(Investigative Ophthalmology and Visual Science. 2001;42:3-9.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Rhodopsin Structure, Function, and Topography The Friedenwald Lecture
Paul A. Hargrave
From the Department of Ophthalmology, School of Medicine, University of Florida, Gainesville, Florida.
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Introduction
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The increase in knowledge of rhodopsin structure and function has
been nothing short of phenomenal over the past 30 years. Although some
of rhodopsin’s properties had been investigated by Kühne as
early as the 1870s, the characterization of rhodopsin’s molecular
properties was still quite rudimentary into the late
1960s.1
Currently, there is information concerning the
complete amino acid sequence of as many as 100 vertebrate visual
pigments, a wealth of information about the function of rhodopsin in
health and disease and a three-dimensional structure of rhodopsin with
a resolution of 5 Å. Moreover, the knowledge of rhodopsin structure
and function has led to an appreciation for how the entire class of
G-protein–linked receptors may function (for reviews see Refs.
2
and 3
).
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Rhodopsin in Rod Cell Function
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The vertebrate rod cell consists of an outer segment that contains
a stack of rhodopsin-containing disc membranes connected to an inner
segment via a ciliary process (Fig. 1)
. The inner segment contains the metabolic machinery of the rod cell.
Rhodopsin is synthesized in the endoplasmic reticulum and passes to the
Golgi membranes where it becomes glycosylated. Rhodopsin-containing
vesicles move from the Golgi to the outer segment where they fuse with
the outer segment plasma membrane. Morphologically separate disc
membranes are formed by evagination and pinching off of the plasma
membrane. The regions of rhodopsin that were facing the outside of the
cell are now facing the inside surface of the disc membrane. The
regions of rhodopsin that were facing the outer segment cytoplasm
remain facing the cytoplasm when the plasma membrane forms disc
membranes. Rhodopsin comprises more than 90% of the protein content of
these disc membranes. It took years of work by many laboratories to
develop a working model of rhodopsin (Fig. 1)
as a bundle of
transmembrane helices that encompass a binding pocket for the
light-sensitive 11-cis-retinal. When light strikes a
molecule of rhodopsin in the rod cell, a photon is absorbed, causing
isomerization of rhodopsin’s 11-cis-retinal to the
all-trans isomer. This causes a change in the conformation
of rhodopsin that stimulates transducin, setting off a biochemical
amplification cascade that results in a drop in cGMP concentration.
That in turn leads to hyperpolarization of the plasma membrane and the
signaling of second-order neurons.
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Figure 1. A vertebrate rod cell, disc membrane, and model for rhodopsin in the
membrane. Rod cell outer segments consist of stacks of disc membranes
containing the photoreceptor protein rhodopsin. The disc membrane shown
in expanded cross section shows the oval shapes of densely packed
rhodopsin molecules that traverse the lipid bilayer. To the front right
of the figure is a drawing of the polypeptide chain of rhodopsin as it
may exist in the lipid bilayer in three dimensions. The N-terminal
exposes two attached oligosaccharide chains at the intradiscal surface.
The cutaway view shows retinal linked to the seventh helix, in a pocket
formed by the inside surface of the helical bundle. Reproduced with
permission from Hargrave PA, Hamm HE, Hofmann KP. Interaction of
rhodopsin with the G-protein, transducin. Bioessays.
1993;15(1):43–50 © 1993 Wiley-Liss, Inc., a subsidiary of John Wiley
& Sons, Inc.
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The molecules involved in these processes are shown in Figure 2 . When rhodopsin (R) becomes photoactivated (R*), the G-protein
transducin (T) can now bind and become activated (T*). This begins the
phototransduction cascade. R* would continue to activate T for a long
time unless there were some mechanism to shut it off. In vivo that
mechanism depends on phosphorylation. Rhodopsin kinase phosphorylates
specific serines and threonines in rhodopsin’s carboxyl-terminal
sequence. This phosphorylation reduces the ability of transducin to
bind, but does not totally eliminate it. However, now arrestin can bind
to the phosphorylated, photoactivated rhodopsin (R*P), and this totally
prevents further activation of transducin. This complex eventually
decays as the all-trans-retinal dissociates, and the
phosphates are removed by a phosphatase. The
all-trans-retinal is regenerated in a series of enzymatic
steps to 11-cis-retinal, and the retinal rebinds to the
protein opsin, regenerating rhodopsin.
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Figure 2. The rhodopsin cycle. Rhodopsin (R) is activated by light, hv,
(to R*). R* activates the G-protein, transducin (T). R* then becomes
phosphorylated by rhodopsin kinase, which allows it to become
inactivated by binding arrestin. The complex R*P-arrestin loses
all-trans-retinal and then arrestin, and the
phosphorylated opsin (OP) becomes dephosphorylated by the action of
protein phosphatase 2A (PrP2A). All-trans-retinal is
transformed, through a series of steps, to
11-cis-retinal, which subsequently rebinds opsin,
forming rhodopsin. Redrawn from and reproduced with permission from
Hargrave PA, McDowell JH. Rhodopsin and phototransduction. Int
Rev Cytol. 1992;137B:49–97 © 1992 Academic Press, Inc.
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Rhodopsin’s Amino Acid Sequence
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Basic to an understanding of the function of any protein is its
amino acid sequence—its primary structure. Such information provides
the groundwork for understanding how the protein carries out its
cellular functions. Currently, the complete amino acid sequence of a
protein is generally determined by inference from its nucleic acid
sequence. When the sequence analysis of rhodopsin was undertaken in the
1970s, one needed to apply chemical and enzymatic methods to the
protein. Automated amino acid sequencing could be applied directly to
an intact protein to determine part of its N-terminal sequence.
However, this was not an option for bovine rhodopsin because its
N-terminal amino acid was blocked. When rhodopsin was enzymatically
digested to a mixture of peptides, a peptide with a blocked N terminus
was identified and purified, and its sequence was
determined.4
The only previously reported sequence of
rhodopsin, a 9-amino acid glycopeptide, was contained within its
sequence.5
However, the larger N-terminal glycopeptide was
found to contain two sites of oligosaccharide attachment, at
Asn2 and
Asn15.4
Because the varieties of oligosaccharides attached to proteins
are assumed to have functional importance, it was of interest to
characterize the nature of these oligosaccharides. In collaboration
with David Papermaster and Michiko Fukuda from the University
of Connecticut, the two sites were found to contain predominantly the
uniquely small GlcNAc3Man3,
with smaller amounts of chains containing Man4
and Man5.6
Similar results were
obtained by others.7
Only recently has a functional
consequence of rhodopsin glycosylation become apparent. Among those
cases of retinitis pigmentosa in which rhodopsin mutations are
implicated as causing the disease process, threonine residues
Thr4 or Thr17 are mutated.
These specific mutations result in a failure of rhodopsin’s
immediately preceding Asn residue to become glycosylated. When a
Thr17 mutant rhodopsin was expressed by Kaushal
et al.8
and its properties were studied, it was found that
the mutant could fold to an apparently correct ground-state structure.
However, its metarhodopsin II photoproduct was unstable and had an
abnormally short lifetime, rendering it defective in signal
transduction. Therefore, glycosylation of Asn15
is required to have a rhodopsin that can function properly in signal
transduction.
The carboxyl-terminal amino acid of rhodopsin was found to be
alanine.9
From tryptic and cyanogen bromide digests of
rhodopsin, the C-terminal peptide (terminating in alanine) was
identified and purified, and its sequence was
determined.9
10
This sequence,
Asp-Asp-Glu-Ala-Ser-Thr-Thr-Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala,
contains many hydrophilic amino acids and is particularly rich in
serine and threonine.
Within the same year, three laboratories independently discovered
that rhodopsin became phosphorylated in a light-dependent reaction.
Because phosphorylation of proteins is frequently associated with
metabolic control processes, it was of interest to further characterize
the site and characteristics of this reaction. When rhodopsin was
phosphorylated with endogenous rod cell kinase(s) using
[32P]ATP, almost all the phosphate could be
found in a single peptide, which was identified as the Ser/Thr-rich
C-terminal peptide.10
It was evident that the sites of
phosphorylation in bovine rhodopsin did not resemble those types of
protein sequences that were know to become phosphorylated by well-known
kinases such as protein kinase A. It became clear that the
phosphorylation was being carried out by a new and different kinase,
rhodopsin kinase,11
and that this kinase had its own
distinct sequence recognition.12
The kinetics of the in
vitro reaction shows a steady progression to high levels of
phosphorylation,13
although the in vivo reaction may be
more limited in its extent. There seems to be general agreement that
the early and preferred sites of phosphorylation are
Ser343 and
Ser338.14
15
16
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Rhodopsin’s Topography
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In parallel with the efforts to determine the sequence of
rhodopsin and characterize its functional sites were efforts to
determine its topography within the rod cell membrane. Transmembrane
proteins like rhodopsin expose different parts of their sequence on
opposite membrane surfaces. It is these surface-exposed regions that
have the potential for functional interactions with other proteins and
with small molecules.
Following earlier work,17
rhodopsin in intact disc
membranes was digested with the proteolytic enzyme thermolysin, as a
probe for these exposed regions. Rhodopsin was converted to a slightly
lower molecular weight, in a rapid reaction.9
This was
accompanied by release of two peptides from its sequence,
Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln and Val-Ala-Pro-Ala, peptides readily
recognized to be from rhodopsin’s newly determined carboxyl-terminal
sequence.9
This provided clear evidence that the
carboxyl-terminal region of rhodopsin was highly exposed on the
cytoplasmic disc membrane surface. Additional digestion of rhodopsin by
thermolysin converted rhodopsin to two lower molecular weight
fragments, F1 and F2. Fragment F1 contained carbohydrate and therefore
derived from the amino-terminal region of rhodopsin. When the digestion
was performed on rhodopsin that had been phosphorylated with
[32P]ATP, fragment F2 contained
32P, demonstrating that fragment F2 originated
from rhodopsin’s carboxyl-terminal region.9
To obtain larger quantities of both fragments for use in structural
studies, the thermolysin digestion of membrane-bound rhodopsin was
performed on a preparative scale. The digested rhodopsin was
solubilized in mild detergent and chromatographically purified as the
noncovalent F1–F2 complex.10
Separation of the fragments
was accomplished by dissolving in concentrated formic acid and
separating by size on a gel filtration column in formic acid/ethanol.
The hydrophobic nature of these large polypeptides, derived from an
intrinsic membrane protein, required the use of such drastic
conditions.
Automated sequencing of the F2 fragment, as well as peptides derived
from it, was successful in completing the amino acid sequence of the
carboxyl-terminal one third of the sequence of bovine rhodopsin, 108
amino acids in length.18
The sequence showed alternating
segments of hydrophilic and hydrophobic amino acids with the two
predominantly hydrophobic stretches of 24- and 27-amino acids in length
being separated by a short hydrophilic sequence. The lengths of the
hydrophobic segments were the correct distance to traverse the
hydrophobic portion of the membrane lipid bilayer. This suggested a
model for the F2 fragment in which its N-terminal and C-terminal were
located on the cytoplasmic surface of the disc membrane (because they
were formed by proteolytic digestion at the membrane surface), whereas
the two hydrophobic sequences would traverse the lipid bilayer and
expose their hydrophilic connecting segment on the opposing membrane
surface.
Evidence was accumulating that rhodopsin’s N-terminal sequence was
exposed to the inside surface of the disc membrane (and the external
surface of the rod outer segment plasma membrane). Chemical evidence
was provided by the reaction of a radioactive nitrene precursor,
nitroazidophenyl taurine (NAP-taurine). When this membrane impermeant
reagent was reacted with rhodopsin in intact disc membranes, peptides
from rhodopsin’s carboxyl terminus became radioactively labeled, but
rhodopsin’s N-terminal peptide was poorly labeled.19
When
the same reaction was performed on reconstituted membranes in which
rhodopsin’s orientation was randomized, the relative modification of
the N terminus was greatly enhanced, suggesting that it had been
previously unavailable because of its exposure on the opposite membrane
surface.
Additional support came from immunocytochemical studies. Grazyna Adamus
in our laboratory prepared anti-rhodopsin mouse monoclonal antibodies
that were specific to N-terminal sequences in rhodopsin20
as well as antisera against rhodopsin’s synthetic N-terminal peptide.
Experiments by Polans and Papermaster,21
using these and
other reagents, demonstrated that rhodopsin’s amino-terminal sequence
was exposed on the outside surface of rod cell outer segments.
Completion of the amino acid sequence of bovine
rhodopsin,22
23
in conjunction with the topographic
information summarized above, made it possible to develop a topographic
model for the organization of rhodopsin within the lipid bilayer (Fig. 3)
. Such a model proposed that rhodopsin’s amino terminal segment was
exposed on the inside surface of the disc membrane (or outer surface of
the plasma membrane) and that the carboxyl-terminal sequence was
exposed on the cytoplasmic surface. The polypeptide chain traversed the
lipid bilayer seven times, embedding hydrophobic sequences in the lipid
side chain environment and exposing hydrophilic linking regions to the
membrane surface aqueous environments. The information derived from
topographic studies placed constraints on construction of the model and
made predictions about the orientation of sequence segments for which
evidence was still to be obtained.
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Figure 3. Topographic model for bovine rhodopsin in the membrane lipid bilayer.
Rhodopsin’s polypeptide chain traverses the lipid bilayer seven times
as the helix segments I through VII. Hydrophilic loop sequences are
exposed to the cytoplasmic surface (i1–i4) and to the intradiscal or
extracellular surface (e1–e3). The amino-terminal sequence is
acetylated at Met1 and contains oligosaccharide chains at
Asn2 and Asn15. Cysteines 110 and 187 are
linked by a disulfide bridge, and Cys322 and
Cys323 are palmitoylated. Retinal is linked via a Schiff
base to Lys296 in helix VII. Reproduced with permission
from Hargrave PA, McDowell JH. Rhodopsin and phototransduction.
Int Rev Cytol. 1992;137B:49–97 © 1992 Academic Press,
Inc.
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The model (Fig. 3)
predicted that rhodopsin’s loop e2 (amino acid
sequence 177–202) should be exposed on the outside surface of the rod
cell outer segment. An antibody raised against proteins from turtle
retina was subsequently identified as an anti-rhodopsin antibody, and
peptide competition demonstrated that its specificity was against the
e2 loop. This antibody labeled the outside surface of intact frog rod
outer segments, and its binding was abolished in the presence of the e2
synthetic peptide, demonstrating that rhodopsin indeed exposed its e2
loop to the extracellular environment.24
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Rhodopsin Function
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Proteins interact with other molecules—substrates, inhibitors,
ions, nucleic acids, carbohydrates, lipids, and other proteins—as ways
in which they carry out their various functions. By the time rhodopsin
reaches its membrane location in the outer segment, it has already
interacted with dozens of proteins involved in such activities as its
biosynthesis, acetylation, glycosylation, and transport. It has bound
two molecules of palmitate and one molecule of 11-cis
retinal and has become embedded in a membrane lipid bilayer. Rhodopsin
is now ready to carry out its role in phototransduction.
After photoactivation, rhodopsin exerts its effect on rod cell
biochemistry by interacting with rod cell proteins through its
cytoplasmic surface. As described previously (Fig. 2)
, photoactivated
rhodopsin (R*) binds and activates the G-protein, transducin,
initiating the phototransduction cascade. R* is then phosphorylated by
rhodopsin kinase, and the resulting R*P is bound by arrestin,
preventing further activation of transducin. Thus, rhodopsin interacts
with three major proteins—transducin, rhodopsin kinase, and
arrestin—and is then dephosphorylated by a phosphatase, as part of its
functional cycle.
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Rhodopsin Interaction with Transducin
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The nature of the interaction with transducin was first explored
using thermolysin-digested rhodopsin. Removal of part of rhodopsin’s
carboxyl-terminal sequence has no effect on the binding of transducin,
indicating that this sequence in the molecule is not involved in
transducin binding.25
However, proteolytic digestion of
the third cytoplasmic loop abolishes binding of transducin, suggesting
involvement of that portion of the rhodopsin sequence in transducin
binding.
Confirmation of the role of the third cytoplasmic loop in transducin
binding was provided by a totally independent set of experiments. In
collaboration with the laboratory of K. Peter Hofmann, Humboldt
University, Berlin, the sites of interaction of photoactivated
rhodopsin (metarhodopsin II) and transducin were probed using a
spectroscopic method. This method depends on the ability to
spectroscopically measure the amounts of metarhodopsins I and II in
their equilibrium mixture. Transducin binds selectively to
metarhodopsin II, shifting the equilibrium and creating more
metarhodopsin II (and if transducin dissociates, this extra amount of
metarhodopsin II is decreased as the metarhodopsin I and II
equilibrium is reestablished). When a synthetic peptide comprising the
sequence of rhodopsin’s third cytoplasmic loop was added to such a
transducin/metarhodopsin II complex, it displaced metarhodopsin II, as
monitored spectroscopically.26
This provided additional
evidence that the third cytoplasmic loop was a site of binding for
transducin on the metarhodopsin II surface. Similar peptide competition
studies showed that the second and fourth cytoplasmic loops (but not
the first loop or the C-terminal sequence) were also sites of binding
for transducin. These and related studies have been
reviewed.27
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Rhodopsin Kinase
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Initial studies on rhodopsin phosphorylation were performed in
vitro using rod outer segments. The properties of the kinase(s)
phosphorylating rhodopsin remained to be determined. Rhodopsin kinase
became recognized as the kinase that catalyzed light-dependent
rhodopsin phosphorylation, and it was found to be a representative of a
new class of kinases—those phosphorylating G-protein–linked receptors
(reviewed in Ref. 28
). Purification of rhodopsin kinase
assisted in determining its properties, and the discovery of conditions
for its stabilization and storage aided in its study.11
One of the ways to determine kinase specificity is to test synthetic
peptides as substrates, and this method was used for rhodopsin kinase.
Rhodopsin kinase can phosphorylate synthetic peptides from rhodopsin’s
carboxyl-terminal sequence, but with
Km values that are approximately three
orders of magnitude higher than that for rhodopsin.12
29
This behavior is in contrast to that of a kinase such as protein kinase
A, which phosphorylates synthetic peptides containing its site of
phosphorylation nearly as well as its entire protein substrate. In
addition, some peptides from rhodopsin’s cytoplasmic surface
(particularly the third cytoplasmic loop) inhibit the phosphorylation
reaction.29
These experiments are done by performing the
phosphorylation of membrane-bound, freshly photolyzed rhodopsin in the
presence of a small amount of synthetic peptide representing sequences
from the cytoplasmic surface of rhodopsin. The amount of peptide is too
small to become phosphorylated, if it were a substrate, but sufficient
to compete for binding sites on rhodopsin by the kinase. Taken together
these findings suggest that more than one part of rhodopsin’s surface
is involved in the interaction between rhodopsin and rhodopsin kinase.
Studies from other laboratories have further implicated cytoplasmic
loop i330
as a binding site for rhodopsin kinase, because
rhodopsin kinase phosphorylates target serines in rhodopsin’s
carboxyl-terminal sequence.
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Rhodopsin Dephosphorylation
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Proteins that become phosphorylated for purposes of
metabolic control become dephosphorylated by the action of
phosphatases. Kühn, in an elegant study,31
demonstrated both the phosphorylation and dephosphorylation of
rhodopsin in vivo in frogs. Because there is great specificity in the
phosphorylation of rhodopsin (rhodopsin kinase phosphorylates only
freshly photolyzed rhodopsin and no other substrate), it is of interest
to determine the specificity of the dephosphorylation reaction. There
is clearly some specificity in this reaction because of the finding
that it is catalyzed by the catalytic subunit of protein phosphatase 2A
but not by protein phosphatases 1, 2B, or 2C.32
33
However, this is a phosphatase that can act on other protein
substrates. The basis of its ability to dephosphorylate phosphorylated
rhodopsin is not known but may derive from a specificity residing in
its noncatalytic subunits. Protein phosphatase 2A represents one more
in the growing list of proteins that interact with rhodopsin, whose
presence and activity are necessary for rhodopsin function. The action
of protein phosphatase 2A on photoactivated, phosphorylated rhodopsin
is blocked in the presence of arrestin.33
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Arrestin
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Although arrestin has had a separate life in the vision immunology
literature as S-antigen, it first came to notice in the vision
biochemistry literature as a MWapp 48-kDa protein
that bound reversibly to photolyzed, phosphorylated
rhodopsin.34
This stimulated an effort to determine the
role and mechanism of action of arrestin in rod cell biochemistry. In
the search for other molecules that might bind to arrestin as part of
its mechanism of action, there were many conflicting reports concerning
arrestin’s biochemical properties. Using a highly purified preparation
of arrestin, it was clear that it did not bind
Ca2+ or the nucleotides ATP or GTP35
and thus was not modulated by fluxes of these important rod cell
compounds. The specificity of arrestin’s binding was localized to its
carboxyl-terminal sequence. Native arrestin binds only to
photolyzed phosphorylated rhodopsin, but arrestin that is
proteolytically truncated at its carboxyl terminus binds to
phosphorylated rhodopsin independent of rhodopsin’s exposure to
light.36
This binding is tighter than the binding of
native arrestin. Thus, arrestin’s acidic C-terminal region appears to
act as a modulator of the binding interaction.
In an effort to learn about the sites of binding of arrestin to
the surface of rhodopsin, synthetic peptides from rhodopsin’s surface
were used as competitors in binding tests. These experiments did not
clearly identify binding regions, although they had done so in similar
experiments with transducin. However, they did lead to identifying the
mechanism of arrestin’s binding to rhodopsin. Because binding of
arrestin had been shown to occur to photolyzed, phosphorylated
rhodopsin but not to photolyzed, unphosphorylated rhodopsin,
the phosphorylated region of rhodopsin was thereby implicated as a site
of binding. The surprise was in the way that this binding interaction
functioned. This was demonstrated by use of a synthetic peptide
representing the fully phosphorylated C-terminal sequence of rhodopsin,
amino acids 331 to 348. In the presence of this
phosphopeptide, arrestin is stimulated to bind to photolyzed,
unphosphorylated rhodopsin.37
Because rhodopsin
itself does not need to be phosphorylated, the phosphopeptide is able
in some manner to substitute for the phosphorylated region of the
protein. Based on a general knowledge of protein function, it was
reasonably hypothesized that arrestin was binding the phosphopeptide
and then undergoing a change in conformation that enabled it to bind to
the (unphosphorylated) surface of photolyzed rhodopsin.
This led to several experiments, the results of which supported this
hypothesis. A K50 of 36 µM was determined for
the binding of the phosphopeptide to arrestin. Limited trypsin
digestion yielded different arrestin fragments in the presence and
absence of phosphopeptide, suggesting that arrestin in the presence of
phosphopeptide assumed a different conformation.37
The
ligand-bound form must have presented a different availability of
peptide bonds for trypsin to cleave. A change in the conformation of
arrestin in the presence of phosphopeptide is also supported by changes
in the chemical reactivity of arrestin’s three sulfhydryl groups
toward dithionitrobenzoic acid (DTNB). In the presence of
phosphopeptide, the rate of reaction of one mole of sulfhydryl group is
greatly enhanced, and the rate of another sulfhydryl is
reduced.38
Here the ligand-bound form of the protein leads
to a different microenvironment surrounding two of the sulfhydryl
groups, affecting their rate of reaction with DTNB. Taken together,
these data suggest that the binding of phosphopeptide to arrestin has
the same effect as the binding of photolyzed phosphorylated rhodopsin,
making it possible for arrestin to then bind to the rhodopsin surface.
In a functional assay, the phosphopeptide substitutes for
phosphorylated rhodopsin. In rod outer segments, phosphodiesterase
activity can be quenched by addition of phosphopeptide in a manner
similar to the quenching achieved by light-stimulated rhodopsin
phosphorylation.38
Although arrestin initially binds to rhodopsin’s phosphorylated
C-terminal region, the carboxyl-terminal is not the final site of its
binding on rhodopsin’s surface. This can be demonstrated by the use of
phosphopeptide to induce binding of arrestin to photolyzed rhodopsin
whose 19-amino acid carboxyl-terminal has been
proteolytically removed.37
Studies in the laboratories of
Weiss39
and Benovic40
have demonstrated that
arrestin, after its "activation" by binding to the phosphorylated
region, then binds to amino acids in rhodopsin’s cytoplasmic loops i1,
i2, and i3.
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Rhodopsin’s Three-Dimensional Structure
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Basic to an understanding of how a protein carries out its
functions is the knowledge of the protein’s three-dimensional
structure. Such structural information provides the necessary framework
for determining mechanisms of protein action. For rhodopsin it would
not only be important to know its structure in its ground state, before
photoactivation, but also to know the conformation that it assumes in
metarhodopsin II, its signaling state, in which it is able to bind and
activate transducin. Knowledge of the signaling state of rhodopsin will
also have important implications in helping to understand how the
entire class of G-protein–coupled receptors may function.
The three-dimensional structures of thousands of proteins have been
solved by crystallizing the proteins and determining their structures
by x-ray diffraction. Only a handful of membrane proteins have had
their structures determined in this way because of technical
difficulties. Membrane proteins must first be solubilized in
detergents, and it has proven difficult to obtain sufficiently large
and well-ordered crystals from the detergent complexes of most membrane
proteins. The prospects for application of these methods to rhodopsin
have been reviewed.41
Incremental progress is currently
being made in several laboratories in the areas of sample preparation,
additives and cryoprotectants for crystallization, production of larger
crystals, use of microfocus beam synchrotron radiation, and improved
methods of data analysis.
Fortunately it has been possible to obtain high-quality structural
information from membrane proteins that can form well-organized
two-dimensional crystals. Frog rhodopsin can be induced to form
two-dimensional crystals in frog disc membranes after extraction with
weak polyoxyethylene sorbitan detergents.42
By optimizing
conditions (detergent type, detergent/protein ratio, pH, buffer
composition, and time of extraction) it has been possible to improve
the reproducibility, yield, and quality of these
crystals.43
Samples of membrane preparations were stained and examined by electron
microscopy to select preparations containing crystalline arrays (Fig. 4)
. Promising membrane preparations were frozen on grids for electron
microscopy at liquid nitrogen temperatures. Electron micrographs were
inspected by optical diffraction, and the diffraction data were
collected and processed for many dozens of crystalline membrane
samples. By these methods it was possible to determine that rhodopsin
molecules have planar dimensions of 28 x 39 Å and are
approximately 64 Å in height.44
Contour cross sections of
the electron density map show the position of electron densities
representing rhodopsin’s transmembrane helices (Fig. 5A
). For the first time it was possible to resolve each of the seven
helices, providing clear biophysical evidence supporting the
seven-helix model.45
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Figure 4. Micrograph of frog disc membranes after mild detergent extraction. Frog
disc membranes were extracted with Tween 80 or a Tween 80/20 mixture
and stained with uranyl acetate.43
Membranes containing
crystals of rhodopsin in the p2 crystal form are shown
in (A) and (C), and rhodopsin crystals in the
p22121 form are
shown in (B) and (D). Reproduced with permission
from Schertler GF, Hargrave PA. Projection structure of frog rhodopsin
in two crystal forms. PNAS. 1995;92(25):11578–11582
© 1995 National Academy of Sciences, USA.
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Figure 5. (A) Structure of frog rhodopsin. The seven helices of the
frog rhodopsin structure. A slice through the center of the density
map (B) is shown. Peaks can be seen for each of
the seven transmembrane helices. The grid spacing is 10 Å. Reproduced
with permission from Unger VM, Hargrave PA, Baldwin JM, Schertler GF.
Arrangement of rhodopsin transmembrane alpha-helices.
Nature. 1997;389(6647): 203–206 © 1997 Macmillan Magazines
Limited. (B) A solid model of the frog rhodopsin projection
map showing a view of helices 1 to 4. The model was constructed from 33
contour sections 2 Å apart. Top: rhodopsin’s cytoplasmic
side; bottom: its intradiscal (extracellular) side.
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By comparing individual slices of the map taken at different
distances within the membrane, it is possible to note differences
between the helices and in their relationships. Four of the helices (4,
6, and 7) are found at nearly the same position at different levels
within the membrane; that is, they are essentially perpendicular to the
plane of the membrane. In contrast, the other helices (and helix 3 in
particular) are tilted. Near the cytoplasmic surface, helices are
packed closely together. Near the intradiscal surface, helices are more
spread apart, creating a larger cross-sectional area. A space is
created between helices 3 through 7 that is the presumptive binding
site for rhodopsin’s light-sensitive chromophore,
11-cis-retinal. From the individual contour cross sections,
a solid model has been constructed (Fig. 5B)
. From this and subsequent
studies with bovine rhodopsin (summarized in Ref. 44
),
a clearer picture of rhodopsin’s three-dimensional structure is
beginning to emerge.
|
Conclusions
|
|---|
Considerable knowledge has been obtained during the past 30
years concerning the structure and function of rhodopsin and its
orientation within the lipid bilayer. This has laid the groundwork for
understanding how rhodopsin functions as a receptor of photons, how
G-protein–coupled receptors may function generally, and how mutations
in rhodopsin lead to a malfunctioning protein in diseases such as
retinitis pigmentosa. A higher resolution, three-dimensional structure
of both rhodopsin and of photoactivated rhodopsin is needed to more
precisely understand the molecular basis of receptor function.
|
Acknowledgements
|
|---|
The author thanks, in particular, J. Hugh McDowell, Anatol Arendt,
Grazyna Adamus, Kris Palczewski, Shao-Ling Fong, Jaime Puig, and our
collaborators the late Hermann Kühn, David Papermaster, K. Peter
Hofmann, Gebhard Schertler, W. Clay Smith, and Vijay Sarthy, among the
many colleagues who have contributed to these studies.
|
Footnotes
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|---|
Supported in part by National Institutes of Health Grants EY06225, EY06226, EY08571 and an unrestricted departmental award from Research to Prevent Blindness. PAH is a Senior Scientific Investigator of Research to Prevent Blindness.
Submitted for publication July 11, 2000; accepted August 2, 2000.
Commercial relationships policy: N.
Corresponding author: Paul A. Hargrave, Department of Ophthalmology, School of Medicine, Box 100284, University of Florida, Gainesville, FL 32610. hargrave{at}ufl.edu
|
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Top
Introduction
Rhodopsin in Rod Cell...
