebrate vision,
invertebrate vision is generally photochromic; a photoactivated invertebrate pigment can be inactivated by absorption of a second photon that induces isomerization to
the ground-state cis-conformation.
In the case of the vertebrate visual system, Gt activation leads to the activation of a cGMP phosphodiesterase (cGMP PDE) and the closing of cGMP-gated cation
channels in the plasma membrane of the rod cell. Light
causes a graded hyperpolarization of the photoreceptor
cell. The amplification, modulation, and regulation of the
light response is of great physiological importance and
has been discussed in detail elsewhere (17, 36, 236, 253).
However, it should be pointed out that despite the fact
that the visual system functions over about a 106-fold
range of light intensity, the retinal rod cell has single
photon detection capability due to extremely low levels of
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backbone structure with the RET chromophore is presented in Figure 3. The structure discussed in this review
is that of the A chain in the crystal unit cell dimer.
As an integral membrane protein, Rho comprises
three topological domains: the extracellular surface, the
membrane-embedded domain, and the intracellular surface. Because of the location of Rho in the disk membrane
of the rod outer segment, the extracellular domain is
sometimes referred to as intradiscal. The amino terminus
of Rho is extracellular and the carboxy terminus is intracellular. The membrane-embedded domain consists of
seven transmembrane segments (H1 to H7), which are
predominantly ␣-helical. The helical segments form a
compact bundle that contains the binding site for the RET
chromophore.
B. Extracellular Surface Domain of Rhodopsin
1. Crystal structure of the extracellular
surface domain
FIG. 3. A molecular graphics ribbon diagram of Rho prepared from
the 2.8-Å crystal structure coordinates. The amino terminus (N) and
extracellular (or intradiscal) surface is toward the top of the figure, and
the carboxy terminus (C) and intracellular (or cytoplasmic) surface is
toward the bottom. Seven transmembrane segments (H1 to H7), which
are characteristic of G protein-coupled receptors, are shown. They are
color coded according to the rainbow spectrum starting with blue and
ending with red. The RET chromophore is shown in magenta, and the
side chains of Lys-296 and Glu-113 are shown to highlight the Schiff base
region of the RET binding pocket. The Schiff base imine nitrogen is
shown in deep blue. The Rho crystal structure does not resolve a small
segment of the C3 loop linking H5 and H6 or a longer segment of the
carboxy-terminal tail distal to H8. The transmembrane segments are
tilted with respect to the presumed plane of the membrane bilayer. They
are generally ␣-helical, but they contain significant kinks and irregularities as described in text. The figures were produced using the program
MOLSCRIPT (130) and represent the A chain of the published crystal
structure coordinates (194).
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.
The extracellular surface domain of Rho comprises
the amino-terminal tail (NT) and three interhelical loops
(E1, E2, and E3) (Fig. 1). There is significant secondary
structure in the extracellular domain and several intraand interdomain interactions. The extracellular domain
essentially provides a stable foundation from which the
transmembrane segments extend (Fig. 3).
NT extends from the amino terminus to Pro-34 and
contains five distorted strands (␤1, ␤2, S3, S4, and S5). It
is located in the crystal structure just outside of loop E3,
with the side chain of Asn-2 close to that of Asp-282 in E3.
The NT domain also seems to be in contact with the
E3 loop in the area near Pro-12. The short segment from
Gly-3 to Pro-12 forms the first two antiparallel strands in
the structure (␤1 and ␤2), which seem to lie roughly
parallel to the plane of the membrane. The segment
Phe-13 to Pro-34 forms strands S3 to S5, and they appear
almost as a right triangle. S3 runs just outside E3 and
parallel to the long axis of the molecule. S4 connects the
dipeptide Ser-14/Asn-15 in NT with Pro-23, which is located close to E1. S5 (Pro-27 to Pro-34) runs along the
surface of the membrane covering the extracellular space
between H1 and H2. NT is glycosylated at Asn-2 and
Asn-15. The oligosaccharides extend away from the extracellular domain and do not seem to interact with any
part of the molecule.
In addition to the NT segment, the extracellular surface domain comprises three extracellular interhelical
loops: loop E1 (amino acids 101–106) connects H2 and
H3, loop E2 (amino acids 174 –199) connects H4 and H5,
and loop E3 (amino acids 278 –285) connects H6 and H7.
The E1 loop runs along the periphery of Rho. Tyr-102
interacts with Pro-23 and Gln-28 to maintain proper orientation between E1 and NT. The E2 loop is extremely
interesting in that it is folded deeply into the core of the
membrane-embedded region of Rho. In addition to contacts with the RET chromophore, E2 forms extensive
contacts with other extracellular regions. Gly-174 and
Met-183 cross the membrane surface. The Met-183 side
chain points toward a hydrophobic pocket around H1,
while the extended side chain of Gln-184 is surrounded by
relatively hydrophilic groups and a water molecule lo-
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partially buried. Solvent access to Asp-190 may be controlled by Arg-177 and His-195, two amino acid residues
with functional side chains that could act to stabilize a
partially buried negative charge. Asp-190 is also strongly
coupled to the chromophore by its flanking residues Ile189 and Tyr-191 (Fig. 4).
One of the most striking and unexpected features
of the Rho structure is the presence and positioning of
the ␤4-strand (Ser-186/Cys-187/Gly-188/Ile-189), which
forms the extracellular floor of the RET binding site,
secluding the ligand from bulk solution on the extracellular surface. The ␤4-strand runs nearly parallel to
the length of the polyene chain from about C9 to the
Schiff base imine nitrogen. The opposite end of RET
from the cyclohexenyl ring to about C10 runs along H3,
which is tilted with respect to the plane of the membrane. The result is that this end of RET seems to be
held very firmly in place by multiple contacts (see
sect. IID).
The E3 loop essentially runs along the periphery of
Rho in similar fashion to E1 and to the connector segment
of E2 (amino acids 191–200). E3 displays at least two
potential interactions with NT. Pro-12 of NT may interact
with the E3 loop, and Asn-2 is close to Asp-282.
FIG. 4. The RET chromophore-binding pocket of bovine Rho. A: a cut-away surface map is shown viewed from the
extracellular surface. All residues above the surface of RET were removed so that the RET pocket can be appreciated.
The protein is colored turquoise, and the RET is colored in red. The side chains of Trp-265 (H6) and Lys-296 (H7) are
labeled for orientation. The kink in RET at the C10-C13 region of the polyene is clearly defined. B: the RET chromophorebinding pocket is shown from within the plane of the membrane bilayer. RET is colored magenta, and amino acid side
chains are colored yellow except for nitrogen atoms (blue), oxygen atoms (red), and sulfur atoms (green). At least 16
amino acid residues are within 4.5 Å of the RET ligand: Glu-113, Ala-117, Thr-118, Gly-121, Glu-122, Glu-181, Ser-186,
Tyr-191, Met-207, His-211, Phe-212, Phe-261, Trp-265, Tyr-268, Ala-269, and Ala-292. Some additional key amino acid
residues are labeled, including the Cys-110/Cys-187 disulfide bond. RET is situated such that its proximal end (approximately C9 to C15) lies along the ␤4-strand and its distal end (approximately C9 to the cyclohexenyl ring) lies along H3.
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cated close to the backbone carbonyl group of Pro-180
and the hydroxyl group of Tyr-192. The segment from
Arg-177 to Glu-181 forms the ␤3-strand, while that from
Ser-186 to Asp-190 forms the ␤4-strand.
The ␤3- and ␤4-strands run antiparallel. The ␤4strand is situated more deeply within the membraneembedded region of Rho than the ␤3-strand. The ␤4strand is adjacent to the RET chromophore and forms the
extracellular boundary, or roof, of the ligand-binding
pocket. A disulfide bond between Cys-187 and Cys-110,
which forms the extracellular end of H3, is highly conserved among all class A GPCRs. The segment from Tyr191 to Asn-200 forms a short surface connector within E2
that is similar to both E1 and E3. The backbone carbonyl
group of Tyr-191 and the amide group of the side chain of
Gln-279, which is at the amino-terminal end of E3, lie in
close proximity. Within the connector portion of E2, Asn199 is near to Trp-175, although it is oriented differently in
the unit cell chain B molecule.
The carboxylic acid side chain of Glu-181 in the ␤3
sheet of the E2 loop points toward the center of the
polyene chain of the retinylidene chromophore (Fig. 4).
Asp-190 at the carboxy-terminal end of the ␤4-strand is
interesting because although it is near to the solventexposed surface of the receptor, its carboxy group is
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1. Genetically engineered animal strains prepared to study rhodopsin biosynthesis,
processing, and physiology
TABLE
Genotype
Species
Phenotypic Characteristics
Mouse
Photoreceptor degeneration seen in both P23H and in strains overexpressing
wild-type human rod opsin.
Photoreceptors develop normally during early postnatal development, but
outer segments remain short. The numbers of rods and cones decreased
progressively with advancing age.
Chimeras (slow photoreceptor degeneration) exhibited mosaicism in coat
color, retinal pigment epithelium, and retina. Photoreceptors in chimeric
retinas degenerated uniformly, independent of the genotype and similar to
photoreceptors in transgenic mice.
Abnormal accumulation of Q334ter Rho in the plasma membrane of the
photoreceptor cell body and 15% longer time-to-peak photoresponse.
Loss of photoreceptors by postnatal day 10 and accumulation of Rho, Gt,
and PDE within the outer plexiform layer of the retina.
Rod-mediated ERGs showed significant reduction in amplitude at 1 mo of
age. Cone-mediated responses were nearly normal for 5 mo, then declined
progressively.
Abnormal prolonged rod photoresponses.
Rhodopsin VPP (V20G/P23H/P27L)
Mouse
Rhodopsin P347S
Mouse
Rhodopsin Q344ter
Mouse
Rhodopsin P23H
Mouse
Rhodopsin VPP (V20G/P23H/P27L)
Mouse
Rhodopsin S334ter (truncated
opsin lacks Rho kinase and PKC
phosphorylation sites)
Rhodopsin K296E (lacks retinal
binding site)
Rhodopsin P347S
Mouse
Rhodopsin P23H
Mouse
Rhodopsin P347S
Mouse
Rhodopsin VPP (V20G/P23H/P27L)
Mouse
Rhodopsin VPP (V20G/P23H/P27L)
Mouse
Rhodopsin P347S
Mouse
Rhodopsin “knockout” (⫺/⫺)
Mouse
Rhodopsin P347L
Rhodopsin P347L
Swine
Swine
Green fluorescent protein (GFP)
under control of rod opsin
promoter
Rhodopsin Q344ter
Xenopus
Rhodopsin P347L
Swine
Rhodopsin T17M, P347S
Mouse
Rhodopsin VPP (V20G/P23H/P27L)
Mouse
Rhodopsin P23H, Q344ter
Mouse
Rhodopsin (⫺/⫺), (⫹/⫺)
Mouse
Mouse
Mouse
Mouse
188
165
99
242
217
77
42
Photoreceptor degeneration. Mutant opsin was invariably phosphorylated
and stably bound to arrestin.
Loss of photoreceptor cells (⬃35%), mistargeting of Rho and elevated levels
of cAMP.
Structural and functional abnormalities leading to apoptosis seen in darkreared transgenic mice.
Accumulation of submicron-sized vesicles laden with Rho consistent with
aberrant transport from the inner segments to the nascent disk
membranes of the outer segments.
Photoreceptor degeneration was significantly higher in VPP mice with albino
phenotype.
Progressive photoreceptor degeneration and increased susceptibility to light
damage.
90% of the nuclei showed chromatin condensation and DNA fragmentation
consistent with apoptosis.
Photoreceptors and rod ERG response lost within 3 mo. Rho (⫹/⫺) retained
photoreceptors, although they showed some structural disorganization.
Early loss of rods with later loss of cones.
Degeneration of rods and cones, but with different time schedules and
different pathological features.
GFP expression noted in retina and pineal.
143
Photoreceptor degeneration was slowed by axokine or leukemia inhibitory
factor.
At birth, rod numbers were normal, but their outer segments were short and
disorganized. Rod synapses of newborns lacked synaptic vesicles and
ribbons and had filopodia-like processes that extended into the outer
plexiform layer. Cones degenerated more slowly than rods. Rod cell death
occurred due to misrouting of mutant Rho.
In vitro, T17M exhibits thermal instability/folding defect and minimal
regeneration with the chromophore and P347S exhibits no abnormal
biochemical activity. High vitamin A diet reduced the rate of decline of
ERG amplitudes in T17M mice but had no significant effect in P347S mice.
T17M mice displayed longer photoreceptor inner and outer segments and
a thicker outer nuclear layer. P347S displayed no significant retina
morphology phenotype.
Slowly progressive degeneration of rods. Photochemical properties
(absorbance spectra, photic sensitivities, and ability of 11-cis-retinal to
regenerate Rho after bleaching) were similar to wild type.
Neovascularization of the retinal pigment epithelium was found very early in
P23H, whereas it occurred much later in Q344ter.
Rho (⫺/⫺) mice retinas initially developed normally, but without the
formation of rod outer segments. Within months of birth, photoreceptor
cells degenerated completely. Rho (⫹/⫺) mice retinas developed normally.
Rod outer segments were of normal size but with half the normal
complement of Rho.
136
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258
167
145
166
254
41
100
200
248
125
146
144
260
182
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Rhodopsin P23H
Reference
Nos.
1665
RHODOPSIN
TABLE
1.—Continued
Genotype
Species
Rat
Rho (⫺/⫺) mice crossed with
mice expressing human Rho
(“rescued mice”)
Rhodopsin P23H
Mouse
Rhodopsin P347L
Swine
Arrestin “knockout” (⫺/⫺)
Mouse
Interphotoreceptor retinoidbinding protein (IRBP)
“knockout” (⫺/⫺)
IRBP “knockout” (⫺/⫺)
Mouse
Rhodopsin S334ter
Rat
Mouse
Mouse
Reference
Nos.
Very rapid photoreceptor degeneration. Increase in caspase-3-like activity
noted.
Extensive structural and functional rescue of the mouse photoreceptors by
the human transgene.
152
P23H rods degenerate when placed under conditions of wild-type retinal
cells in culture. Coculture of P23H rods with wild-type retinal cells
rescues mutant rods. Rescue was also observed upon treatment with
retinoic acid receptor antagonist. A diffusible factor found in normal cells
has a protective effect on photoreceptor cell survival, and the factor is
absent in the transgenic retinal cells.
Physiology of cone photoreceptor was normal for months before cone cell
death ultimately occurred. Cone circuitry maturation failed throughout
postnatal development.
Minor effects on visual cycle kinetics (Rho recovery ⬃80%, 11-cis-retinal
recovery modestly delayed).
IRBP necessary for photoreceptor survival but not essential for visual
pigment turnover.
233
Significant loss of photoreceptor nuclei and gross changes in the structure
and organization of ROS.
Mutant Rho expressed at inappropriately high levels in the plasma
membrane and cytoplasm of photoreceptors. Missorting of Rho caused
apoptosis by interfering with normal cellular machinery.
214
158
13
195
195
79
Studies are arranged in chronological order of publication. This table focuses primarily on rhodopsin and does not include all animal strains,
transgenic strains, or “knockout” strains prepared to study phototransduction, retinal degeneration, or the chromophore regeneration pathway. Rho,
rhodopsin; Gt, transducin; PDE, phosphodiesterase; ERG, electroretinogram.
2. Structure-activity relationships in the extracellular
surface domain
The role of the extracellular surface domain in Rho
function has been elucidated by a number of studies
involving site-directed mutagenesis. The extracellular
loops and NT of bovine Rho have been shown in a deletion analysis to be important for proper folding of the
receptor that allows cellular processing and chromophore
binding (47). Insertional mutagenesis was also used in a
related study to probe the topology of Rho and to correlate the location of epitope insertion to stability and cell
trafficking (27). Interestingly, several mutations that interfered with the formation of a correct tertiary structure
on the intradiscal surface resulted in mutant opsins that
appeared to be retained in the endoplasmic reticulum
during heterologous expression and were complexed
with molecular chaperones (7). Antibody accessibility
studies suggested that the NT domain constitutes a defined tertiary structure that contributes to the overall
extracellular domain (35). This conclusion is supported
by the crystal structure.
Rho is known to be glycosylated at Asn-2 and Asn-15
of the NT. A nonglycosylated Rho, which was prepared in
the presence of tunicamycin, was defective in light-dependent activation of Gt (114). Site-directed opsin mutants
with replacements of these two residues or of neighboring
consensus sequence residues were studied as well. It was
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concluded that glycosylation at Asn-15 was required for
full signal transduction activity, but apparently not for
correct biosynthesis or folding (114). The structural basis
of this findings is not clear from the Rho crystal structure
since the oligosaccharide chains point away from the
molecule and do not seem to engage in intramolecular
interactions.
Future studies of mutant opsins with defects in folding, membrane insertion, or cell trafficking will be facilitated by the use of a methodology to purify regenerated
pigment from nonregenerated opsin. The paradigm was
developed by using the opsin mutant containing only
three intradiscal Cys residues (112). It was shown that the
nonregenerated opsin, which could not bind RET, was
misfolded and was likely to have an incorrect disulfide
bond pairing (213). This is additional evidence pointing to
the role of the intradiscal domain, and in particular the
early formation of a correct disulfide bond linkage between Cys-110 and Cys-187, in the proper folding of the
opsin apoprotein (101).
The two conserved cysteine residues on the extracelluar domain, Cys-110 and Cys-187, are essential for proper
folding of opsin expressed in COS cells (112). These two
residues were shown to form a disulfide linkage in an
elegant study in which the four intracellular and three
membrane-embedded Cys residues were removed by sitedirected mutagenesis to create a mutant receptor with
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Rhodopsin S334ter
Phenotypic Characteristics
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log that lacks the C19-methyl group) (72). Otherwise, the
RET C19-methyl group might interact with another part of
Rho during activation, the RET C19-methyl group-Ile-189
contact being only a ground state interaction. A mutant
such as I189W might have a decreased ability to bind RET,
as would any mutant with a substitution at the neighboring Gly-188 residue. If they did bind RET, the phenotypes
of these mutants with respect to Gt activation would also
be interesting.
In Rho, Glu-181, which arises from the linker between ␤3 and ␤4 but points toward the polyene chain,
may serve a role in spectral tuning as discussed below. In
addition, Glu-181 may serve to influence the electron
density of the conjugated polyene system of the retinal
chromophore so that photoisomerization occurs exclusively at the C11-C12 double bond. The corresponding His
residue in the green and red cone pigments constitutes a
part of a binding site for a chloride ion. Chloride binding
causes a spectroscopic red shift in the visible absorption
spectra of green and red pigments.
C. Membrane-Embedded Domain of Rhodopsin
1. Crystal structure of the membrane-embedded
domain
The crystal structure of Rho suggests that 194 amino
acid residues make up the seven transmembrane segments (H1 to H7) included in the membrane-embedded
domain: H1 (amino acids 35– 64), H2 (amino acids 71–
100), H3 (amino acids 107–139), H4 (amino acids 151–
173), H5 (amino acids 200 –225), H6 (amino acids 247–
277), and H7 (amino acids 286 –306). The crystal structure
of this domain is remarkable for a number of kinks and
distortions of the individual transmembrane segments,
which are otherwise generally ␣-helical in secondary
structure. Many of these distortions from idealized secondary structure were not accounted for in molecular
graphics models of Rho based on projection density
maps obtained from cryoelectron microscopy (12, 131,
227, 249).
H1 has a very slight kink at Pro-53, its sole Pro
residue. H2 begins with Pro-71, and it is also kinked
around vicinal Gly residues, Gly-89 and Gly-90 so that this
region of H2 is brought closer to H3 than to H1. This
feature is interesting in that Gly-90 comes into close proximity to the RET Schiff base counterion, Glu-113, on H3.
H3 contains only a single Pro residue at its amino
terminus, Pro-107. Consecutive Gly residues in H3 at Gly120 and Gly-121 do not seem to distort H3 to the degree
that Gly-89 and Gly-90 distort H2. The cytoplasmic end of
H3 contains the highly conserved Glu/Arg/Tyr motif that is
known to regulate Gt coupling. The Glu-134/Arg-135/Tyr136 tripeptide exhibits a slight deviation from a regular
helical structure. Several hydrophobic residues surround
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only the three extracellular Cys residues remaining (111).
In a related study, the double mutant C110A/C187A was
shown to bind RET to form a Rho-like pigment (45).
However, the meta-II-like photoproduct of the mutant
pigment, which could activate Gt in response to light, was
considerably less stable than native meta-II (45). In general, mutations on the extracellular surface that might
interfere with the formation of a disulfide bond between
residues Cys-110 and Cys-187 correlate with a loss of
function phenotype. Namely, mutations affecting the ability of these two Cys residues to juxtapose during translation or membrane translocation affect expression level,
transport to the plasma membrane, and the ability of the
mutant opsin to bind RET chromophore to form a pigment with normal stability. The Cys-110/Cys-187 disulfide
bond seems to stabilize the ground state structure of the
chromophore-binding pocket, but the adjacent side
chains must be able to acquire their proper positions even
without the disulfide.
Several point mutations that result in amino acid
substitutions in the NT domain are linked to autosomal
dominant retinitis pigmentosa (ADRP), including positions Pro-23 and Gln-28. ADRP is an inherited human
disease that causes progressive retinal degeneration, loss
of dim-light vision, loss of peripheral vision, and eventual
blindness. Pro-23 and Gln-28 interact with Tyr-102, which
is in the E1 loop. This interaction might maintain an
essential structural orientation between NT and E1 that is
disrupted in the NT of the ADRP mutants. Thus Tyr-102
interacts with Pro-23 and Gln-28 to maintain proper orientation between E1 and NT. The roles of specific amino
acid residues in the NT domain were also studied in
various transgenic mice strains harboring point mutations
that correspond to sites linked to ADRP. These transgenic
strains are listed in Table 1.
The amino acids of the ␤4-strand are highly conserved among vertebrate opsins. Of 130 opsins considered, 128 contained Ser and 2 contained Ala at position
186, 130 contained Cys at position 187, and 130 contained
Gly at position 188. There was more variability at position
189, where most opsins contained an Ile, but some contain Pro or Val instead. In class A GPCRs that do not bind
RET, for example, adrenergic receptors, this region does
not seem to be highly conserved, except for the Cys-187
residue. Therefore, the ␤4-strand might serve specifically
to define the RET ligand-binding pocket in vertebrate
opsins.
One particularly interesting interaction in this region
is that of Ile-189 with RET C19 (the methyl group bonded
to C9 of the polyene chain). Because the C19-methyl group
is required for light-dependent activation of Rho, it would
be interesting to determine the phenotype of the mutant
I189A. The prediction would be that I189A would fail to
activate Gt and would have a phenotype similar to that of
19-desmethyl Rho (opsin regenerated with the RET ana-
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RHODOPSIN
2. Structure-activity relationships in the
membrane-embedded domain
The membrane-embedded domain of Rho is characterized by the presence of several intramolecular interactions that may be important in stabilizing the ground state
structure of the receptor (Fig. 5). One of the hallmarks of
the molecular physiology of Rho is that it is essentially
silent biochemically in the dark. The RET chromophore
serves as a potent pharmacological inverse agonist to
minimize activity. The result is that the rod cell can attain
single photon sensitivity (94). The Rho structure reveals
numerous potentially stabilizing intramolecular interactions, some mediated by the RET chromophore and some
arising mainly from interhelical interactions that do not
involve the RET-binding pocket directly.
The phenyl rings of the side chains of Phe-293 and
Phe-294, both in H7, interact with Leu-40 in H1 and Cys264 in H6, respectively. This interaction seems to be facilitated by the slight distortion of H6 in the region near
Ile-263. In addition to these core interactions, there appear to be four H-bond networks that provide stabilizing
interhelical interactions at or near the cytoplasmic surface of the receptor.
Physiol Rev • VOL
FIG. 5. Interhelical interactions in Rho. The structure of Rho is
remarkable for several interhelical interactions that are likely to stabilize the ground state conformation of the receptor. In addition to several
hydrophobic interactions and interactions mediated by RET in the membrane-embedded domain, three H-bond networks exist toward the cytoplasmic side of the helical core of the receptor. H-bond networks 1 and
3 involve highly conserved Asn/Pro/X/X/Tyr and Glu/Arg/Tyr motifs,
respectively. A detailed description of the residues involved in the
H-bond networks is presented in the text. A: Rho is viewed from above
the cytoplasmic surface of the receptor and roughly perpendicular to the
putative plane of the membrane. The cytoplasmic loops and the carboxyterminal tail distal to H8 have been removed. Transmembrane helices H1
to H7 are shown as discontinuous cylinders and are color coded as in
Fig. 3. B: Rho is viewed from within the membrane plane, and only the
cytoplasmic half of the transmembrane domain is presented. As in A, the
cytoplasmic loops and the carboxy-terminal tail distal to H8 have been
removed. Transmembrane helices H1 to H7 are shown as discontinuous
cylinders and are color coded as in Fig. 3.
H-bond network 1 links H1, H2, and H7. The helical
structure of H7 is elongated in the region from Ala-295 to
Tyr-301, which permits the backbone carbonyl group of
Ala-299 to H-bond with the side chains of Asn-55 in H1 and
Asp-83 in H2. Asn-55 is a very highly conserved residue
that plays the central role in the H-bond network 1 since
it H-bonds to both Asp-83 and the backbone carbonyl of
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the cytoplasmic end of H3 (Pro-71, Leu-72, Phe-148, Leu226, Val-230, Val-250 and Met-253), which might form a
binding site for Gt.
Although H4 contains two consecutive Pro residues
(Pro-170 and Pro-171), significant distortion is only appreciated near the extracellular end of the segment. The
cytoplasmic end of H4 is near to H2, but the two segments
seem to diverge beyond the level of Trp-161, which is
highly conserved in class A GPCRs. Irregular helicity is
also noted in the cytoplasmic end of H4.
H5 appears to be nearly straight despite the presence
of Pro-215, which is approximately in the middle of the
segment. However, helix irregularity is noted, especially
in the region near His-211. H5 contains six Phe residues
and two Tyr residues. Tyr-223, which partially fills the
interhelical region between H5 and H6, is highly conserved.
The presence of Pro-267 creates a significant bend in
H6 at about the level of the Schiff base with respect to the
putative membrane boundaries. The boundary between
C3 and H6 at the cytoplasmic surface is highly basic due
to presence of Lys-245, Lys-248, and Arg-252.
H7 is the most highly distorted of the seven transmembrane helical segments. There are kinks at two Pro
residues, Pro-291 and Pro-303. In addition, the helix is
irregular around the region of residue Lys-296, which is
the chromophore attachment site. Pro-303 is a part of the
highly conserved Asn/Pro/X/X/Tyr motif (Asn-302/Pro303/Val-304/Ile-305/Tyr-306 in Rho).
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mutant opsins. A decrease in interaction between Met-257
and Asn-302 might relieve an interhelical constraint that
stabilizes the ground state structure of Rho. However, the
most highly constitutively active Met-257 mutants were
M257Y, M257N, and M257S, which are all theoretically
capable of forming H-bonds with the adjacent Asn-302. It
is conceivable that the amino acid residue at position 257
in a mutant receptor forms H-bond interactions that stabilize the active state structure of the receptor as well.
Whether constitutive activity is caused simply by a lack of
H6/H7 interactions, or whether a gain of active state
stabilizing interactions is required could be determined by
testing mutant receptors with alterations of the Asn/Pro/
X/X/Tyr motif, for example, N302A and I305A in Rho, or
analogous mutations in other class A GPCRs.
It should be noted that the highly conserved Asn/Pro/
X/X/Tyr (H7) and Glu/Arg/Tyr (H3) motifs play central
roles in several of the core-stabilizing interactions described above. Their precise roles in the regulation of
receptor activation will require knowledge about any
structural reorientations that occur with respect to these
residues in the formation of R*.
D. Chromophore-Binding Pocket
1. Crystal structure of the chromophore-binding pocket
The RET chromophore is a derivative of vitamin A1
with a total of 20 carbon atoms (Fig. 1). The carbon atoms
of the cyclohexenyl ring are numbered C1 to C6. The
polyene carbons extend from C7 to C15. Two methyl
groups (C16 and C17) are bonded to C1, and single methyl
groups are attached at each of three other carbons: C5
(C18 methyl), C9 (C19 methyl), and C13 (C20 methyl). The
structural conformation of the bound chromophore in the
Rho crystal structure appears to be 6-s-cis, 11-cis, 12-strans. The protonated Schiff base bond appears to be in
the anti conformation. A higher resolution Rho structure
would be required for a crystallographic determination of
the precise chromophore structure. Although a variety of
spectroscopic studies support the 6-s-cis, 11-cis, 12-strans RET conformation, recent NMR experiments suggested a 6-s-trans conformation (80).
The binding site of the RET chromophore lies
within the membrane-embedded domain of the receptor (Fig. 4). All seven transmembrane segments and
part of the extracellular domain contribute interactions
with the bound chromophore. The chromophore is located closer to the extracellular side of the transmembrane domain of the receptor than to the cytoplasmic
side. The chromophore polyene from C6 to C11 runs
almost parallel to H3, which provides many of the
amino acid side chains that form the chromophorebinding pocket: Glu-113, Gly-114, Ala-117, Thr-118, Gly120, and Gly-121. The polyene chain facing toward the
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Ala-299. Asp-83, in turn, is connected to the backbone
carbonyl of Gly-120 in H3 through a water molecule.
H-bond network 2 links H2, H3, and H4. This network
involves Asn-78 as the key residue, which H-bonds to the
hydroxyl functions of Ser-127 (H3), Thr-160 (H4), Trp-161
(H4), and the backbone carbonyl of Phe-159. Mutant pigments S127A and T160V displayed normal ground-state
spectral properties consistent with a lack of direct contact with RET (103). Another possible interhelical interaction in this region might involve Glu-122 (H3), Met-163
(H4), and His-211 (H5). An indirect functional interaction
between Glu-122 and His-211 has been demonstrated experimentally (18).
H-bond network 3 links H3 and H6. This network
involves the conserved Arg-135, which interacts with Glu134 and with the hydroxyl group of Thr-251 and side chain
of Glu-247. The carboxylate of Glu-134 seems to be in
position to form a salt bridge with the guanidinium group
of Arg-135. This would be consistent with the hypothesis
that Glu-134 is unprotonated in Rho and becomes protonated during the transition to R* (9, 59). It is interesting to
note the three consecutive Val residues (Val-137, Val-138,
and Val-139) are situated to form a cytoplasmic cap to H3
so that the Glu-134/Arg-135 dipeptide is between the receptor core and the Val tripeptide. This Val cap might act
to stabilize the Glu-134/Arg-135 salt bridge, which in turn
acts to keep the receptor in its off state in the dark. It is
also interesting to note that Thr-251 in Rho is in the
position equivalent to Ala-293 in the ␣1B-adrenergic receptor. Mutation of Ala-293 causes the receptor to become
constitutively active (121). The Asp(Glu)/Arg/Tyr(Trp)
motif at the cytoplasmic border of H3 is one of the most
highly conserved structural motifs in class A GPCRs.
Finally, H-bond network 4 links H6 and H7. The key
interaction here is between Met-257 and Asn-302. The
precise functional importance of the highly conserved
Asn/Pro/X/X/Tyr motif (Asn-302/Pro-303/Val-304/Ile-305/
Tyr-306 in Rho) is unclear. However, one key structural
role is to mediate several interhelical interactions. The
side chains of Asn-302 and Tyr-306 project toward the
center of the helical bundle. The hydroxyl group of Tyr306 is close to Asn-73 (cytoplasmic border of H2), which
is also highly conserved. A key structural water molecule
may facilitate an H-bond interaction between Asn-302 and
Asp-83 (H2). A recent mutagenesis study of the human
platelet-activating factor receptor showed that replacement of amino acids at the positions equivalent to Asp-78
and Asn-302 in Rho with residues that could not H-bond
prevented agonist-dependent receptor internalization and
G protein activation (137).
The interaction between Met-257 and the Asn/Pro/X/
X/Tyr motif was predicted earlier to explain the results of
a mutagenesis study in which Met-257 was replaced by
each of 19 other amino acid residues (91). Nearly all
Met-257 replacements caused constitutive activity of the
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2. Structure-activity relationships
in the chromophore-binding pocket
A number of experimental approaches have been
employed to investigate RET-protein interactions in the
membrane-embedded domain of bovine Rho. Several
spectroscopic methods such as resonance Raman spectroscopy (98, 128, 148), Fourier-transform infrared
(FTIR)-difference spectroscopy (60), and NMR spectroscopy (52, 80, 81, 251) have been reported. Other approaches have included reconstitution of opsin apoprotein with synthetic retinal analogs (97, 154) and
photochemical cross-linking (26, 170, 266). Early work on
the structure and function of recombinant bovine Rho
focusing on the use of techniques of molecular biology
has been reviewed (116, 172).
Lys-296 and Glu-113 are two of the key amino acid
residues that define the structure and function of the
retinal chromophore in Rho. The Schiff base linkage of
the chromophore to Lys-296 is a key feature of Rho structure (92). Light-dependent Schiff base deprotonation is
required for the formation of the active state of the receptor R* (117, 153). However, it has been shown that
light can induce the receptor active state in the absence of
a Schiff base chromophore linkage to the opsin (268). In
this experiment, mutant opsin K296G was treated with a
model retinal Schiff base compound prepared from 11cis-retinal and n-butyl amine. A visible pigment was
formed, although no linkage was present between the
model compound and the opsin. Photoisomerization of
the model chromophore led to receptor activation as
judged by Gt activation. This demonstrates that opsin can
employ a diffusible ligand, which is analogous to liganddependent GPCRs, such as biogenic amine receptors (189).
Glu-113 in bovine Rho serves as the counterion to the
positive charge of the RET protonated Schiff base (171,
225, 267). Glu-113 is unprotonated and negatively charged
in the ground state of Rho (57). It becomes protonated
upon light-dependent formation of meta-II and is the net
proton acceptor for the Schiff base proton (104). The
Glu-113-protonated Schiff base interaction serves to stabilize the Schiff base proton such that its acid dissociation
constant (pKa) in Rho is estimated to be ⬎12, compared
with a value of ⬃7 for a model compound in aqueous
solution, although the mechanism of protonated Schiff
base stabilization is not entirely clear from the crystal
structure. The stable interaction between Glu-113 and the
protonated Schiff base may also inhibit hydrolysis of the
Schiff base linkage in darkness. For example, hydroxylamine does not react with the Schiff base of Rho, but
readily reacts with that of meta-II or with Rho mutants in
which Glu-113 is replaced by a neutral amino acid residue
by mutagenesis (225).
This is an important consideration since the opsin
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extracellular side of the receptor is covered, or capped,
by the amino acid residues from the ␤4-sheet (Ser-186
to Ile-189) of the E2 loop as described above. The
carboxylic acid side chain of Glu-181 in the ␤3-sheet of
the E2 loop points toward the center of the RET polyene chain.
Glu-113 serves as the RET Schiff base counterion.
A number of other amino acid side chains surround the
imine moiety, including Tyr-43, Met-44, and Leu-47 in
H1, Thr-94 in H2, and Phe-293 in H7. In particular,
Met-44 and Leu-47, in addition to the peptide bond
between Phe-293 and Phe-294, help to orient the side
chain of Lys-296 in the direction of the long axis of Rho.
The phenyl rings of Phe-293 and Phe-294 also interact
with side chains of adjacent helices. The two oxygen
atoms of the Glu-113 carboxylate side chain of Glu-113
are located 3.3 and 3.5 Å from the imine nitrogen. The
hydroxyl group of Thr-94 is also ⬃3.4 Å from one of the
Glu-113 carboxylate oxygens. Thr-92 and Thr-93 are
also in the vicinity of the Schiff base imine but may not
be close enough to contribute significantly to stabilization of its protonated ground state. The presence of
water molecules in the Schiff base region has been
postulated, but the crystal structure at the reported
resolution does not contain defined water in this region (168).
The position of the cyclohexenyl ring of the chromophore is largely constrained on the cytoplasmic side of
the binding pocket by three residues: Glu-122 (H3), Phe261 (H6), and Trp-265 (H6). The indole side chain of
Trp-265 points inward from the more cytoplasmic position of the Trp-265 backbone and comes within ⬃3.8 Å of
the RET C20. Side chains from Met-207, His-211, and Phe212 on H5 and Tyr-268 and Ala-269 on H6 further constrain the chromophore ring.
Gly-121 interacts with RET in a direct steric manner
and lies closest to the C18-methyl group bonded to C5 of
the cyclohexenyl ring (Gly-121 C␣-RET C18 distance 3.5
Å). Gly-121 is close to Phe-261 (H6) in the Rho crystal
structure. They pair to form one boundary of the RET
binding site, defining the C4-C5-C18 orientation (Phe-261
Cz-Gly-121 C␣ distance 5 Å; Phe-261 Cz-RET C4 distance
3.7 Å). This portion of the RET binding pocket around H3
and H6 appears to be held rigidly together by tight van der
Waals interactions.
H4 contributes only one residue, Cys-167, directly to
the chromophore-binding pocket.
In summary, at least 16 amino acid residues are
within 4.5 Å of the RET moiety: Glu-113, Ala-117, Thr118, Gly-121, Glu-122, Glu-181, Ser-186, Tyr-191, Met207, His-211, Phe-212, Phe-261, Trp-265, Tyr-268, Ala269, and Ala-292. The most striking feature of the RET
binding pocket is the presence of many polar or polarizable groups to coordinate an essentially hydrophobic
ligand (Fig. 4).
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the C11-C12 double bond. The potential ionic interaction
between RET and Glu-181 also suggests that it may have
a role in the mechanism of the opsin shift (see below).
Perturbation of the electron distribution near the center
of the polyene chain is one mechanism to facilitate spectral tuning (97).
Glu-181 is highly conserved among vertebrate opsins,
blue and ultraviolet (UV) cone pigments. The corresponding position in green and red cone pigments is His-197,
which forms part of a chloride ion-binding site. Chloride
binding causes a red shift in absorption of the green and
red pigments. Interestingly, the H197E/R200Q mutant of
the human green cone pigment displays a visible ␭max
value of 500 nm (257), which is the same as the ␭max value
of Rho, suggesting that perturbation of the polyene by
chloride may be the only element in the green cone pigment responsible for its spectral difference from Rho. The
negative charge of the chloride ion bound to His-197 in the
long-wavelength sensing cone pigments might be brought
closer to RET than the charge of the carboxylate of
Glu-181 in Rho.
The position of the cyclohexenyl ring of the chromophore is largely constrained on the cytoplasmic side of
the binding pocket by three residues: Glu-122 (H3), Phe261 (H6), and Trp-265 (H6). The indole side chain of
Trp-265 points inward from the more cytoplasmic position of the Trp-265 backbone and comes within ⬃3.8 Å of
the RET C20. Trp-265 is close enough to RET that it can
serve as an intrinsic probe of the chromophore conformation (149). Side chains from Met-207, His-211, and Phe212 on H5 and Tyr-268 and Ala-269 on H6 further constrain the chromophore ring. Replacements of Phe-261 by
Tyr or Ala-269 by Thr produce bathochromic spectral
shifts in the ␭max values of the resulting mutant pigments
(38). These residues are also responsible in part for the
spectral shift in red cone pigments. Whereas red pigments
have Thr and Tyr at the positions corresponding to 261
and 269 in Rho, green cone pigments have Phe and Ala
(11, 181).
The interaction between Gly-121 and RET is consistent with mutagenesis experiments in which replacement
of Gly-121 caused blue-shifted ␭max values and decreased
RET binding that corresponded to the bulk of the substituted side chain (86). Second-site replacement of Phe-261
by Ala caused a reversion of the loss of function Gly-121
mutant phenotypes, which was interpreted to mean that
Gly-121 and Phe-261 interacted to form a part of the RET
binding pocket (85). Gly-121 and Phe-261 are indeed very
close together in the Rho crystal structure. They pair to
form one boundary of the RET binding site to define the
C4-C5-C18 orientation (Phe-261 Cz-Gly-121 C␣ distance 5 Å;
Phe-261 Cz-RET C4 distance 3.7 Å). Interestingly, Gly-121
is conserved among all vertebrate and invertebrate visual
pigments (204), and Phe-261 is strictly conserved among
nearly all GPCRs (3). As described above, in long-wave-
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alone, without the RET chromophore, has a small but
measurable activity (31, 207, 215, 244). The high sensitivity of the rod cell depends on an extremely low intrinsic
level of signaling in darkness. Dark noise can be generated by thermal isomerization events in Rho (14, 15, 262),
by the presence of opsin lacking the RET chromophore,
which acts as an inverse agonist, or by mutant opsins that
display the property of constitutive activity (215). Constitutive activity refers to the ability of an opsin to activate
Gt in the absence of any chromophore. Generally, a mutation that disrupts a putative salt bridge between Glu-113
and Lys-296 in the opsin apoprotein leads to constitutive
activity. Replacement of either Glu-113 or Lys-296 by a
neutral amino acid results in a mutant opsin with constitutive activity.
Other mutations such as G90D or A292E also result in
constitutive activity, presumably because the introduction of the negatively charged residue into the membraneembedded domain of the receptor affects the stability of
the Glu-113/Lys-296 salt bridge (44, 207). The crystal
structure shows that H2 is kinked around vicinal Gly
residues, Gly-89 and Gly-90, so that this region of H2 is
brought closer to H3 than to H1. This feature is interesting
in that Gly-90 comes into close proximity to the retinylidene Schiff base counterion, Glu-113, on H3. A mutation
that results in the replacement of Gly-90 by an Asp residue
causes congenital stationary night blindness in humans,
probably because of destabilization of the ionic interaction between Glu-113 and the Schiff base (269), or because of constitutive activity of the mutant opsin apoprotein that results from a disruption of a salt bridge between
Glu-113 and Lys-296 (207). The mechanism of constitutive
activity of opsins and the potential relevance of constitutive activity to visual diseases such as congenital night
blindness has been reviewed (208).
Opsin activity was reported to be only ⬃10⫺6 as
much as the activity of meta-II, a level much lower than
previously estimated (159). Therefore, the role of opsin
activity in bleaching desensitization and the pathophysiology of retinal degeneration may be much less than
previously suggested. Opsin can also be activated without
light by the addition of all-trans-retinal or various retinal
analogs (31, 220). Expressed mutant opsins can also be
activated by various retinal analogs (88). These results
show that diffusible ligands can activate opsin and provide information about the active-state conformation
of Rho.
Glu-113 is not located near C12 of the retinal polyene
as predicted by two-photon spectroscopy and NMR spectroscopy of retinal analogs and semiempirical quantum
mechanical orbital calculations (24, 82, 89, 90). However,
the C12 of RET does interact with Glu-181 from the E2
loop. Glu-181 may serve to influence the electron density
of the conjugated polyene system of the RET chromophore so that photoisomerization occurs exclusively at
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E. Cytoplasmic Surface Domain of Rhodopsin
1. Crystal structure of the cytoplasmic surface domain
The cytoplasmic domain of Rho comprises three cytoplasmic loops and the carboxy-terminal tail: C1 (amino
acids 65–70), C2 (amino acids 140 –150), C3 (amino acids
226 –246), and CT (amino acids 307–348). Loops C1 and
C2 are resolved in the crystal structure, but only residues
226 to 235 and 240 to 246 are resolved in C3. CT is divided
into two structural domains. C4 extends from the cytoplasmic end of H7 at Ile-307 to Gly-324, just beyond two
vicinal Cys residues (Cys-322 and Cys-323), which are
posttranslationally palmitoylated. The remainder of CT
extends from Lys-325 to the carboxy terminus of Rho at
Ala-348. The crystal structure does not resolve residues
328 to 333 in CT.
Loop C1 displays a rigid structural organization that
includes the basic side chains of His-65, Lys-66, Lys-67,
and Arg-69. The Lys-66 and Arg-69 side chains point toward the bilayer. His-65 sits close to the C4 loop at its H8
border. Lys-67 points toward the solvent and seems to
interact with CT, which runs nearly parallel to C1.
Loop C2 resembles an L-shaped structure when it is
viewed from a vantage point parallel to the putative membrane plane. A short ␤-barrel structure extends from Met143 to Phe-146 along the main long axis of Rho. A distinct
cytoplasmic border seems to be formed by the four polar
side chains of Lys-141, Ser-144, Asn-145, and Arg-147. C2
and C3 extend approximately to the same level above
what appears to be the cytoplasmic border of the receptor.
H5 probably extends into the cytoplasmic region but
breaks at the start of C3 at Leu-226, which is immediately
followed by an S-shaped loop structure that lies nearly on
the membrane surface. The loop extends nearly to the
lipid-facing side of H6 at Ala-235 and leaves a part of the
cytoplasmic surface of the seven helical bundle uncovered. The region from Gln-236 to Glu-239 is absent in the
crystal structure. The polar side chains of Ser-240 and
Physiol Rev • VOL
Thr-242 come close to a portion of CT around Ser-334 to
form a small cluster of hydroxyl groups. The tetrapeptide
Thr-243 to Ala-246 is ␣-helical.
The hallmark of the C4 loop is an ␣-helical stretch,
H8. H8 is connected to H7 by the Met-309/Asn-310/Lys-311
tripeptide that acts as a short linker. H8 lies nearly perpendicular to H7 and together with the Asn/Pro/X/X/Tyr
motif in H7 is one of the most highly conserved long
stretches of primary structure in Rho. The environment
around H8 is mainly hydrophobic, which may lead to
increased helical stability. H8 might be best described as
a cationic amphipathic ␣-helix, with Lys-311 and Arg-314
on one face of the helix and Phe-313, Met-317, and Leu-321
buried in the hydrophobic core of the bilayer between H1
and H7. H8 points away from the center of Rho, and it
appears that the palmitoyl groups linked to Cys-322 and
Cys-323 by thioester bonds may be anchored in the membrane bilayer, although this is not resolved in the crystal
structure. The helical structure of H8 is terminated by
Gly-324.
The residues at the extreme carboxy-terminal end of
CT compose the most solvent exposed region of Rho. CT
folds back over a small portion of the helical bundle at H1
and H7.
2. Structure-activity relationships in the cytoplasmic
domain of rhodopsin
A number of cytoplasmic proteins are known to interact exclusively with R*. Because the crystal structure
depicts the inactive Rho structure that does not interact
significantly with cytoplasmic proteins, the structure can
provide only indirect information about the relevant R*
state. In addition, two regions of the cytoplasmic surface
domain of Rho (amino acid residues 236 –239 and 328 –
333) are not fully resolved in the crystal structure. Potentially important structural information relevant to understanding protein-protein interactions in the visual
transduction cascade may be lacking in the reported
structure. It should also be noted that the borders between the transmembrane helical segments and the cytoplasmic loops do not necessarily represent the boundary
between aqueous phase and membrane bilayer. The helical segments generally tend to extend into the cytoplasmic aqueous phase. A number of amino acid residues that
are involved in Gt binding or activation, such as Glu-134 or
Lys-248, are situated in the helical segments.
Perhaps the most extensively studied receptor-G protein interaction is that of bovine Rho with Gt (96). Detailed biochemical and biophysical analysis of the R*-Gt
interaction has been aided by mutagenesis of the cytoplasmic domain of bovine Rho. Numerous Rho mutants
defective in the ability to activate Gt have been identified
(69). Several of these mutant receptors were studied by
flash photolysis (55, 68), light scattering (54), or proton
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length sensing cone pigments, Phe-261 is replaced by a
tyrosine that is involved in spectral tuning (38).
The Rho structure shows no interaction between
Gly-121 and RET C19 as was suggested by data from
regeneration of mutants with various chromophore analogs (83, 84). However, the structure does not rule out the
possibility that the two are coupled together during activation, or that they may come into proximity in R*, especially considering that Gly-121 replacement mutants exhibit dark activity even with bound RET (83, 87). In these
mutant opsins, RET displays partial agonist activity. Regeneration of opsin with C20-desmethyl retinal also produces a pigment with an increased level of basal activity
in the dark.
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MENON, HAN, AND SAKMAR
III. MECHANISM OF THE OPSIN SHIFT AND
SPECTRAL TUNING IN VISUAL PIGMENTS
The molecular interactions between the opsin protein and RET define the spectral properties of a particular
visual pigment. The broad visible absorption peaks of
visual pigments represent a collection of overlapping energy states. The absorbance at a particular wavelength
can be viewed as the energy difference between the chromophore ground state energy and the energy of its first
excited state. An increase in the energy gap causes a blue
Physiol Rev • VOL
shift (hypsochromic shift) from a particular reference
value. A decrease in the energy gap causes a red shift
(bathochromic shift) from a particular reference value.
Rho has been used as a model pigment for a variety
of chemical and spectroscopic studies to elucidate the
mechanism of the opsin shift. In this context, the opsin
shift may be defined as the difference between the ␭max
value of a RET Schiff base model compound (⬃440 nm) in
solution and that of Rho (500 nm). How is the positive
charge on the protonated Schiff base, which can be delocalized by the conjugated polyene system, stabilized by
the protein to create a unique envelope of chromophore
electronic distribution? One important result to arise from
the study of mutant bovine Rho pigments was the identification of Glu-113 as the RET Schiff base counterion
(171, 225, 267). This result combined with the characterization of other membrane-embedded carboxylic acid residues by both UV-visible spectroscopy (169, 171, 225, 267)
and microprobe resonance Raman spectroscopy (151)
suggested that the retinal-binding pocket in bovine Rho
was electrostatically neutral (24). Additional studies including the use of photoaffinity reagents (170, 266), retinal
analogs regenerated with site-directed mutants (212), or
site-directed mutant pigments (85, 86, 149, 169) led to a
more complete picture of the amino acid residues in the
membrane-embedded domain of Rho that interact with
the retinal chromophore.
The crystal structure now provides a clear picture of
the RET chromophore binding pocket in Rho (Fig. 4). One
of the key surprises with respect to previous models of
the opsin shift mechanism is Glu-181, which lies in the
extracellular linker between ␤3 and ␤4 and points toward
the center of the polyene chain. Glu-181 is highly conserved among vertebrate opsins, and blue and UV cone
pigments. However, the corresponding position in green
and red cone pigments is His-197. An extensive mutagenesis study of positively charged amino acids in human
middle (M-) and long (L-) wavelength sensitive pigments
identified His-197 and Lys-200 in E2 as the chloride ion
binding site in these pigments (257). Chloride binding in
the M- and L-sensing pigments causes a significant spectral red shift. It seems plausible that a chloride ion held in
place by His-197 and Lys-200 might interact with the
central portion of the polyene of RET in these pigments.
Although the role of Glu-181 in the opsin shift in Rho
has not yet been elucidated, data available to date suggest
that the dominant mechanism responsible for the opsin
shift is the interaction of dipolar amino acid residues with
both the ground-state and excited-state charge distributions of the chromophore (127, 150). This general mechanism is supported by the crystal structure in that the
RET binding pocket contains a large number of dipolar or
polarizable amino acid residues.
Because the visual pigments of many species have
been studied by absorption spectroscopy or microspec-
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uptake assays (9). The key overall result of these studies
is that C2, C3, and H8 are involved in R*-Gt interaction.
This finding is consistent with other approaches including
peptide competition studies (129, 155).
Although several biochemical studies implicated CT
in Gt activation (201, 247), alanine-scanning mutagenesis
failed to confirm that the proximal portion of CT was
required (191, 259). Recently, a combination of site-directed mutagenesis and peptide binding studies clearly
showed that the C4 loop region, which includes H8, is
involved in Gt binding and activation (55, 120, 155). Direct
evidence for the interaction between H8 and Gt comes
from studies using a synthetic peptide corresponding to
Asn-310 to Leu-321 of Rho, which was shown to bind to Gt
(155).
H8 is a cationic amphipathic helix that may bind a
phospholipid molecule, especially a negatively charged
phospholipid such as phosphatidylserine. In fact, spectroscopic evidence has been reported to show an interaction
between Rho and a lipid molecule that is altered in the
transition of Rho to meta-II (19, 103). High conservation
of Phe-313 and Arg-314 suggests that the amphipathic
character of H8 may be functionally important. H8 points
away from the center of Rho, and the area of the membrane surface covered by the entire cytoplasmic surface
domain appears to be roughly large enough to accommodate Gt in a one-to-one complex.
The CT distal to the palmitoylated Cys-322 and Cys323 residues appears to be highly disordered and dynamic
based on the results of site-directed electron paramagnetic resonance (EPR) spin labeling (135). However, the
CT appears to encode essential information concerning
Rho sorting and targeting. Mutation of Pro-347 is associated with a biosynthetic cellular transport defect that
leads to ADRP (243). In addition, CT interacts with a
cytoplasmic dynein light-chain Tctex-1. The Rho-dynein
interaction controls the polarized targeting of Rho in the
rod cell and in polarized epithelial cells when ectopically
expressed (246).
Conformational changes in the cytoplasmic surface
domain that are coupled to chromophore isomerization
are discussed in detail in section IV.
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RHODOPSIN
Physiol Rev • VOL
long as chromophore isomerization occurs). Only the efficiency of photon capture varies with photon energy and
the dynamic output relates only to the rate of photon
capture. For example, in the simplest case of a human
retina with only three individual cone cells, one cell of
each class, chromatic lights would be perceived to be
identical if they produced the same absorptions in all
three cells, and different if they did not. Thus human
trichromatic vision is the result of three independent
comparisons of rates of photon absorption by the three
cone types.
Three genomic and cDNA clones encoding the apoproteins of the human cone pigments were cloned and
characterized (177). The amino acid sequences of these
opsins are ⬃41% identical to that of human Rho. The Mand L-sensing opsins are ⬃96% identical to each other and
⬃43% identical to the S-sensing opsin. Previously, the
spectral properties of human cone pigments had been
studied by a variety of techniques ranging from psychophysical color matching to microspectrophotometry (29).
More recently, the human cone pigment genes were expressed in tissue culture cell lines, reconstituted with
RET, and studied by UV-visible spectroscopy (160, 190).
The ␭max values reported in two studies were as follows:
S 426 nm, M 530 nm, L 552 nm and 557 nm for polymorphic variants (160); and S 424 nm, M 530 nm, and L 560 nm
(190). The ␭max value of the human S-sensing pigment was
recently reported to be 414 nm from a measurement of its
absolute spectrum (66). These studies also confirmed the
correct functional assignments of the pigments that had
been based on genetic analysis of the cloned pigment
genes.
Analysis of the arrangement of the cone opsin genes
on the X chromosome has led to a preliminary understanding of the molecular genetics of inherited variations
in color vision (176). In males with normal color vision it
was proposed that a single L-sensing opsin gene resides
with one or more M-sensing opsin genes in a head-to-tail
tandem array. Thus, in one type of color vision defect,
anomalous trichromacy, unequal intragenic recombination can theoretically result in an opsin gene that is a
hybrid between M- and L-sensing genes. It was proposed
that these hybrids would have anomalous spectral properties (176, 179). This hypothesis was confirmed experimentally at the level of the photoreceptor pigment by
obtaining absorption spectra for heterologously expressed hybrid pigments responsible for anomalous
trichromacy (161). Quantitative polymerase chain reaction (PCR) methods have been applied to evaluate in
more detail the numbers and ratios of M- and L-wavelength sensing cone opsin genes in males with normal
color vision. It was found that many subjects had more
numerous cone opsin genes than previously suggested
and that in many cases more than one L-wavelength opsin
gene was present on the X chromosome (178, 180). The
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trophotometry of visual organs, it is straightforward to
classify visual pigments solely on their visible ␭max values.
Another common classification system is to group visual
pigments based on the photoreceptor cell type of the
retina in which they are found (29). Rod cells, responsible
for dim-light (scotopic) vision, contain Rho (“red” opsin).
Cone cells, responsible for bright-light (photopic) and
color vision, contain iodopsins (“violet” opsins), also
known as cone pigments or color vision pigments. The
cloning and characterization of opsins from a variety of
species have allowed further comparisons and phylogenetic classifications based on their structural, spectral,
and biochemical properties (8). The homology in the opsin family of genes indicates that divergent evolution
occurred from a single precursor RET-binding protein to
form short (S-) and L-wavelength absorbing ancestors.
The L-sensing ancestor diverged to form L- and M-sensing
pigments. The S-wavelength sensing ancestor then diverged to form an S-sensing pigment and the family of
Rhos and Rho-like M-sensing pigments. These evolutionary models can be tested experimentally by ancestral
gene reconstruction using gene synthesis methodology
(39, 40).
Whereas all Rho pigments generally absorb maximally at ⬃500 nm, the ␭max values of cone cell pigments
vary. Individual cone pigments display ␭max values from
the near UV (⬃380 nm) to the far visible red (⬃600 nm).
Different vertebrate species may have more or fewer cone
types, but all cone pigments share the same RET (or
11-cis-3,4-didehydroretinylidene) chromophore. Therefore, any differences in spectral properties among a collection of cone pigments must be related to differences in
the structures of their RET binding pockets, which is
ultimately related to amino acid sequence, although not
necessarily strictly related. Cone pigments also display a
number of other distinct biochemical properties, including fast rates of R* decay and chromophore regeneration
compared with those of Rho. Specific amino acid residues
in the chromophore-binding pocket seem to control these
biochemical properties independently of spectral tuning (102).
Despite two centuries of study, the genetics, physiology, and psychophysics of human color vision remain of
great interest. Human trichromatic color vision requires
the presence of three physiological detection systems,
which correspond to the three classes of photoreceptor
cone cells. Each cone class (S-wavelength sensing, Mwavelength sensing, and L-wavelength sensing) contains a
different pigment molecule. The peaks of absorbance are
different for the three cone-cell pigments, but their absorbance spectra overlap considerably so that a combination
of one, two, or three cone types reacts more or less
strongly to a given light stimulus. A key element is that the
response of a particular cone cell is the same no matter
what the energy is of the photon that it captures (i.e., as
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MENON, HAN, AND SAKMAR
molecular genetics of blue cone monocromacy have also
been investigated (173). A genetic model to account for
the absence of the green and red genes has been tested in
transgenic mice (256). The results suggest that a conserved 5⬘-region interacts with the green or red gene
promoter to determine which gene is expressed in a given
cone cell.
IV. MOLECULAR MECHANISM OF RECEPTOR
ACTIVATION
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Although the crystal structure of Rho does not provide direct information about the structure of R* or about
the dynamics of the Rho to R* transition, it does provide
a wealth of information that should help to design experiments using existing methods to address specific questions regarding the molecular mechanism of Rho activation. An inactive receptor conformation must be capable
of changing to an active conformation, which catalyzes
nucleotide exchange by a G protein. In Rho, the RET
chromophore is in its “off” state, but switches to the “on”
state 11-trans geometry by photoisomerization leading to
the R* conformation of the receptor. Any amino acid side
chain that is involved in such a conformational change
must be able to exist in two different sterical and/or
electrostatic states, which can be designated as on or off
depending on whether the particular state is observed in
the active or inactive receptor, respectively (62, 245).
Because receptor activation involves conformational
changes at different topological locations, an individual
amino acid or a structural domain in the chromophorebinding pocket may be in its on state without necessarily
locking the Gt interaction domain into its active conformation, and vice versa. Thus a concerted transition of
individual groups may create the overall active conformation, but it may not be clear whether or not all of the
measurable structural changes are actually essential. A
minimal subset of molecular groups may be able to govern the transition to an active receptor conformation
irrespective of the binary states (off/on) of all possible
groups usually participating in receptor activation. The
functional hierarchy of individual group transitions may
become obvious only in mutant, or in otherwise modified
receptors, in which the state of an individual amino acid
side chain can be influenced, or even locked, into one of
two states. Ideally, it should be possible to alter the
receptor activation pathway such that certain transitions
become uncoupled from other transitions. It has been
argued that the simplified binary model of individual molecular transitions provides a framework to analyze functional data obtained from recombinant Rhos (62, 91).
According to this argument, only molecular changes
occurring between dark Rho and the R* state are relevant.
Thus FTIR-difference spectroscopy has proven to be a
well-suited technique for the study of light-induced conformational changes in retinal proteins (60, 218, 229).
FTIR-difference spectroscopy measures frequency shifts
of only those vibrational modes that are affected during photoproduct formation, irrespective of whether
they are caused by the protein constituents or by the
chromophore. In contrast, resonance Raman spectroscopy measures specifically chromophore vibrations and
thereby helps to define the chromophore geometry and
the Schiff base protonation state without the potential
drawback of overlapping absorptions of amino acid side
chains. Both techniques have contributed significantly to
knowledge of molecular changes occurring during meta-II
formation. Application of these techniques to site-directed Rho mutants has allowed the identification of a
number of on/off states of particular amino acids during
R* formation.
For example, among the membrane-embedded carboxylic acid groups, light-induced changes of protonation
states or hydrogen-bond strengths were deduced from
characteristic frequency shifts of C⫽O stretching vibrations of protonated carboxylic acid groups in FTIR-difference spectra. Their assignment to specific Asp or Glu
residues was based on the disappearance of specific difference bands in site-directed mutants and revealed that
Asp-83 (57, 209) and Glu-122 (57) are protonated in both
dark Rho and meta-II, whereas Glu-113 is ionized in the
dark state and becomes protonated in meta-II (104).
Evidence for the importance of steric interactions
distal to the Schiff base comes from FTIR studies using
ring-modified retinal analogs. Increased flexibility, as in
5,6-dihydro (71) or 7,8-dihydro analogs (196), reduces the
usually observed torsions along the retinal chain in the
intermediate trapped at 80 K where bathorhodopsin
would normally be stable. In addition, the protein conformational changes observed at temperatures that stabilize
the meta-I or meta-II intermediates differ from those observed in native Rho. In an extreme case, illumination of
a pigment regenerated with a retinal analog lacking the
cyclohexenyl ring fails to induce the complete set of
infrared absorption changes typical of the meta-II conformation and results in reduced Gt activation (105). Therefore, the cyclohexenyl ring must transmit important steric
changes to the protein. This model seems consistent with
the location of the RET ring in the crystal structure
of Rho.
Movement of ␣-helical domains is known to be involved in the signal transduction mechanisms of some TM
receptor proteins, such as the bacterial chemoreceptors
(162), and has been shown to occur during the proton
pumping cycle following retinal isomerization in bacteriorhodopsin (BR), the seven-transmembrane segment lightdriven proton pump (237–239). Recent studies have suggested that steric and/or electrostatic changes in the
ligand-binding pocket of Rho may cause changes in the
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RHODOPSIN
Physiol Rev • VOL
The functional interaction of H3 and H6 was further probed in a study in which metal ion binding sites
were introduced between the cytoplasmic surfaces of
transmembrane helices with the aim of restraining specific activation-induced conformational changes (228).
Pairs of His residues are capable of chelating metal ions
such as Zn(II) if the distance and geometry between the
residues is appropriate. His residues substituted for the
native amino acids at the cytoplasmic ends of H3 and
H6, but not H5 and H7, created mutant proteins that
were able to activate Gt in the absence, but not in the
presence, of metal ions. It was concluded that specific
metal-ion cross-links between positions 138 and 251, or
141 and 251, on H3 and H6, respectively, prevented
receptor activation. These results indicated a direct
coupling of receptor activation to a change in the spatial disposition of H3 and H6. This could occur if movements of H3 and H6 were coupled to changes in the
conformation of the connected intracellular loops,
which are known to contribute to binding surfaces and
tertiary contacts of Rho with Gt (68, 69).
More direct evidence for changes in interhelical interactions upon receptor activation were provided by extensive site-directed spin labeling and EPR spectroscopy
studies of the transition of Rho to R* in modified, or
expressed mutant pigments. The results suggested a requirement for rigid body motion of transmembrane helices, especially H3 and H6, in the activation of Rho (65). A
slight reorientation of helical segments upon receptor
activation is also supported by experiments using polarized attenuated total reflectance infrared difference spectroscopy (46). Finally, movement of H6 was also detected
by site-specific chemical labeling and fluorescence spectroscopy (51). The structural rearrangement of helices
upon activation might not result in an R* structure that is
drastically different from that of Rho since an engineered
receptor with four disulfide bonds (between the cytoplasmic ends of H1 and H7 and H3 and H5, and the extracellular ends of H3 and H4 and H5 and H6) was still able to
activate Gt (235).
Because the arrangement of the seven transmembrane segments is likely to be evolutionarily conserved
among the family of GPCRs, the proposed motions of H3
and H6 may be a part of a conserved activation mechanism shared among all receptor subtypes (74, 110). In
other class A GPCRs, agonist ligand binding would be
coupled to a change in the orientations of H3 and H6.
However, it should also be noted that the helix movement
model of GPCR activation has been demonstrated so far
only in the class A GPCR family. The recent crystal structure of the extracellular amino-terminal domain of a
metabotropic glutamate receptor (mGluR) indicates that
ligand-mediated receptor dimerization most likely plays a
role in the activation mechanism (132). Other GPCRs in
the class C family include the GABA receptor, the cal-
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relative disposition of TM helices within the core of the
receptor. These changes may be responsible for transmitting a “signal” from the membrane-embedded binding site
to the cytoplasmic surface of the receptor. Trp mutagenesis (149), mutagenesis of conserved amino acid residues
on H3 and H6 (85, 86), and the introduction of pairs of His
residues at the cytoplasmic borders of TM helices to
create sites for metal chelation (228) have recently provided insights regarding the functional role of specific
helix-helix interactions in Rho.
The indole group of a Trp amino acid residue is often
used as a noninvasive environment-sensitive probe of
protein structure because of its unique absorption and
fluorescence properties. UV-absorption spectroscopy has
suggested that the local protein environment around Trp
residues changes during the conversion of Rho to meta-II
(205). In addition, a linear dichroism study of UV-difference bands indicated a reorientation of an indole side
chain during the meta-I to meta-II conversion (37). To
determine which Trp residues were responsible for the
absorbance changes previously reported, each of the five
Trp residues in Rho was replaced by either a phenylalanine or a tyrosine. Replacements of Trp-126 and Trp-265
resulted in a decrease in the magnitudes of the UV-difference peaks at 294 and 302 nm. These results were consistent with a change of Trp-126 and Trp-265 to more
polar environments during activation of the receptor
(149). It was further suggested that the photoactivation of
Rho involved a change in the relative disposition of H3
and H6, which contain Trp-126 and Trp-265, respectively,
within the ␣-helical bundle of the receptor.
H3 of Rho contributes many amino acid side chains
that interact with RET, including Glu-113 and Gly-121.
Replacement of Gly-121 resulted in a relative change of
opsin selectivity for reconstitution with all-trans-retinal
over RET in membranes. All-trans-retinal could efficiently
bind and activate the mutant opsins, even though none of
them displayed constitutive activity. The G121L replacement also caused RET to become a partial agonist in the
G121L pigment (87). These results showed that H3, and
Gly-121 in particular, were important in determining the
specificity of the chromophore-binding pocket in Rho.
The cyclohexenyl ring portion of the RET binding site is
bordered by Gly-121 and Phe-261, consistent with the
notion that functional interactions between H3 and H6
mediated by the chromophore are crucial for receptor
photoactivation. Phe-261 was also found to be involved in
the control of the decay of an early photointermediate
(blue shifted intermediate) in a study of site-directed
mutants by laser photolysis and rapid time-resolved absorption difference spectroscopy (106). Similar measurements of recombinant expressed Rho and Glu-113 counterion mutants were also recorded to provide a basis for
understanding the relative movement of H3 and H6 required for receptor activation (107).
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MENON, HAN, AND SAKMAR
Physiol Rev • VOL
plex revealed an infrared difference band that could be
assigned to protonation of Glu-134 (56, 61, 183).
Other techniques have been successful in monitoring
light-dependent structural alterations occurring during
meta-II formation as well. As measured by hydrogendeuterium exchange of buried peptide groups using timeresolved FTIR spectroscopy, the kinetics of exchange in
R* is increased. This result implies that buried regions of
the peptide backbone structure become more accessible
to solvent in forming meta-II (210). Light-dependent intramolecular charge movements in Rho can be monitored
kinetically in vivo by recording an electroretinogram
(ERG). The early phase of the ERG represents electrostatic potential in the oriented photoreceptor molecule. In
fact, Rho displays a light-induced early receptor potential
(ERP) akin to “gating currents” in voltage-dependent ion
channels. Remarkably, the ERP can be recorded directly
in giant cells that heterologously express Rho (223, 240).
This method in combination with site-directed mutants
may be able to identify specific amino acid side chains
that move or exchange protons during the Rho photocycle.
Extensive site-directed spin labeling and EPR spectroscopy have been employed over the past 5 years to
map the cytoplasmic domain of Rho (6, 33). EPR can
provide information about side chain mobility, secondary
structure, and solvent accessibility in both Rho and metaII. Ultimately, this information can be assembled to form
a map of structural changes that accompany receptor
activation. Time-resolved (63) and static EPR spectroscopy studies (211) on site-specific spin-labeled Rho
showed that the cytoplasmic terminations of H3 and H7
undergo structural rearrangements in the vicinities of
Cys-140 and Cys-316, respectively. These changes have
been specifically assigned to the meta-II conformation.
Cys-140 is close to the highly conserved Glu-134/Arg-135/
Tyr-136 tripeptide at the cytoplasmic border of H3.
Site-directed spin labeling of the amino acid residues
from Tyr-306 to Leu-321 was also carried out. The information obtained regarding conformational changes in H8
upon meta-II formation was limited by the relative lack of
reactivity of the Cys residues engineered into positions
317, 318, 320, and 321. However, structural changes were
detected at positions 306, 313, and 316, consistent with
movements of the nearby H6 and with biochemical evidence for a light-dependent interaction between Cys-65
and Cys-316 in native Rho (4, 32). Consistent with the
notion of light-dependent structural changes in the vicinity of the cytoplasmic end of H7 is the observation that a
monoclonal antibody with an epitope that was mapped to
the amino acid sequence 304 –311 bound only to R* and
not to Rho (1). Only relatively small light-dependent structural changes were noted in and around the C1 loop when
residues 56 –75 were individually probed by site-directed
spin labeling (5).
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cium-sensing receptor, and some pheromone receptors.
Currently, the activation mechanism in the class B
GPCRs, including the glucagon receptor and related peptide receptors, is not clear (250). It remains likely in both
class B and C receptors that agonist binding, which requires the NT, causes TM helix movement to transmit a
signal across the membrane.
Some conformational changes must occur at the cytoplasmic surface of Rho to produce R* that can activate
Gt. Does the Rho structure provide any potential insights
that might help to predict the identity of these conformational changes? Does H8 unwind or come off the membrane surface activation? Do C2 and C3, which are known
to be important for Gt activation, move? What are the
active state conformations of the conserved Glu-134/Arg135/Tyr-136 in H3 and the conserved Asn-302/Pro-303/Val304/Ile-305/Tyr-306 in H7?
Synthetic Rho-derived peptides have been shown to
compete with native Rho for Gt binding (129, 155). This
has allowed the identification of the C2, C3, and the
proximal portion of CT as Gt binding sites. Site-directed
mutagenesis has further characterized groups of amino
acids in these regions implicated in Gt binding and activation (55, 68, 69, 155). Mutagenesis experiments can be
designed to elucidate the light-dependent alterations of
physical or chemical states of specific amino acids required for Gt activation. For example, structural changes
in the cytoplasmic surface domain of Rho were suggested
by changes in the reactivities of Cys residues introduced
at various positions by site-directed mutagenesis (123).
Conformation-dependent interhelical interactions and tertiary contacts on the cytoplasmic surface were also
probed biochemically using site-directed disulfide bond
formation (234) and/or expression of split receptors (264,
265). With the use of the core of Rho as a scaffold,
cytoplasmic loops of other GPCRs were substituted for
those of Rho; the results of G protein activation experiments suggested that C2 and C3 might have distinct roles
in Gt activation and G protein subtype specificity (261).
These results also indirectly support the general activation mechanism of helix movement that transmits a signal
from the membrane-embedded cored to the surface loops
of the receptor.
Additional biophysical methods have been employed
to probe conformational changes at the cytoplasmic surface domain concomitant with receptor activation. FTIR
spectroscopic determination of protonation states or hydrogen bonding of specific amino acid side chains on the
cytoplasmic surface of Rho is less advanced than for
residues in the hydrophobic core of the receptor. This is
in part due to the relatively small contribution of waterexposed amino acids to infrared absorption changes in
typical FTIR samples (hydrated films) (70). Recently, attenuated total reflectance (ATR) FTIR difference spectroscopy of the R*-Gt (or peptides derived from Gt) com-
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RHODOPSIN
V. THE RHODOPSIN PHOTOCYCLE AND THE
CHROMOPHORE REGENERATION PATHWAY
As outlined above, photoisomerization of the RET chromophore induces the active receptor conformation R* from
the inactive conformation Rho (30, 192, 263). R* is spectroscopically indistinct from the Rho photoproduct meta-II,
which is characterized by an unprotonated Schiff base chromophore linkage and a ␭max value of 380 nm. At low temperature, a number of photointermediates that characterize
Physiol Rev • VOL
the transition of Rho to R* can be trapped and studied by a
variety of spectroscopic techniques. The classical photolysis
pathway of Rho at low temperatures proceeds according to
the following scheme: Rho (500 nm) 3 bathorhodopsin
(batho) (540 nm) 3 lumirhodopsin (lumi) (497 nm) 3 metaI (478 nm) 7 meta-II (380 nm).
Laser flash photolysis coupled with nanosecond
time-resolved UV-visible spectroscopy has identified a related photocycle that occurs at or near physiological temperature (124): Rho (500 nm) 3 batho (543 nm) 7 blueshifted intermediate (BSI) (477 nm) 3 lumi (497 nm) 3
meta-I (480 nm) 7 meta-II (380 nm). Meta-II decays to
metarhodopsin III (meta-III) (450 nm) and finally to opsin
plus free 11-trans-retinal (141). The chromophore photoisomerization occurs on an ultrafast time scale and was
observed to be a vibrationally coherent process (255). The
Rho photocycle is summarized in Figure 6.
Significant progress has been made in understanding
structure-function relationships in the photointermediates
of Rho through optical, resonance Raman, FTIR, NMR, and
other spectroscopic studies on a variety of native visual
pigments, chemically modified pigments, and artificial pigments (native opsins reconstituted with synthetic retinal
analogs; Refs. 25, 57, 62, 67, 89, 122, 124, 142, 149, 151, 196,
203, 224, 229). These types of studies provide specific detailed information about chromophore structures and about
dynamic chromophore-opsin interactions.
In addition to probing the chromophore, specific absorbance changes by aromatic residues can also be measured in
early Rho photointermediates (140). Trp-265 is a useful
probe of specific chromophore-protein interactions during
the Rho photocycle, especially given its proximity to the
RET chromophore. It can be probed by UV absorption spectroscopy, fluorescence spectroscopy, or UV resonance Raman spectroscopy (126). The photocycle can also be studied
under a variety of conditions that might provide a basis for
identifying specific amino acid residues that might be involved in intramolecular proton transfer reactions. For example, the pH dependency was determined for the formation of Rho photoproducts from lumi to meta-II (109). These
types of experiments could be carried out with site-directed
mutants as well in future studies.
R* becomes phosphorylated by Rho kinase, and its
ability to activate Gt is diminished by the binding of
arrestin to phosphorylated R*. The RET Schiff base linkage also becomes accessible to water in its R* conformation. Through an autocatalytic mechanism that involves
Glu-113 (225), the RET Schiff base is hydrolyzed and
all-trans-retinal (ATR) is released in the outer segment of
the rod cell. ATR is reduced to all-trans-retinol in the
outer segment by ATR dehydrogenase and NADPH. The
all-trans-retinol is transported out of the rod cell where it
binds to the interphotoreceptor retinoid-binding protein
(IRBP). IRBP carries the all-trans-retinol to the retinal
pigmented epithelium (RPE) where the chromophore is
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An EPR study of Rho mutants with a substitution of
Glu-134 showed that the mutant receptors displayed an
EPR signature consistent with a partially activated conformational state in the dark (118). This finding seems to
be consistent with extensive earlier studies of Glu-134
replacement mutants. The structural change detected by
EPR spectroscopy may be directly related to the apparent
requirement for protonation of Glu-134 upon R* formation. A rearrangement of neighboring hydrogen-bonding
partners may be necessary for protonation of Glu-134 to
occur. The pH profile of Gt activation (59) as well as the
abolishment of the uptake of two protons in mutant
E134Q (9) suggests the existence of other titratable
groups influenced by Glu-134. Glu-134 interacts primarily
with Arg-135 in Rho, but it is not clear whether Glu-134
would interact with other side chains in R*, or simply be
in a position to interact with bound Gt. The Glu-134/Arg135 dipeptide may form a functional microdomain that is
responsible for inducing the release of GDP from R*bound Gt (2, 147).
Any model of receptor activation has to account for
the fact that the chemical environment of the Schiff base
is altered so that net proton transfer occurs between the
Schiff base imine and Glu-113 (104). This change in the
RET binding pocket must be transmitted to the cytoplasmic surface of the receptor. The Rho crystal structure is
consistent with the helix movement model of receptor
activation (65, 228) since it provides a structural basis to
explain how chromophore isomerization could lead to
displacement of H3 and H6 that would subsequently result
in a change in orientation of Glu-134 at the cytoplasmic
border. The structure suggests possible contacts between
the cyclohexenyl ring of the chromophore and H3, which
should change upon photoisomerization (26). At the
Schiff base end of the chromophore, the C20 methyl group
seems to interact with Trp-265, and this interaction
should also change upon isomerization. Other key interhelical constraints are expected to be directly sensitive to
chromophore isomerization, including those mediated by
Phe-294, Ala-299, Asn-302, and Tyr-306. Any concerted
disruption of stabilizing interhelical interactions may be
expected to lead to helix movement and rearrangement of
the helical bundle.
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MENON, HAN, AND SAKMAR
released and transported into the RPE cell. The all-transretinol (pro-R at C15) is palmitoylated by lecithin retinol
acyl transferase (LRAT). Isomerohydrolase acts on the
all-trans-retinyl ester to form 11-cis-retinol (pro-S at C15).
The 11-cis-retinol is oxidized to RET by 11-cis-retinol
dehydrogenase in the RPE cell endoplasmic reticulum
lumen. The RET is released at the apical pole of the RPE
cell where it binds to IRBP and is transported to the rod
cell. The RET binds to the opsin apoprotein to regenerate
a Rho pigment. The details of the chromophore regeneration pathway are presented elsewhere (206, 219).
How does the retinal chromophore enter or exit the
membrane-embedded core of the opsin apoprotein? The
extracellular surface domain is very well organized, with
many half-buried hydrophobic cores as well as H-bond
networks. Considering this fact and the hydrophobicity of
RET, it seems reasonable to assume that the chroPhysiol Rev • VOL
mophore enters and exits the membrane-embedded core
of the receptor via the membrane phospholipid bilayer.
This implies that the chromophore would have to pass
between two adjacent transmembrane helices. There is a
much wider gap between H5 and H6 than between any of
the other helix pairs, largely due to the numerous Phe
residues along one face of H5. One or two of the Phe
residues might be imagined to move back and forth to
allow the ligand to enter or exit.
VI. INTERACTION OF R* WITH MOLECULES
INVOLVED IN VISUAL TRANSDUCTION
Although studies of the photocycle intermediates of
Rho provide insights into dynamic changes in protein
conformation and RET-protein interactions, R* is defined
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FIG. 6. The Rho photocycle. Photoisomerization of the RET chromophore to its 11-trans form is the only lightdependent event in vertebrate vision. The RET chromophore is a derivative of vitamin A1 with a total of 20 carbon atoms
(Fig. 1). Photoisomerization in Rho occurs on an ultrafast time scale with photorhodopsin as the photoproduct formed
on a femtosecond time scale (255). The photolyzed pigment then proceeds through a number of well-characterized
spectral intermediates (141). As the protein gradually relaxes around 11-trans-RET, protein-chromophore interactions
change and distinct ␭max values are observed. The Schiff base imine remains protonated, presumably due to stabilization
by its counterion, Glu-113, through metarhodopsin I (meta-I). Meta-I is in a dynamic equilibrium under physiological
conditions with metarhodopsin II (meta-II), which is characterized by an unprotonated Schiff base imine and a
dramatically blue-shifted ␭max value (380 nm). Meta-II is the photoproduct that is able to catalyze guanine nucleotide
exchange by Gt. Meta-II eventually decays to free 11-trans-RET and opsin apoprotein. Important photochemical
properties of Rho in the rod cell disk membrane include a very high quantum efficiency (⬃0.67 for Rho versus ⬃0.20 for
RET in solution) and an extremely low rate of thermal isomerization. Some fishes, reptiles, or aquatic mammals use a
derivative of vitamin A2 (11-cis-3,4-didehydroretinylidene), which contains an additional carbon-carbon double bond in
the ring. In contrast to vertebrate vision, invertebrate vision is generally photochromic; a photoactivated invertebrate
pigment can be inactivated by absorption of a second photon that induces isomerization to the ground state cisconformation.
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RHODOPSIN
surface domain of R* and at least two regions of Gt, the
carboxy-terminal tail of the ␣-subunit and the carboxyterminal tail of the ␥-subunit (73, 95, 119). In fact, the
binding of Gt, or certain peptides derived from Gt, can
stabilize R* (53, 120). The stabilization of R* by Gt is
related to the so-called “GTP effect” originally described
for hormone receptors in hepatocyte membrane preparations (216). The molecular basis of the GTP effect is that
receptors are stabilized in their high-affinity agonist conformation by the binding of G proteins. The addition of
excess GTP causes G protein dissociation, and receptors
return to a low-affinity agonist binding conformation. R*
is stabilized in its high affinity 11-trans chromophorebound state by interaction with Gt.
FIG. 7. Role of Rho in the biochemical cycle of visual transduction. Key protein-protein interactions take place on
the disk membrane of the photoreceptor rod cell. Photoisomerization of the RET chromophore to all-trans produces R*.
R* interacts with Gt to catalyze guanine nucleotide exchange by Gt. The GTP-bound ␣-subunit of Gt activates cGMP
phosphodiesterase (not shown). Rod cGMP levels fall and a plasma membrane cGMP-gated cation channel closes to
cause hyperpolarization of the rod cell. R* is turned off in ⬍1 s by phosphorylation by Rho kinase and binding of arrestin
to the phosphorylated R*. R* also decays more slowly due to Schiff base hydrolysis and loss of all-trans-retinal, which
is then reduced to all-trans-retinol by retinal dehydrogenase and NADPH. The biochemical cycle of chromophore
regeneration is largely carried out in the retinal pigment epithelial cells. Protein phosphatase 2A can dephosphorylate
phospho-opsin to produce opsin apoprotein. One key feature of the vertebrate visual system is that each Rho molecule
is used only once.
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as the conformation that catalyzes guanine nucleotide
exchange by Gt and propagates and amplifies the signal of
photon capture. R* is the key to the biochemical amplification cascade of the vertebrate visual system (Fig. 7). R*
is spectrally indistinct from the meta-II photoproduct of
Rho. Meta-II displays a ␭max value of 380 nm due in part to
the presence of an unprotonated RET Schiff base chromophore linkage. It is now widely accepted that meta-II
exists in two isospectral forms (meta-IIa and meta-IIb)
that differ at least in their protonation states (9, 10).
Whereas meta-IIa cannot bind to Gt, meta-IIb may be
identical to R*.
The transfer and amplification of signal from R* to Gt
involves specific interactions between the cytoplasmic
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MENON, HAN, AND SAKMAR
Physiol Rev • VOL
play calcium-dependent conformational changes (e.g., recoverin or S-modulin, isolated from bovine and frog rods,
respectively), the mechanistic role of calcium, at least in
terms of its effect on Rho phosphorylation, is not yet
understood (115).
The biochemical mechanisms of background and
light adaptation are known to involve light-dependent
changes in intracellular concentrations of calcium as well
as cGMP. Calcium levels influence the rate of accumulation of R* (133). However, the precise effects of changes
of calcium and cGMP concentrations on the rate of recovery during adaptation remain to be determined (78).
The details of the regulation of the biochemical activities
of Rho kinase, phosducin, recoverin, Gt, and cGMP phosphodiesterase and their exact roles in signal termination
and signaling system recovery and adaptation also remain
to be elucidated in detail.
VII. RHODOPSIN MUTATIONS AS A CAUSE
OF HUMAN DISEASE
Defects in G protein-mediated signaling are involved
in the pathogenesis of a number of human diseases, both
acquired and congenital (64, 231). The specific defects are
often not fully understood, but two common themes of
molecular pathophysiology generally emerge. First, the
signal transduction machinery may be present and in its
proper cellular location, but not properly functioning.
Second, some element of the machinery may have failed
to be properly synthesized, processed, or transported.
These two paradigms appear to apply to Rho in cases of
congenital stationary night blindness and retinitis pigmentosa, respectively.
Retinitis pigmentosa is a group of hereditary progressive blinding diseases with variable clinical presentations
(21). One form of the disease, ADRP, was linked to a
mutation in the gene for Rho (50). This first mutation
discovered to be linked to ADRP was at codon 23 and
would result in the change of a Pro residue to a His
residue in the NT of Rho. Over 70 sites of rhodopsin
mutation have been reported subsequently in ADRP patients (22, 28, 34, 48 –50, 76, 156, 163, 197, 202, 226, 241).
Recently, enough detailed phenotypic information has
also been collected to attempt to group genotypes according to disease classes (43, 163).
Several studies have been carried out in which sitedirected mutant opsin genes corresponding to ADRP genotypes were prepared (113, 164, 243). When expressed in
tissue culture, the mutant opsins display a heterogeneity
of spectral properties, biochemical properties, and cellular transport behavior. Some mutants are defective in
chromophore binding, others in cellular transport and
insertion into the plasma membrane. However, some mutations have no apparent effect in vitro. One particularly
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Perhaps the most extensively studied receptor-G protein interaction is that of bovine Rho with bovine Gt (96),
which can both be purified readily from bovine retinas.
Detailed biochemical and biophysical analysis of the
R*-Gt interaction has been aided by mutagenesis of the
cytoplasmic domain of bovine Rho. Numerous Rho mutants defective in the ability to activate Gt have been
identified (69). Several of these mutant receptors were
studied by flash photolysis (68), light scattering (54), proton uptake assays (9), or photoregeneration assays (55,
120). The salient results of these studies were that C2, C3,
and CT (especially in the region involving H8) were involved in R*-Gt interaction. These findings were consistent with other approaches including peptide competition
studies using native Rho (129). A class of interesting
receptor mutants was also identified in which spectrally
normal meta-II-like pigments were formed upon illumination. The photolyzed mutants bound Gt in the presence of
GDP, but failed to release the bound Gt in the presence of
GTP (54, 68). These results were consistent with the idea
that Gt binding and Gt activation were discrete steps
mechanistically, which could be uncoupled by specific
amino acid substitutions on the cytoplasmic surface of
the receptor.
A number of cytoplasmic proteins in addition to Gt
are known to interact with R*. These include Rho kinase,
which phosphorylates R* at specific serine residues (Ser334, Ser-338, and Ser-343) (157, 184, 198). Reconstitution
experiments suggest that light-dependent phosphorylation of Rho is catalyzed primarily by Rho kinase, and not
by protein kinase C (185). Phosphorylation decreases the
effective lifetime of R*, but paradoxically shifts the metaI-meta-II equilibrium toward meta-II (75). Signal quenching requires that arrestin binds to phosphorylated R* to
prevent it from activating Gt. The role of the distal carboxy-terminal tail in the shut-off of the light signal in the
rod cell was shown convincingly in a study using a transgenic mouse strain. The mice, whose rod cells expressed
a Rho transgene with a truncated carboxy-terminal tail,
were studied by electrophysiological techniques. Phosphorylation of the carboxy-terminal tail was required for
termination of light-induced signaling (42). A summary of
some engineered animal models used to study rod cell
physiology is presented in Table 1.
A detailed discussion of the use of physiological
methods to address questions related to the termination
of the flash response and to adaptation mechanisms has
been presented (14, 15). For example, it appears that light
adaptation cannot depend on high levels of phosphorylation of nonbleached Rho in rod cells (23). Light adaptation is regulated by intracellular calcium concentration.
Intracellular calcium concentration is high in the dark but
low in light-adapted rod cells. Although it is likely that the
change in intracellular calcium concentration is detected
by several cytoplasmic calcium-binding proteins that dis-
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RHODOPSIN
Physiol Rev • VOL
VIII. CONCLUSION
The recent report of the crystal structure of bovine
Rho provides a unique opportunity to address questions
related to the structural basis of Rho physiology in the
vertebrate visual transduction cascade. Over the past decade a remarkable amount of information about structure-activity relationships in Rho, and other GPCRs, has
been obtained using techniques of molecular biology. The
crystal structure of Rho also provides a chance to evaluate the quality of this information. Site-directed mutant
pigments have been employed to elucidate key structural
elements, the opsin-shift mechanism, and the mechanism
of receptor photoactivation. Much of the past work on
Rho was aided by reasonable molecular models based on
projection density maps of the membrane-embedded domain of Rho obtained from cryoelectron microscopy of
Rho in reconstituted bilayers.
Perhaps the biggest surprise in the crystal structure
was the role of the extracellular surface domain in defining the RET binding pocket. Whether or not this feature
carries over to other GPCRs remains to be determined.
One particular advantage of studying Rho has been the
opportunity to employ various spectroscopic methods,
especially in combination with site-directed mutagenesis.
Optical spectroscopy and resonance Raman spectroscopy
are possible because of the presence of the RET chromophore, which is probed as a sensor of chromophoreprotein interactions. Difference spectroscopy techniques,
such as FTIR and UV-visible difference spectroscopy,
make use of the chromophore as an optical switch. Overexpression of recombinant Rho allows a variety of biophysical methods to be used to address particular questions related to protein and chromophore conformational
changes. The recent use of engineered animal models to
study not only rod cell development and cellular pathophysiology but structure-physiology relationships as well
has been unusually productive.
The important work of the future will involve questions related to the precise molecular mechanism of signal transduction by Rho. This will require some understanding of the dynamic changes in protein conformation,
not only in Rho, but in the other proteins of the vertebrate
visual cascade.
We gratefully acknowledge the members of the Laboratory
of Molecular Biology and Biochemistry at The Rockefeller University, including students, postdoctoral fellows, and associates,
who have worked on visual phototransduction over the past 10
years. In addition, productive collaborations with several laboratories have been greatly appreciated.
Support was provided by National Institutes of Health
Training Grants GM-07739 and EY-07138, the Charles H. Revson
Foundation, the Aaron Diamond Foundation, the Arts and Letters Foundation, and the Allene Reuss Memorial Trust. T. P.
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interesting mutation linked to retinal degeneration is a
replacement of the Schiff base Lys-296 by Glu (K296E).
This mutation should prevent chromophore Schiff base
formation. Interestingly, the corresponding mutation in
bovine Rho was shown to cause constitutive activity of
the mutant opsin in Gt activation assays in vitro (215).
Significant work in this area has continued using
transgenic mouse technology to produce mouse models
of retinal degeneration. A summary of recent transgenic
animal models is presented in Table 1. There appears to
be no early developmental defect involving the retina in
RP mouse models, and the work has potential relevance
in understanding the molecular and cellular pathophysiology of some forms of ADRP (48, 50, 113, 241, 243).
Transgenic mice expressing the P23H mutation on the
intradiscal domain of Rho have been characterized, and
results indicate that retinal degeneration may be due to
improper mutant opsin expression and cellular transport
(188, 217). Other interesting transgenic mouse models,
such as those corresponding to mutations of the conserved residues Val-343 or Pro-347, point to a defect in
targeting of the defective gene product to the rod cell
outer segment (242). Rho mutants with alterations or
deletions of these residues near the carboxy-terminal tail
bind RET to form a pigment. In response to light, they
activate Gt and are phosphorylated normally.
Further work is needed to clarify how the various
Rho mutations associated with ADRP lead to photoreceptor cell death and to degeneration of the many cell layers
of the retina. However, in the vast majority of cases it
appears that the inherited single allelic mutation in the
Rho gene allows the synthesis of a Rho protein that is in
some way abnormal in structure such that its normal
cellular processing or transport is prevented. Over time
this defect leads to the activation of cellular apoptotic
pathways that eventually result in rod cell death. The
progression from rod cell death to retinal degeneration
may involve local inflammatory reactions. The effects of
nutritional vitamin A supplementation, at least in animal
models, suggest a potential therapeutic benefit by stabilizing mutant opsins through increased chromophore
availability (144).
Constitutive activity of mutant opsin G90D has also
been implicated as a cause of congenital night blindness
(207), a defect in scotopic vision in which the dark noise
level of the rod cell is increased (230). The mutant pigment associated with congenital night blindness has been
characterized in some detail (207, 269). A discussion of
activating mutations of Rho and other GPCRs has also
been reported (208). Other inherited mutations in the
vicinity of the Schiff base in Rho might affect chromophore stability or cause constitutive activity without
necessarily leading to rod cell apoptosis. Polymorphisms
might also be expected to result in some heterogeneity in
scotopic visual sensitivity among the general population.
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Sakmar is an Associate Investigator of the Howard Hughes
Medical Institute and an Ellison Foundation Senior Scholar.
Address for reprint requests and other correspondence:
T. P. Sakmar, Rockefeller University, 1230 York Ave., New York,
NY 10021 (E-mail: [email protected]).
23.
24.
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