Review articles
Ca2‡-binding proteins in the retina:
structure, function, and the etiology
of human visual diseases
Krzysztof Palczewski,1,2,3* Arthur S. Polans,4 Wolfgang Baehr,5 and
James B. Ames6
Summary
The complex sensation of vision begins with the
relatively simple photoisomerization of the visual pigment chromophore 11-cis-retinal to its all-trans configuration. This event initiates a series of biochemical
reactions that are collectively referred to as phototransduction, which ultimately lead to a change in the
electrochemical signaling of the photoreceptor cell. To
operate in a wide range of light intensities, however, the
phototransduction pathway must allow for adjustments
to background light. These take place through physiological adaptation processes that rely primarily on Ca2‡
ions. While Ca2‡ may modulate some activities directly,
it is more often the case that Ca2‡-binding proteins
mediate between transient changes in the concentration
of Ca2‡ and the adaptation processes that are associated with phototransduction. Recently, combined
genetic, physiological, and biochemical analyses have
yielded new insights about the properties and functions
of many phototransduction-specific components, including some novel Ca2‡-binding proteins. Understanding these Ca2‡-binding proteins will provide a more
complete picture of visual transduction, including the
mechanisms associated with adaptation, and of related
degenerative diseases. BioEssays 22:337±350, 2000.
ß 2000 John Wiley & Sons, Inc.
1
Department of Ophthalmology, University of Washington, Seattle,
Washington.
2
Department of Pharmacology, University of Washington, Seattle,
Washington.
3
Department of Chemistry, University of Washington, Seattle,
Washington.
4
Department of Ophthalmology and Visual Sciences, and the
Department of Biomolecular Chemistry, University of Wisconsin,
Madison, Wisconsin.
5
Moran Eye Center, University of Utah Health Science Center, Salt
Lake City, Utah.
6
The Center for Advanced Research in Biotechnology, University of
Maryland Biotechnology Institute, Rockville, Maryland.
Funding agencies: NIH; Foundation Fighting Blindness, Inc. (FFB);
Research to Prevent Blindness, Inc. (RPB), New York, NY; E.K. Bishop
Foundation; Grant numbers: EY08061 EY08123.
*Correspondence to: Krzysztof Palczewski, PhD, University of
Washington, Department of Ophthalmology, Box 356485, Seattle,
WA 98195-6485. E-mail: [email protected]
BioEssays 22:337±350, ß 2000 John Wiley & Sons, Inc.
Introduction
A vertebrate rod photoreceptor consists of an outer segment
connected by a thin modified cilium to an inner segment
(Fig. 1A). The inner segment contains the organelles typical
of a eukaryotic cell, while the outer segment is designed to
convert light, electromagnetic energy, into the electrochemical signals that eventually are interpreted by the visual
centers of the brain. The outer segment consists of a plasma
membrane, which encloses a series of double-membranous
disks or flattened saccules. Each outer segment contains
1000±2000 of these disks, which are physically separate
from the plasma membrane. Each disk contains 104 ±106
visual pigment molecules, rhodopsin, as integral membrane
proteins (Fig. 1B). In the dark, the chromophore 11-cis-retinal
is bound to the protein moiety. The only direct effect of light in
vision is to cause the isomerization of 11-cis-retinal to its alltrans configuration (Fig. 1C).
In the human retina, 11-cis-retinal is bound to four related
proteins which are collectively referred to as opsins. These
four visual pigment complexes are distributed between rod
photoreceptors, responsible for visual detection under
conditions of low illumination, and the three spectral types
of cone photoreceptors that provide information about color.
While most of what we know about phototransduction is
derived from studies of rod photoreceptors, the same
elements encoded by homologous genes appear to operate
in cone cells. The absorption of light by the rod photopigment
molecule, rhodopsin, initiates a chain of molecular events
culminating in the hydrolysis of cGMP (reviewed in Ref. 1)
(Fig. 2A). Briefly, photoactivation of rhodopsin leads to the
replacement of GDP by GTP on a heterotrimeric G-protein,
often referred to as transducin (T). The GTP-bound subunit
of transducin then activates a phosphodiesterase (PDE) by
dislodging two inhibitory g subunits that otherwise are bound
to the two catalytic subunits of the enzyme. The activated
PDE then hydrolyzes cGMP to 5'-GMP. Cyclic nucleotide
gated (CNG) cation channels in the plasma membrane of the
photoreceptor outer segment are held in an open state by
cGMP, so that the activation of PDE by light and the
subsequent reduction in the intracellular concentration of
cGMP results in the closure of these channels.(2) The cell
then hyperpolarizes, decreasing the amount of synaptic
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Figure 1. The rod photoreceptor and the photoisomerization of 11-cis-retinal to all-trans-retinal. A. The outer segment (OS) of the rod
photoreceptor cell is the primary site of phototransduction. Other portions of the cell are the inner segment (IS), nucleus (N), and
synaptic region (S). B. Rhodopsin is an integral membrane protein within the disks of the outer segment. The retinal chromophore is
embedded in a hydrophobic pocket of rhodopsin, coupled via a Schiff base to a Lys residue located in the transmembrane portion. C.
The initial step in phototransduction is the isomerization of the chromophore 11-cis-retinal to all-trans-retinal. Oxygen is shown in red.
transmitter released from the cell, thereby conveying the
capture of the initial light signal to the next order of retinal
neurons.
Once the transduction pathway has been activated by
light, additional mechanisms quench the underlying biochemical reactions and restore the system to its initial
conditions, ready for the next visual event. In the recovery
phase, photoactivated rhodopsin is phosphorylated by a
specific kinase and subsequently binds arrestin, a protein
that blocks further interaction with transducin. All-trans
retinal, converted to its 11-cis configuration, is reassociated
with opsin, and the regenerated rhodopsin is poised to
capture the next photon. The hydrolysis of GTP bound to the
a-subunit of transducin, coupled with other events, allows the
inhibitory subunit of the PDE to reassociate with its catalytic
subunits and thereby stop the further hydrolysis of cGMP.
The enzyme guanylate cyclase (GC) is then stimulated to
enhance its synthesis of cGMP, which is followed by the
reopening of the channels and reestablishment of the normal
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dark potential of the cell (Fig. 2A). Additional steps contributing to recovery of the dark condition of the photoreceptors are described elsewhere.(3±5)
The cation channels of the outer segment are heterotetrameric and open when 3±4 cyclic nucleotides are bound
per channel. One can calculate that at a concentration of 5±
10 mM cGMP, a K1/2 of 35 mM, and a cooperativity of 3, only
a small number of channels are open at a given time in the
dark, perhaps 5% of the total. At a typical dark potential
(ÿ40 mV), however, and assuming a conductance of 0.1±0.2
pS, a given rod channel will pass approximately 30,000
cations/sec. While the majority of this current is carried by
Na‡ ions, 15% of the current is comprised of Ca2‡ ions
(reviewed in Ref. 6). Thus, even small decreases in the
cytoplasmic concentration of cGMP induced by light can
cause major changes in the influx of Ca2‡ ions (Fig. 2A). In
the dark, Ca2‡ homeostasis is maintained by its continued
extrusion via a light-insensitive plasma membrane Na‡/
Ca2‡-K‡ exchanger, termed NCKX(7) (Fig. 2A). When cation
Review articles
Figure 2. Phototransduction and adaptation of rod photoreceptor cells. A. Phototransduction. In rods, a photon causes the
isomerization of a single molecule of the
visual chromophore, 11-cis-retinal,
coupled to opsin, referred to in the figure
as rhodopsin (R). A cascade of events is
set into motion culminating in the activation of the G-protein, transducin (T*),
followed by activation of PDE (PDE*) and
the hydrolysis of cGMP. Broken arrows
originating at rhodopsin (R), transducin
(T), and phosphodiesterase (PDE) indicate this flow of light activation. R* is
generally assumed to be metarhodopsin II.
T* consists of the Ta subunit charged with
GTP, dissociated from Tbg. PDE* is a
complex of T* and PDE in which the two
PDEg subunits are dislodged from their
inhibitory sites. Activation of PDE leads to
the closure of cyclic-nucleotide-gated
(CNG) cation channels and subsequently
the influx of cations, including Ca2‡, due to
continuos Ca2‡ extrusion by the Na‡/
Ca2‡-K‡ exchanger (NCKX). Return to
the dark state (heavy black arrows) occurs
in three major phases: 1. At the photopigment level: phosphorylation of photolyzed
rhodopsin by rhodopsin kinase (RK), and
the subsequent binding of arrestin, is
followed by dephosphorylation of R* and
regeneration of R with 11-cis-retinal (the
visual cycle). 2. At the transducin/PDE
level: GTP hydrolysis mediated by a Gprotein activating protein (GAP, which in
rods is RGS9/Gb5), is followed by recombination of the three T subunits and reinhibition of PDE. 3. At the cGMP/Ca2‡
level: Re-synthesis of cGMP by GCAP
stimulation of GC (GC*), is followed by reopening of CNG cation channels, return of
the dark current to its original level, and
replenishment of cations, particularly Na‡
and Ca2‡. B. Adaptation of rod photoreceptor cells. The voltage response of a rod cell to a flash of light is depicted as a function of increasing light intensity (solid line). The
result of the same treatment after Ca2‡ has been restricted to its concentration in the dark is also shown (circles). The slope of the
response has increased, indicating that the useful range of light intensity has narrowed, owing to the loss of the Ca2‡ flux that normally
occurs upon illumination.
channels in the outer segment plasma membrane close in
response to light, the amount of Ca2‡ entering the cell is
reduced, while the exchanger continues to operate. Thus,
light not only lowers cytoplasmic cGMP, but also decreases
the free concentration of Ca2‡ from approximately 500±
700 nM in the dark to about 30 nM upon illumination. This
simple feature of modulating the concentration of Ca2‡
underlies much of the process of photoreceptor adaptation.
First, what is meant by adaptation? This actually is a
rather complex topic which can be presented here only in a
very simplified form; other reviews are recommended for
more comprehensive treatments.(6,8) If one measures the
voltage response of the photoreceptor cell to various
intensities of light, a sigmoidal relationship is obtained (Fig.
2B). This relationship indicates that the absorption of each
photon is less effective in eliciting a response than the
preceding photon. The precise shape of the response curve,
indicating the range of light intensities over which discern-
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able responses can be obtained, is determined by changes
in the intracellular concentration of Ca2‡. If one interferes
with changes in the concentration of Ca2‡, however, the
response curve steepens, reducing the discernible responses of the cell by several orders of light intensity (Fig.
2B). Photoreceptor cells also adapt to background light,
shifting their response range to match the intensity of the
background illumination, and thereby extending the useful
range of lighting conditions in which they can function. This
phenomenon underlies the common experience of adjusting
to dark conditions when entering a movie theater or adjusting
to the sunlight upon exiting the theater. Recently, the
mechanism by which Ca2‡ intercedes during phototransduction to mediate these adaptation events has been revealed.
As we shall describe here, changes in the intracellular
concentration of Ca2‡ counteract the effect of light at several
stages of phototransduction, thus extending the range of
informative light intensities.
In principle, changes in the concentration of Ca2‡ ions
could effect portions of the phototransduction cascade
directly or indirectly through specific Ca2‡-binding proteins.
Research in the last few years has shown that Ca2‡-binding
proteins play an essential role during the recovery phase of
phototransduction (Fig. 2A). One of the most significant
effects of Ca2‡ is its regulation of GC. As the concentration
of Ca2‡ decreases upon illumination, it leads to the activation
of GC by means of a specific family of Ca2‡-binding proteins
termed guanylate cyclase-activating proteins (GCAPs)
(Fig. 3). GCAPs are members of the EF-hand superfamily
of Ca2‡-binding proteins which includes calmodulin (CaM),
parvalbumin, troponin C, and a host of small acidic proteins
collectively referred to as S100 Ca2‡-binding proteins. In
addition to their normal functions, several human pathologies
have been linked to mutations in genes encoding different
retinal Ca2‡-binding proteins and their targets. In light of the
importance of these proteins, we will present the most recent
information pertaining to the function, structure and related
pathologies of GCAP and similar Ca2‡-binding proteins of
the retina.
Biochemistry and molecular genetics of
photoreceptor guanylate cyclases (GCs),
guanylate cyclase activating proteins
(GCAPs), and GCAP-like proteins (GLPs)
The diversity of GCs, GCAPs, and GLPs
Two major GC families, soluble and particulate (membraneassociated), have been identified in different organisms. In
mammals, more than six distinct membrane forms have been
extensively characterized, at least three of which have been
identified in retinal photoreceptors.(9 ± 11) Two of these,
termed GC1 and GC2 possess a multidomain structure
composed of an extracellular receptor-like binding domain, a
single transmembrane segment, a kinase like domain, a
dimerization motif, and a catalytic domain (Fig. 3).(9) In
photoreceptor cells, GCs are mostly associated with the disk
membranes, with the result that the ``extracellular'' domain is
actually sequestered in the lumen of the disks. This orients
Figure 3. Model of GC stimulation by GCAP.
A. Model of the arrangement of GC in the rod
outer segment disk membrane. In the basal
state (nonactivated), GC is thought to be a
monomer with a single transmembrane domain.(54) The extracellular portion (ext) of
photoreceptor GC is exposed extracellulary
or sequestered within the intracellular disks.
The function of this domain in photoreceptor
GC is unknown. The intracellular part has a
kinase domain (kin) and a catalytic domain
(cat), separated by a dimerization motif (dim).
GCAP is associated with GC regardless of the
Ca2‡ concentration, and for simplicity has
been located next to the kin domain and the
membrane, although the precise interfaces
are currently unknown. Note that GCAP in the
Ca2‡-bound form (three functional EF loops)
is compact. After reduction of Ca2‡ levels, GC
is converted into an active form (right). Ca2‡free GCAP assumes a more open conformation interacting with GC through the kin and
cat domains. GC is also thought to dimerize, the dimer presumably interacting with 2 GCAPs. The resulting acceleration of cGMP
synthesis is five to tenfold.
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Review articles
Figure 4. Physical and genetic properties of GCAPs, GLPs, S100, and CaM. A. Physical constants, properties, targets, major tissue
distribution, and gene loci of GCAPs, GLPs, S100, and CaM. A.A., number of amino acids in the unprocessed proteins; myr, presence or
absence of myristoyl in the mature protein; EF1-EF4, EF hand motifs; MW, calculated molecular weight based on cDNA cloning; Pi,
calculated isoelectric point; KD, Ca2‡ dissociation constant; Hill, observed Hill coefficient; v-retina, vertebrate retina; h-retina, human
retina; f-cones, frog cones; GC1, guanylate cyclase 1. Except for GCIP (guanylate cyclase inhibitory protein (frog)), the amino acid
numbers are based on the human proteins. B. Illustration of gene structure of GCAPs and GLPs. GCAP1 and GCAP2 are in a tail-to-tail
array on 6p, while GCAP3 is located on 3q and recoverin on 17q. The approximate lengths of the human introns a±c are shown. The two
introns of the recoverin gene are located at positions homologous to introns b and c of the GCAP genes. The CaM gene structures (a
rare case of three distinct genes encoding an identical protein) are unrelated to those of GCAPs and GLPs. A dendrogram shown on the
left was generated on the basis of the amino acid sequences (PC/Gene).
the other functional domains to the cytoplasmic space. The
sequestration of the extracellular domain prevents regulation
by extracellular peptides, characteristic of GCs in other cell
types. The photoreceptor GCs are regulated intracellularly by
GCAPs, however (Fig. 3).
GCAPs are 23 kDa Ca2‡-binding proteins belonging to
the CaM superfamily with 4 EF hand motifs (discussed
below). Three isoforms of mammalian GCAPs, namely
GCAP1,(12) GCAP2,(13,14) and GCAP3,(15) have been characterized to date (Fig. 4). Only GCAPs, in their Ca2‡ free
forms, have been shown to activate photoreceptor GC. In
biochemical assays, a third GC activator, S100b (also termed
CD-GCAP) has recently been shown to activate GC1 in the
Ca2‡-occupied form in-vitro.(16) In addition to GCAPs, a
number of GCAP-like proteins have been described, among
them recoverin (S-modulin), the first photoreceptor-specific
Ca2‡-binding protein characterized,(17,18) its cone variant
S26,(19) and GCIP, a GC inhibitory protein identified in frog
cones.(20)
EF hand Ca2‡-binding proteins
The EF-hand Ca2‡-binding motif, a 29-residue helix-loophelix structure containing a 12 residue Ca2‡-binding loop,
was first discovered in the crystal structure of parvalbumin.(21) Over 200 Ca2‡-sensing proteins in eukaryotes
contain one or more EF-hand motifs and thus belong to the
EF-hand superfamily.(22) EF-hand proteins are structurally
and functionally quite diverse. The 17-kDa protein CaM
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serves as a Ca2‡ sensor in nearly all eukaryotic cells. CaM
stimulates a wide array of enzymes, pumps and other target
proteins, such as CaM-dependent protein kinases, calcineurin and the NMDA receptor.(23) EF-hand proteins are also
highly specialized. GCAPs, recoverin and other GCAP-like
proteins, for example, are expressed almost exclusively in
retinal photoreceptor cells and serve as Ca2‡ sensors in
vision.(1)
Regulation of phototransduction by
Ca2‡-binding proteins
Ca2‡-sensitive portions of the phototransduction cascade
contribute to the recovery phase of the photoresponse and to
light adaptation in photoreceptors. In the current model of
recovery, photon absorption lowers intracellular Ca2‡,
[Ca2‡]i, which in turn increases the sensitivity of the channel
to cGMP and accelerates the recovery of the dark current by
uncoupling CaM from the channel.(24,25) Calmodulin controls
the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the b-subunit.(25)
Lowering [Ca2‡]i also accelerates the synthesis of cGMP
through GC stimulation by the Ca 2‡-free form of
GCAPs.(12,13) (Fig. 2, 3). Two additional Ca2‡-sensitive steps
are still poorly understood and their physiological significance is unclear.(26) Lowering [Ca2‡]i may shorten the
lifetime of R* by interfering with rhodopsin kinase (RK)
inhibition through the action of recoverin (S-modulin),
another retina-specific Ca2‡-binding protein.(27) The lightinsensitive NCKX exchanger may also be regulated by Ca2‡binding protein(s).
Gene loci and gene organizations
The GC1 gene (retGC-1 in human, or GC-E in mouse) is
located on human chromosome 17p. Defects in this gene
have been linked to Leber's congenital amaurosis type 1(28)
and dominant cone-rod dystrophy.(29) The GC2 gene (GC-F
in mouse) is located on Xq22(30) and has not been linked to a
disease phenotype. GCAP1 and GCAP2 genes are organized in a tail-to-tail array in vertebrates.(31) In humans, the
array is located on the short arm of chromosome 6 (p21.1),
while the GCAP3 gene is located on 3q13.1.(15) The GCAP1
gene has been linked to autosomal dominant cone dystrophy
(see below). The structures of the GCAP genes (4 exons)
are identical (Fig. 4). The positions of the two introns of the
human recoverin gene (located on 17q) are exactly as those
of the second and third intron of the GCAP genes,
suggesting that these genes were generated by duplication
from a common ancestor. While a complex gene cluster on
chromosome 1 encodes many S100 proteins, the S100
gene is located on 21q. S100 has been found to be
overexpressed in Alzheimer's disease, Down's syndrome,
and some tumor tissues.(32)
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Expression of photoreceptor GCs,
GCAPs, and GLPs
An important criterion for involvement of specific gene
products in physiological processes is their colocalization.
Many independent studies have been carried out to
determine the cellular and subcellular distribution of GCs,
GCAPs, and GLPs in the vertebrate retina. GC1 and GC2
have been localized to mammalian photoreceptors by using
monospecific antibodies.(11,33) The teleost appears to express two kinds of GCs in rods and a third one in cones.(34)
In-situ hybridization studies in bovine and monkey retinas
revealed nearly identical expression patterns of GCAP1 and
GCAP2 mRNAs in the myoid regions of rod and cone
photoreceptors.(35) Immunocytochemical studies demonstrated that GCAP1 and GCAP2 are present in rods and
cones, and that GCAP2 is also present in cells of the inner
retina.(31,36) Among different species, GCAP2 apparently is
more prevalent in rods, while GCAP1 is expressed at high
levels in cones.(35±37) GCAP2, however, could not be isolated
biochemically from bovine rod outer segments (ROS).(14,38)
Apart from inner and outer segments, both GCAPs are also
present in photoreceptor synaptic regions but their role in
these regions is unknown. The cellular localization of GCAP3
has not been determined and its mRNA translation rate is
very low; it is thought to be a component of blue cones.
Recoverin (S-modulin) is present in mammalian rod and
cone photoreceptors and certain types of bipolar cells.(17,39)
S26 is thought to be a homologue of S-modulin present in
frog cones.(19) GCIP, a GLP that interacts with GC and
inhibits its stimulation, is expressed exclusively in frog
cones.(20) S100b has not been identified in ROS but may
play a role in synaptic terminals, which are also immunopositive for GC1.(40)
Deletion of GCAP and GC genes in
animal models
When both GCAP1 and GCAP2 genes were deleted in
mouse knock-out models, the resulting rod phenotype
consisted of a delayed return to the dark-adapted state, a
result consistent with a defect in GC stimulation.(41) Absence
of GCAPs in rods, however, did not cause morphological
changes in photoreceptors, indicating that GCAP genes are
not essential for the development or the survival of rods. In a
naturally occurring GC1 null mutant (rd chicken), photoreceptors develop normally, but there is no detectable
phototransduction in rods or cones. In contrast to GCAP1/
GCAP2 knockout mice, in the absence of GC1, both cell
types degenerate rapidly, starting one week post-hatch.(42)
Thus, expression of a functional GC1 in chicken retina is
essential for the survival of rods and cones. In GC1 knockout
mouse models, at least some rod photoreceptor function is
preserved, while cones are nonfunctional and degenerate
rapidly. In the mouse, therefore, GC1 is essential only for
Review articles
Figure 5. Biochemistry of GC stimulation by GCAPs and GCAP mutants. A. Ca2‡-titration of GCAP1/3.(15) The Ca2‡ titration of
recombinant GC1 activity in the presence of 4 mg of GCAP3 (gray squares) or GCAP1 (dark circles) is shown. Inset: Dose dependence of
GC1 activity in insect cell membranes stimulated by GCAP1 (dark circles) and GCAP3 (gray squares). GCAPs stimulate GC activity only
at low, nanomolar free [Ca2‡]. B. Inhibition of a GCAP1-tm (GCAP1(E75D, E111D, E155D)), a constitutive activator of GC, by GCAP1
and GCAP3.(15) The inhibition of GC stimulation by GCAP1-tm (1.5 mM) at [Ca2‡]free ˆ 2 mM by GCAP1 (dark circles) and GCAP3 (gray
squares). C. Binding of GCAP1 to ROS membranes.(14) Washed ROS were incubated with increasing amounts of purified GCAP1. The
membranes were collected by centrifugation. A portion of each sample was assayed for GC activity, while GCAP1 was identified by
western blot analysis by using a monoclonal antibody (inset). D. Persistent stimulation of GC by GCAP1(Y99C). This mutation was linked
to autosomal dominant cone dystrophy in a British family.(60) Ca2‡-titration of GC activity in washed bovine ROS in the presence of 2 mM
bovine GCAP1 (squares) and bovine GCAP1(Y99C) (circles). A double arrow shows the difference in activities between the mutant and
GCAP1 at physiological dark [Ca2‡]. The shaded areas show the approximate physiological range of Ca2‡ in dark and light adapted
photoreceptors.(76)
cones, while GC2 or yet another unidentified GC may
substitute, in part, for the loss of GC1 in rods.(43)
Specificity of GC stimulation
GCAPs stimulate GC activity only in low [Ca2‡]i, while at high
[Ca2‡]i they inhibit GCs (Fig. 5A). In vitro, GCAPs display
restricted specificities toward photoreceptor GCs. For example, GCAP1 effectively stimulates only GC1.(13) (Fig. 5A),
while GCAP2 and GCAP3 stimulate both GC1 and GC2.(15)
Recently, a mutation associated with a cone-rod dystrophy in
the human GC1 gene (R838C) was shown to dramatically
reduce stimulation by GCAP2, while increasing the affinity
for GCAP1 and altering the Ca2‡-sensitivity.(44) These and
other results suggest that GCAP1 and GCAP2 may have
distinct but overlapping contact sites on GC1. GCAPs inhibit
GCs when [Ca2‡]free is elevated (Fig. 5A and 5B). The
physiological significance of GC inhibition by GCAPs is
unclear, since photoreceptors in GCAP1/GCAP2 null mice
display a dark current that is indistinguishable from that in
wild-type mice.(41) These data suggest that the dark
photoreceptor [Ca2‡] is not high enough to inhibit GC. GCIP
from frog cones does not stimulate GC in low Ca2‡, but
inhibits GC in high Ca2‡, and is, therefore, termed GCIP
(guanylate cyclase-inhibitory protein). GCIP and GCAPs
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have diverged substantially, but conserved domains present
in all vertebrate GCAPs are also present in GCIP (Fig. 4).
The physiological role of this protein is, however, unknown.
EF hand mutations
Specific mutations in the EF-hand motifs of GCAP1 (similarly
in CaM(45) or GCAP2(46)) render the protein Ca2‡-insensitive.
For GCAP1, Glu(E) at the invariant 12th residue in the Ca2‡binding loop was replaced in one mutant by Asp(D),(47) which
did not perturb significantly the structure of GCAP1 but
drastically lowered the affinity for Ca2‡. Addition of native
GCAP1 or GCAP3, GCIP(20) or GCAP2(47) (Fig. 5B)
competed out the GCAP mutant, suggesting that the binding
site on GC is at least partially overlapping. These data are
consistent with the results of Gorczyca et al.,(14) which
showed that once ROS membranes are saturated with one
GCAP, addition of the second GCAP did not increase the
activity of GC.
Interfaces of GCs/GCAPs
Gorczyca et al.(14) first found that GCAPs and GC formed a
stable complex at low and high [Ca2‡] (Figs. 4 and 5C).
These findings are consistent with the dual ability of GCAPs
to activate and inhibit GC(46) (Fig. 5B). In three-dimensional
models, GCAPs expose an acidic/hydrophilic side and a
more hydrophobic side upon Ca2‡ binding.(48) It is still
unclear which side of GCAP interacts with GC. Attempts to
identify the protein face that forms a complex with GC
included peptide competition assays(48) and the use of
chimeric proteins comprised of GCAPs and related proteins
that do not bind to GC.(49,50) Neither approach is definitive,
however, as GCAP±GC interactions have a relatively low
affinity (KD1 mM), and peptide inhibition requires up to 1 mM
concentration, conditions which may produce several artifacts. The results of the chimera approach appear to be even
less clear without structural information, as exemplified in
recent studies where GCAP could be converted to the
activator at high [Ca2‡].
The mechanism of photoreceptor GC stimulation is
fundamentally distinct from hormone peptide stimulation of
other cyclase receptors. GCAP1 is believed to interact with
an intracellular domain of GC, because a mutant ROS-GC in
which the extracellular domain was deleted was stimulated
by GCAP1 in a manner indistinguishable from native ROSGC. Deletion of the intracellular kinase-like domain diminished stimulation by GCAP1, suggesting that this domain is
involved in Ca2‡ modulation.(51,52) A protease protection
assay was used to localize regions of the intracellular
domains of GCs important for the interaction with
GCAP2.(53) GCAP2 reduces the access of trypsin to a site
in the kinase homology domain of GC1. Furthermore, the
region within GC1 that comprises the interacting domain
with GCAPs corresponds to a loop between b-strand 3 and
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a-helix 4. When this region was replaced by the corresponding sequence of GCAP-insensitive GC type A (guanylate
cyclase- linked atrial natriuretic peptide receptor), GCAPs did
not stimulate the GC1 mutant.(48) The corresponding loop in
adenylyl cyclase (AC) is involved in the activating interaction
with Gs. The results further support the idea that both AC and
GC, despite differences in overall topology and activating
proteins, may be activated by similar mechanisms that
involve conformational changes in corresponding regions of
each protein.(48) Furthermore, dimerization of the catalytic
domains of GC1 and AC may be an essential step for
activation.(54)
Three-dimensional structure of EF-hand
Ca2‡-binding proteins
Structure of the EF hand
The three-dimensional structure of the EF-hand motif in
various Ca2‡-binding proteins is highly conserved. The EFhand is formed by helices E and F (named after helices in
parvalbumin), which are positioned like the forefinger and
thumb of the right hand. The Ca2‡-binding site is formed by a
12-residue loop between these helices. The main-chain
structures of individual EF-hand motifs of CaM, recoverin,
and GCAPs are very similar, even though their amino acid
sequences appear quite different. The CaM EF-hands are
less than 25% identical in primary sequence to the
corresponding EF-hands of recoverin and GCAPs.
The EF-hand Ca2‡-binding site contains a consensus
sequence of residues in the 12-residue binding loop.
Residues at positions 1,3, and 5 contain carboxylate oxygen
atoms in their side-chains (residues D, N, Q, or E), although,
in some cases, the residues serine and threonine are
present. A glycine residue is conserved at position 6 and is
structurally necessary for forming a tight b-turn in the middle
of the loop. The residue at position 7 contributes its mainchain carbonyl oxygen to coordinate Ca2‡ and, therefore,
can be any residue. Glutamate (or sometimes aspartate) is
required at position 12, because its side-chain carboxylate
oxygen atoms serve as a bidentate ligand to the bound
Ca2‡ ion.
Three-dimensional structures of
CaM and recoverin
The full three-dimensional structures of CaM and recoverin
are illustrated in Fig. 6. Each of these structures contains
four EF-hand motifs (shown in different colors in Fig. 6) that
form very different spatial arrangements. In each structure,
the four EF-hands form two domains: EF-1 and EF-2 interact
with one another to form the N-terminal domain, and EF-3
and EF-4 form the C-terminal domain. The structural
organization of the two domains is very different in the
structures of CaM and recoverin. In CaM, the two domains
Review articles
Figure 6. Ca2‡-induced structural changes in
CaM (A), recoverin (B), and GCAP2 (C). The
three-dimensional structures of the Ca2‡-free (left)
and Ca2‡-bound (right) forms are shown. Exposed
hydrophobic residues in the target-binding site are
highlighted magenta. The Protein Data Bank
accession numbers are 1dmo.pdb (Ca2‡-free
CaM(77)), 3cln.pdb (Ca2‡-bound CaM(78)), 1iku.pdb
(Ca2‡-free recoverin(79)), 1jsa.pdb (Ca2‡-bound
recoverin.(57)). Schematic ribbon representation of
the structure of Ca2‡-bound GCAP2 has been
published recently.(81) The four EF-hands (green,
red, cyan, and yellow) and three bound Ca2‡ ions
(orange) are highlighted. The side-chain atoms of
residues at the domain interface (Ala 63, Ala 67, Ile
103, and Ile 120) are shown as ball-and-stick
representation.
BioEssays 22.4
345
Review articles
are separated by a long central helix, giving rise to an overall
dumbbell-shaped appearance (Fig. 6A). In contrast, recoverin contains a short U-shaped inter-domain linker that
positions the two domains in close contact with one another,
forming a bilobed, globular shape (Fig. 6B). A structural
comparison between CaM and recoverin reveals a rather
large root-mean-square deviation (10 AÊ) in comparing the
main-chain atoms of each structure. As seen here for CaM
and recoverin, the structurally conserved EF-hand motif is
arranged into very different three-dimensional configurations
which are necessary for providing their diverse and
nonoverlapping functions.
Conformational changes induced by Ca2‡
The X-ray crystal structure of troponin C (with Ca2‡ bound to
EF1 and EF2 and not bound to EF3 and EF4) first revealed
the structures of both the Ca2‡-free and Ca2‡-bound EFhand motifs.(55) In the Ca2‡-free state, the EF-hand structure
exhibits a ``closed conformation'' in which helices E and F are
somewhat parallel (helix packing angle ˆ 130±180 ). The
Ca2‡-bound EF-hand adopts an ``open conformation'' in
which helices E and F are nearly perpendicular (helix packing
angle ˆ 90±110 ). Similar Ca2‡-induced structural changes
are observed in the EF-hands of CaM and recoverin (Fig. 6).
In addition to these internal changes within the EF-hands, the
binding of Ca2‡ also markedly influences the overall topology
and three-dimensional arrangement of the two domains.
The Ca2‡-induced rearrangement of the domains of CaM
is quite striking. The root-mean-square deviation of the main
Ê , when the Ca2‡-free
chain atoms of CaM is more than 10 A
2‡
and Ca -bound structures are compared (Fig. 6A). The
Ca2‡-free form of CaM exhibits a dumbbell shape with the
Ê . The binding of
two domains separated by more than 11 A
2‡
Ca causes an opening of the EF-hands that leads to the
exposure of many hydrophobic residues (Ala 15, Leu 32, Met
36, Phe 68, Ala 88, Met 109, and Met 145). The two domains
of Ca2‡-bound CaM contain large, exposed hydrophobic
patches which are flanked by negatively charged regions.
These are complementary to the positively charged amphipathic helices of target proteins. The central linker of CaM
serves as a flexible tether that allows the two domains to
come together to form a contiguous binding site that can
accommodate a wide variety of target proteins. The dumbbell
shape of Ca2‡-free CaM changes into a globular form in the
Ca2‡-bound state in which the target peptide sits in a
hydrophobic channel surrounded by many side-chains
(magenta in Fig. 6A) from both domains.
The structures of Ca2‡-free and Ca2‡-bound recoverin
are compared in Fig. 6B. A striking feature of these
structures is the large rotation of the two domains. The Cterminal domains of the two forms are quite similar, apart
from minor changes in the Ca2‡-binding loop and entering
the helix of EF-3. The N-terminal domain, by contrast,
346
BioEssays 22.4
undergoes a striking rearrangement that leads to the
extrusion of the amino-terminal myristoyl group. Extrusion
of the myristoyl group requires the binding of Ca2‡ to EF-2
and EF-3. The binding of Ca2‡ to EF-3 decreases its
interhelical angle, similar to the Ca2‡-induced ``opening'' of
EF-hands seen in CaM and troponin C. Ca2‡-binding to EF-2
does not change its interhelical angle much but instead
causes the exiting helix to twist clockwise about its central
helical axis.(56) This Ca2‡-induced helical twisting in EF-2 is
novel and has not been observed previously in other
members of the superfamily. The Ca2‡-induced conformational changes in EF-3 and EF-2 alter the interaction of these
EF-hands at the domain interface and promote a conformational change near Gly 96 in the interdomain linker. The
interface between the two domains is rearranged completely
by rotation at Gly 96, leading to a 45 rotation of one domain
with respect to the other, in which many hydrophobic
residues are exposed. The Ca2‡-induced exposure of the
myristoyl group, termed the Ca2‡-myristoyl switch,(57) may
enable recoverin to bind to membranes at high Ca2‡.
Structure of GCAP2
The structure of GCAP2 (Fig. 6C) contains four EF-hand
motifs arranged in a compact array like that seen in
recoverin. The overall main-chain structure of Ca2‡-bound
GCAP2 is very similar to that of recoverin and neurocalcin.
Three Ca2‡ ions are bound to GCAP-2 (EF2, EF3, and EF4),
as anticipated on the basis of its amino acid sequence and
site-directed mutagenesis.(46) Ca2‡ is not bound to EF-1
because the binding loop is distorted from a favorable Ca2‡binding geometry by Pro 36 at the fourth position of the 12residue loop. Also, the third residue in the loop (Cys 35) is not
suitable for ligating Ca2‡. A prominent exposed patch of
hydrophobic residues formed by EF1 and EF2 (Leu 24, Trp
27, Phe 31, Phe 45, Phe 48, Phe 49, Tyr 81, Val 82, Leu 85,
and Leu 89) resembles the hydrophobic target binding sites
in the structures of Ca2‡-bound CaM (highlighted magenta in
Fig. 6). The GCAP-2 structure is likely to be similar to that of
GCAP-1 (40% sequence identity), GCAP-3 (35% identity),
and GCIP (37% identity). Most of the hydrophobic residues in
the hydrophobic core and in the exposed patch are highly
conserved. Also conserved are the residues that chelate
Ca2‡ in the EF-hand loops. The structure of physiologically
relevant Ca2‡-free GCAPs is currently unknown.
Ca2‡-binding proteinsÐat the crossroads
of life and death
Recoverin and CAR
Ca2‡-binding proteins mediate between changes in the
intracellular concentration of Ca2‡ and a host of cellular
activities elicited by those changes. Interference in either the
expression or function of these proteins can lead to cell death
Review articles
and manifest as a human disease. Ca2‡-binding proteins are
directly involved in two degenerative diseases of the retina.
The first, cancer-associated retinopathy (CAR), is an
autoimmune-mediated disease initiated by the aberrant
expression of the Ca2‡-binding protein recoverin in some
primary neoplasms (for review see Ref. 1). Recoverin
normally is expressed in the rod and cone photoreceptor
cells of the retina. Its expression in tumors outside of the
eye leads to an immune response, which then inadvertently destroys retinal photoreceptor cells, thus causing
visual impairment or completes blindness. The loss of
vision often precedes the diagnosis of cancer so that the
detection of anti-recoverin antibodies in the patient's serum
is not only indicative of CAR but also acts as an early
warning sign for the presence of a tumor. The disease can
be reproduced in an animal model either by inoculation
with recoverin or by the transfer of lymphocytes from an
immunized animal to a naive recipient. The precise role of
the humoral and cellular components of the immune response are unknown, although the presence of recoverin
antibodies can induce programmed cell death (apoptosis) in
photoreceptor cells.(58)
GCAP1 and cone dystrophy
Degenerative events also occur in photoreceptor cells as the
result of mutations in the gene encoding GCAP1.(59,60) A
mutation (Y99C) that results in a tyrosine to cysteine change
is found in humans afflicted with an autosomal dominant
cone dystrophy.(60,61) The cone-specific degeneration is
consistent with high expression levels of GCAP1 and the
absence of significant amounts of other GCAPs in the outer
segment of this cell type. The Y99C mutation has been
shown to alter the Ca2‡ sensitivity of GCAP1, leading to the
constitutive stimulation of GC1 at high [Ca2‡]i, limiting its
ability to fully inactivate GC1 under physiological dark
conditions. (Fig. 5D). An increase in the concentration of
cGMP is expected to ensue, and such alterations have been
linked in other studies to the degeneration of photoreceptor
cells.
Early in vitro studies demonstrated that PDE inhibitors,
causing elevated levels of cGMP, promote photoreceptor cell
death.(62) Mutations in the b-subunit of PDE also result in
elevated cGMP and the subsequent degeneration of photoreceptor cells.(63,64) How is cGMP linked to cell death? As
cGMP levels rise in the photoreceptor, they open cation
channels in the plasma membrane of the photosensitive
outer segment (Fig. 1A). Approximately 15% of the current
that enters through these channels is carried by Ca2‡ ions.(6)
Therefore, conditions that elevate cGMP would be expected
to increase the intracellular concentration of Ca2‡. Aberrant
concentrations of Ca2‡, in turn, can induce apoptosis
through pathways that are either dependent on or independent of Ca2‡-binding proteins.
Ca2‡ and apoptosis
Ca2‡ ions generally enter a cell through channels or by
means of an exchanger. Homeostasis is maintained in part
by returning Ca2‡ to the extracellular environment through
the action of pumps and the expenditure of energy (for
review see Ref. 65). The majority of Ca2‡ that remains within
the cell is either bound to lipid or sequestered in compartments that include the endoplasmic reticulum (ER), mitochondria, and the nucleus. The major storage site for Ca2‡ is
within the ER where it is necessary for normal protein
synthesis and processing and for cellular signaling. Ca2‡
signals can arise either by the entry of Ca2‡ from the
extracellular space or by the release of sequestered Ca2‡.
These signals temporarily increase the concentration of
intracellular Ca2‡ and usually are offset by mitochondrial
uptake. Eventually, the balance between Ca2‡ in the ER and
the mitochondria is re-established. In contrast to Ca2‡
signals that are transient, sustained intracellular concentrations of Ca2‡ beyond approximately 200 mM lead to cell
death(66) either by apoptosis or necrosis depending, at least
in part, on the sustained levels of Ca2‡.
Substantial evidence supports diverse roles for Ca2‡
during apoptosis,(67) some aspects of which are mediated by
Ca2‡-binding proteins. CaM antagonists, for example, can
interfere with the activation of cell death, while overexpression of CaM enhances apoptosis.(67) Cyclosporin A
blocks a Ca2‡/CaM-dependent serine/threonine phosphatase, calcineurin, and hinders Ca2‡-induced apoptosis.
Calcineurin appears to function by dephosphorylating BAD,
a pro-apoptotic member of the Bcl-2 family, thereby allowing
BAD to interact with Bcl-x and promote cell death.(68) DAPkinase is another Ca2‡/CaM dependent enzyme linked to cell
death.(69)
The over-expression of the Ca2‡-binding protein calbindin
protects some cells from signals that increase intracellular
Ca2‡ and induce apoptosis. The buffering capacity of
calbindin also limits the extent of cell death induced by
Ca2‡ ionophores. ALG-2 is a newly identified Ca2‡-binding
protein that participates in cell death, perhaps by regulating
signal transduction pathways that depend on MAP kinase.(70)
Independent of these binding proteins, elevated Ca2‡ also
can alter the permeability transition pore of mitochondria,
releasing cytochrome c and activating caspases, the major
executioners of cell death.(71±73) The enhanced entry of
Ca2‡ ions into photoreceptor cells due to elevated levels of
cGMP can activate any of the enzymes and pathways just
described and thereby induce cell death.
The vertebrate photoreceptor cell, however, offers a
unique situation owing to the compartmentalization of its
organelles and the spatial distribution of the molecules
governing its movement of ions. Normally, Ca2‡ ions that
enter through CNG channels of the outer segment are
returned to the extracellular space by the action of a NCKX
BioEssays 22.4
347
Review articles
also localized to the outer segment. This differs from the
route followed by Na‡ ions, which form a current loop by
exiting the inner segment via a Na‡/K‡ ATPase (Fig. 1A). No
measurements have been made to determine whether a
``pathological'' increase in the concentration of Ca2‡ in the
outer segment can affect the flux of Ca2‡ through the inner
segment. The organelles and pathways associated with
Ca2‡-induced apoptosis are physically separated into the
inner segment, so a ``Ca2‡ connection'' must exist in order for
pathological changes in the concentration of cGMP in the
outer segment to be translated into a Ca2‡-induced signal for
apoptosis in the inner segment. Interestingly, a Ca2‡ channel
blocker that also acts on CNG channels of photoreceptors
has recently been demonstrated to protect rod and cone cells
from degeneration in the rd mouse.(74) This mutant provides
an animal model of the human genetic disease retinitis
pigmentosa, in which a mutation in the PDEb gene leads to
elevated levels of cGMP, and presumably, Ca2‡. These
results are consistent with the hypothesis that Ca2‡ is a
mediator of cell death in a variety of degenerative diseases of
the retina initiated by very disparate mutations or environmental insults.(75)
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