Ca 2 -binding proteins in the retina: structure, function, and the etiology of human visual diseases

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1 Ca 2 -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. Ames 6 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 Ca 2 ions. While Ca 2 may modulate some activities directly, it is more often the case that Ca 2 -binding proteins mediate between transient changes in the concentration of Ca 2 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 Ca 2 -binding proteins. Understanding these Ca 2 -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, 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 EY *Correspondence to: Krzysztof Palczewski, PhD, University of Washington, Department of Ophthalmology, Box , Seattle, WA palczews@u.washington.edu 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 10 4 ±10 6 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 BioEssays 22:337±350, ß 2000 John Wiley & Sons, Inc. BioEssays

2 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 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 K 1/2 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 ( 40 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 Ca 2 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 Ca 2 ions (Fig. 2A). In the dark, Ca 2 homeostasis is maintained by its continued extrusion via a light-insensitive plasma membrane Na / Ca 2 -K exchanger, termed NCKX (7) (Fig. 2A). When cation 338 BioEssays 22.4

3 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 Ca 2, due to continuos Ca 2 extrusion by the Na / Ca 2 -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 G- protein 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/ca 2 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 Ca 2. 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 Ca 2 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 Ca 2 flux that normally occurs upon illumination. channels in the outer segment plasma membrane close in response to light, the amount of Ca 2 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 Ca 2 from approximately 500± 700 nm in the dark to about 30 nm upon illumination. This simple feature of modulating the concentration of Ca 2 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- BioEssays

4 able responses can be obtained, is determined by changes in the intracellular concentration of Ca 2. If one interferes with changes in the concentration of Ca 2, 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 Ca 2 intercedes during phototransduction to mediate these adaptation events has been revealed. As we shall describe here, changes in the intracellular concentration of Ca 2 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 Ca 2 ions could effect portions of the phototransduction cascade directly or indirectly through specific Ca 2 -binding proteins. Research in the last few years has shown that Ca 2 -binding proteins play an essential role during the recovery phase of phototransduction (Fig. 2A). One of the most significant effects of Ca 2 is its regulation of GC. As the concentration of Ca 2 decreases upon illumination, it leads to the activation of GC by means of a specific family of Ca 2 -binding proteins termed guanylate cyclase-activating proteins (GCAPs) (Fig. 3). GCAPs are members of the EF-hand superfamily of Ca 2 -binding proteins which includes calmodulin (CaM), parvalbumin, troponin C, and a host of small acidic proteins collectively referred to as S100 Ca 2 -binding proteins. In addition to their normal functions, several human pathologies have been linked to mutations in genes encoding different retinal Ca 2 -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 Ca 2 -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 Ca 2 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 Ca 2 -bound form (three functional EF loops) is compact. After reduction of Ca 2 levels, GC is converted into an active form (right). Ca 2 - 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. 340 BioEssays 22.4

5 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; P i, calculated isoelectric point; K D,Ca 2 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 Ca 2 -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 Ca 2 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 Ca 2 -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 Ca 2 -binding protein characterized, (17,18) its cone variant S26, (19) and GCIP, a GC inhibitory protein identified in frog cones. (20) EF hand Ca 2 -binding proteins The EF-hand Ca 2 -binding motif, a 29-residue helix-loophelix structure containing a 12 residue Ca 2 -binding loop, was first discovered in the crystal structure of parvalbumin. (21) Over 200 Ca 2 -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 BioEssays

6 serves as a Ca 2 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 Ca 2 sensors in vision. (1) Regulation of phototransduction by Ca 2 -binding proteins Ca 2 -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 Ca 2, [Ca 2 ] 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 [Ca 2 ] 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 Ca 2 -sensitive steps are still poorly understood and their physiological significance is unclear. (26) Lowering [Ca 2 ] i may shorten the lifetime of R* by interfering with rhodopsin kinase (RK) inhibition through the action of recoverin (S-modulin), another retina-specific Ca 2 -binding protein. (27) The lightinsensitive NCKX exchanger may also be regulated by Ca 2 - 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) 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 342 BioEssays 22.4

7 Figure 5. Biochemistry of GC stimulation by GCAPs and GCAP mutants. A. Ca 2 -titration of GCAP1/3. (15) The Ca 2 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 [Ca 2 ]. 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 [Ca 2 ] 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) Ca 2 -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 [Ca 2 ]. The shaded areas show the approximate physiological range of Ca 2 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 [Ca 2 ] i, while at high [Ca 2 ] 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 Ca 2 -sensitivity. (44) These and other results suggest that GCAP1 and GCAP2 may have distinct but overlapping contact sites on GC1. GCAPs inhibit GCs when [Ca 2 ] 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 [Ca 2 ] is not high enough to inhibit GC. GCIP from frog cones does not stimulate GC in low Ca 2, but inhibits GC in high Ca 2, and is, therefore, termed GCIP (guanylate cyclase-inhibitory protein). GCIP and GCAPs BioEssays

8 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 Ca 2 -insensitive. For GCAP1, Glu(E) at the invariant 12th residue in the Ca 2 - 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 Ca 2. 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 [Ca 2 ] (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 Ca 2 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 (K D 1 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 [Ca 2 ]. 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 ROS- GC. Deletion of the intracellular kinase-like domain diminished stimulation by GCAP1, suggesting that this domain is involved in Ca 2 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 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 G s. 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 Ca 2 -binding proteins Structure of the EF hand The three-dimensional structure of the EF-hand motif in various Ca 2 -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 Ca 2 -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 Ca 2 -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 Ca 2 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 Ca 2 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 344 BioEssays 22.4

9 Figure 6. Ca 2 -induced structural changes in CaM (A), recoverin (B), and GCAP2 (C). The three-dimensional structures of the Ca 2 -free (left) and Ca 2 -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 (Ca 2 -free CaM (77) ), 3cln.pdb (Ca 2 -bound CaM (78) ), 1iku.pdb (Ca 2 -free recoverin (79) ), 1jsa.pdb (Ca 2 -bound recoverin. (57) ). Schematic ribbon representation of the structure of Ca 2 -bound GCAP2 has been published recently. (81) The four EF-hands (green, red, cyan, and yellow) and three bound Ca 2 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

10 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 Ca 2 The X-ray crystal structure of troponin C (with Ca 2 bound to EF1 and EF2 and not bound to EF3 and EF4) first revealed the structures of both the Ca 2 -free and Ca 2 -bound EFhand motifs. (55) In the Ca 2 -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 Ca 2 -bound EF-hand adopts an ``open conformation'' in which helices E and F are nearly perpendicular (helix packing angle ˆ 90±110 ). Similar Ca 2 -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 Ca 2 also markedly influences the overall topology and three-dimensional arrangement of the two domains. The Ca 2 -induced rearrangement of the domains of CaM is quite striking. The root-mean-square deviation of the main chain atoms of CaM is more than 10 AÊ, when the Ca 2 -free and Ca 2 -bound structures are compared (Fig. 6A). The Ca 2 -free form of CaM exhibits a dumbbell shape with the two domains separated by more than 11 AÊ. The binding of Ca 2 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 Ca 2 -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 Ca 2 -free CaM changes into a globular form in the Ca 2 -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 Ca 2 -free and Ca 2 -bound recoverin are compared in Fig. 6B. A striking feature of these structures is the large rotation of the two domains. The C- terminal domains of the two forms are quite similar, apart from minor changes in the Ca 2 -binding loop and entering the helix of EF-3. The N-terminal domain, by contrast, undergoes a striking rearrangement that leads to the extrusion of the amino-terminal myristoyl group. Extrusion of the myristoyl group requires the binding of Ca 2 to EF-2 and EF-3. The binding of Ca 2 to EF-3 decreases its interhelical angle, similar to the Ca 2 -induced ``opening'' of EF-hands seen in CaM and troponin C. Ca 2 -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 Ca 2 -induced helical twisting in EF-2 is novel and has not been observed previously in other members of the superfamily. The Ca 2 -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 Ca 2 -induced exposure of the myristoyl group, termed the Ca 2 -myristoyl switch, (57) may enable recoverin to bind to membranes at high Ca 2. 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 Ca 2 -bound GCAP2 is very similar to that of recoverin and neurocalcin. Three Ca 2 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) Ca 2 is not bound to EF-1 because the binding loop is distorted from a favorable Ca 2 - binding geometry by Pro 36 at the fourth position of the 12- residue loop. Also, the third residue in the loop (Cys 35) is not suitable for ligating Ca 2. 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 Ca 2 -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 Ca 2 in the EF-hand loops. The structure of physiologically relevant Ca 2 -free GCAPs is currently unknown. Ca 2 -binding proteinsðat the crossroads of life and death Recoverin and CAR Ca 2 -binding proteins mediate between changes in the intracellular concentration of Ca 2 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 346 BioEssays 22.4

11 and manifest as a human disease. Ca 2 -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 Ca 2 -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 Ca 2 sensitivity of GCAP1, leading to the constitutive stimulation of GC1 at high [Ca 2 ] 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 Ca 2 ions. (6) Therefore, conditions that elevate cgmp would be expected to increase the intracellular concentration of Ca 2. Aberrant concentrations of Ca 2, in turn, can induce apoptosis through pathways that are either dependent on or independent of Ca 2 -binding proteins. Ca 2 and apoptosis Ca 2 ions generally enter a cell through channels or by means of an exchanger. Homeostasis is maintained in part by returning Ca 2 to the extracellular environment through the action of pumps and the expenditure of energy (for review see Ref. 65). The majority of Ca 2 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 Ca 2 is within the ER where it is necessary for normal protein synthesis and processing and for cellular signaling. Ca 2 signals can arise either by the entry of Ca 2 from the extracellular space or by the release of sequestered Ca 2. These signals temporarily increase the concentration of intracellular Ca 2 and usually are offset by mitochondrial uptake. Eventually, the balance between Ca 2 in the ER and the mitochondria is re-established. In contrast to Ca 2 signals that are transient, sustained intracellular concentrations of Ca 2 beyond approximately 200 mm lead to cell death (66) either by apoptosis or necrosis depending, at least in part, on the sustained levels of Ca 2. Substantial evidence supports diverse roles for Ca 2 during apoptosis, (67) some aspects of which are mediated by Ca 2 -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 Ca 2 /CaM-dependent serine/threonine phosphatase, calcineurin, and hinders Ca 2 -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 Ca 2 /CaM dependent enzyme linked to cell death. (69) The over-expression of the Ca 2 -binding protein calbindin protects some cells from signals that increase intracellular Ca 2 and induce apoptosis. The buffering capacity of calbindin also limits the extent of cell death induced by Ca 2 ionophores. ALG-2 is a newly identified Ca 2 -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 Ca 2 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 Ca 2 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, Ca 2 ions that enter through CNG channels of the outer segment are returned to the extracellular space by the action of a NCKX BioEssays

12 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 Ca 2 in the outer segment can affect the flux of Ca 2 through the inner segment. The organelles and pathways associated with Ca 2 -induced apoptosis are physically separated into the inner segment, so a ``Ca 2 connection'' must exist in order for pathological changes in the concentration of cgmp in the outer segment to be translated into a Ca 2 -induced signal for apoptosis in the inner segment. Interestingly, a Ca 2 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, Ca 2. These results are consistent with the hypothesis that Ca 2 is a mediator of cell death in a variety of degenerative diseases of the retina initiated by very disparate mutations or environmental insults. (75) References 1. Polans A, Baehr W, Palczewski K. Turned on by Ca 2! The physiology and pathology of Ca 2 -binding proteins in the retina. Trends Neurosci 1996;19:547± Dzeja C, Hagen V, Kaupp UB, Frings S. Ca 2 permeation in cyclic nucleotide-gated channels. EMBO J 1999;18:131± He W, Cowan CW, Wensel TG. RGS9, a GTPase accelerator for phototransduction. Neuron 1998;20:95± Makino ER, Handy JW, Li T, Arshavsky VY. The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit. Proc Natl Acad Sci USA 1999;96: 1947± Gaudet R, Savage JR, McLaughlin JN, Willardson BM, Sigler PB. A molecular mechanism for the phosphorylation-dependent regulation of heterotrimeric G proteins by phosducin. Mol Cell 1999;3:649± Koutalos Y, Yau KW. Regulation of sensitivity in vertebrate rod photoreceptors by calcium. Trends Neurosci 1996;19:73± Schnetkamp PPM, Tucker JE, Szerencsei RT. Ca 2 influx into bovine retinal rod outer segments mediated by Na /Ca 2 /K exchange. Am J Physiol 1995;269:C1153±C Lyubarsky A, Nikonov S, Pugh EN, Jr. The kinetics of inactivation of the rod phototransduction cascade with constant Ca 2. J Gen Physiol 1996; 107:19± Garbers DL, Lowe DG. Guanylyl cyclase receptors. J Biol Chem 1994; 269:30741± Seimiya M, Kusakabe T, Suzuki N. Primary structure and differential gene expression of three membrane forms of guanylyl cyclase found in the eye of the teleost Oryzias latipes. J Biol Chem 1997;272: 23407± Yang RB, Garbers DL. Two eye guanylyl cyclases are expressed in the same photoreceptor cells and form homomers in preference to heteromers. J Biol Chem 1997;272:13738± Palczewski K, Subbaraya I, Gorczyca WA, Helekar BS, Ruiz CC, et al. Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase activating protein (GCAP). Neuron 1994;13:395± Dizhoor AM, Olshevskaya EV, Henzel WJ, Wong SC, Stults JT, et al. Cloning, sequencing, and expression of a 24-kDa Ca 2 -binding protein activating photoreceptor guanylyl cyclase. J Biol Chem 1995;270: 25200± Gorczyca WA, Polans AS, Surgucheva I, Subbaraya I, Baehr W, et al. Guanylyl cyclase activating protein: a calcium-sensitive regulator of phototransduction. J Biol Chem 1995;270:22029± Haeseleer F, Sokal I, Li N, Pettenati M, Rao N, et al. Molecular characterization of a third member of the guanylyl cyclase- activating protein subfamily. J Biol Chem 1999;274:6526± Duda T, Goraczniak RM, Sharma RK. Molecular characterization of S100A1-S100B protein in retina and its activation mechanism of bovine photoreceptor guanylate cyclase. Biochemistry 1996;35: 6263± Dizhoor AM, Ray S, Kumar S, Niemi G, Spencer M, et al. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science 1991;251:915± Kawamura S, Murakami M. Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods. Nature 1991;349: 420± Kawamura S, Kuwata O, Yamada M, Matsuda S, Hisatomi O, et al. Photoreceptor protein s26, a cone homologue of S-modulin in frog retina. J Biol Chem 1996;271:21359± Li N, Fariss RN, Zhang K, Otto-Bruc A, Haeseleer F, et al. Guanylate cyclase inhibitory protein is a frog retinal Ca 2 binding protein related to mammalian guanylate cyclase activating proteins. Eur J Biochem 1998; 252:591± Kretsinger RH, Nockolds CE. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem 1973;248: 3313± Kawasaki H, Nakayama S, Kretsinger RH. Classification and evolution of EF-hand proteins. Biometals 1998;11:277± Crivici A, Ikura M. Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct 1995;24:85± Hsu Y-T, Molday RS. Modulation of the cgmp-gated channel of rod photoreceptor cells by calmodulin. Nature 1993;361:76± Weitz D, Zoche M, Muller F, Beyermann M, Korschen HG, et al. Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the beta-subunit. EMBO J 1998;17:2273± Otto-Bruc AE, Fariss RN, Van Hooser JP, Palczewski K. Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin. Proc Natl Acad Sci USA 1998;95:15014± Kawamura S. Photoreceptor light-adaptation mediated by S-modulin, a member of a possible regulatory protein family of protein phosphorylation in signal transduction. Neurosci Res 1994;20:293± Perrault I, Rozet J-M, Calvas P, Gerber S, Camuzat A, et al. Retinalspecific guanylate cyclase gene mutations in Leber's congenital amaurosis. Nature Genet 1996;14:461± Kelsell RE, Gregory-Evans K, Payne AM, Perrault I, Kaplan J, et al. Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant con-rod dystrophy. Hum Mol Genet 1998;7:1179± Yang RB, Fulle HJ, Garbers DL. Chromosomal localization and genomic organization of genes encoding guanylyl cyclase receptors expressed in olfactory sensory neurons and retina. Genomics 1996;31:367± Howes KA, Bronson JD, Dang YL, Li N, Zhang K, et al. Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2. Invest Ophthalmol Vis Sci 1998;39:867± Schafer BW, Heizmann CW. The S100 family of EF-hand calcium-binding proteins: functions and pathology. Trends Biochem Sci 1996;21: 134± Liu X, Seno K, Nishizawa Y, Hayashi F, Yamazaki A, et al. Ultrastructural localization of retinal guanylate cyclase in human and monkey retina. Exp Eye Res 1994;59:761± Hisatomi O, Honkawa H, Imanishi Y, Satoh T, Tokunaga F. Three kinds of guanylate cyclase expressed in medaka photoreceptor cells in both retina and pineal organ. Biochem Biophys Res Commun 1999;255: 216± Otto-Bruc A, Fariss RN, Haeseleer F, Huang J, Buczylko J, et al. Localization of guanylate cyclase activating protein 2 in mammalian retinas. Proc Natl Acad Sci USA 1997;94:4727± Cuenca N, Lopez S, Howes K, Kolb H. The localization of guanylyl cyclase-activating proteins in the mammalian retina. Invest Ophthalmol Vis Sci 1998;39:1243± BioEssays 22.4