Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina

Cellular Signalling 12 (2000) 711 ± 719

Review Article

Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina Alexander M. Dizhoor* Department of Ophthalmology/Kresge Eye Institute, K-456, 4717 St. Antoine Boulevard, Detroit, MI 48201, USA Department of Pharmacology, Detroit, MI 48201, USA Department of Anatomy and Cell Biology, Detroit, MI 48201, USA Received 19 July 2000; accepted 1 September 2000

Abstract Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction. The decrease in the free intracellular Ca2 + concentrations caused by illumination stimulates two isoforms of retinal membrane guanylyl cyclase (RetGC) via Ca2 + -sensor proteins and thus contributes to photoreceptor recovery and light adaptation. Unlike other members of the membrane guanylyl cyclase family, retinal guanylyl cyclases do not have identified extracellular peptide ligands. Recoverin-like proteins, GCAP-1 and GCAP-2, interact with the intracellular portion of the cyclases and stimulate its activity through dimerization of the cyclase subunits. Several mutations that affect the function of photoreceptor guanylyl cyclase and the activator protein have been linked to various forms of congenital human retinal diseases, such as Leber congenital amaurosis, cone and cone ± rod dystrophy. D 2000 Elsevier Science Inc. All rights reserved. Keywords: Retina; Photoreceptor; cGMP; Guanylyl cyclase; Calcium; GCAP; Cone dystrophy

1. Cyclic GMP and calcium feedback in phototransduction and light adaptation As a first step in visual signaling in vertebrate animals, light induces hyperpolarization of photoreceptor plasma membrane and thus inhibits the release of neuromediator, glutamate, from the synaptic end of the photoreceptor. Photoexcitation of rhodopsin in rods (or related visual pigments in cones) decreases the inward Na + /Ca2 + current across the photoreceptor plasma membrane by a process called phototransduction (Fig. 1). Cyclic GMP plays central role in phototransduction because it directly controls the permeability of Na + / Ca2 + plasma membrane channels. It has been estimated that the free cGMP concentrations in outer segments of dark-adapted photoreceptors are near 2± 4 mM, which is sufficient to keep a few percentage of the cGMP-gated channels in the open state. Photoisomerized rhodopsin triggers the hydrolysis of cGMP by activation of a G protein, transducin, which stimulates cGMP phosphodies-

* Tel.: +1-313-577-1573; fax: +1-313-577-7635. E-mail address: [email protected] (A.M. Dizhoor).

terase and thus causes cGMP-gated channels to close (see Refs. [1± 3] for review). Due to the high gain in the PDE cascade [4], even a single absorbed photon can generate a detectable rod photoresponse [5]. Both rods and cones quickly return to their resting potential after the excitation induced by a short non-saturating flash of light. Exposure of photoreceptors to a constant illumination at first saturates their response, but as a result of a process called light adaptation, the cells can partially restore their light sensitivity. Compared to the darkadapted retina, light-adapted photoreceptors decrease the amplitude and accelerate the kinetics of their photoresponses depending on the intensity of the background light (see Ref. [6] for review). Multiple reactions that occur in the cells during the recovery and light adaptation lead to reopening of the cGMP-gated channels. These reactions include inactivation of the photoexcited rhodopsin, turning off the light-sensitive PDE cascade and resynthesis of cGMP (Fig. 1) [6± 9]. Along with hyperpolarization of the cell plasma membrane, photoexcitation gives rise to a significant change in the intracellular Ca2 + concentrations in rods and cones, which provides a powerful feedback essential for normal recovery and light adaptation (Fig. 2). In dark-adapted

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Fig. 1. Phototransduction and recovery in vertebrate photoreceptors. (A) The excitation phase. Plasma membrane of photoreceptor outer segment contains cation channels. In the dark, some of the channels are opened by bound cGMP and allow influx of Na + and Ca2 + into the outer segment. Photoactivated rhodopsin (Rh*) converts a G-protein, transducin (T), into its active GTP-bound state (T-GTP) that stimulates the effector enzyme, phosphodiesterase (PDE), by displacing its inhibitory subunit. The hydrolysis of cGMP by the activated PDE* shuts down the cGMP-gated channels and thus results in hyperpolarization of the plasma membrane. (B) The recovery phase. Left to right. The Rh* undergoes phosphorylation (Rh*-P) by rhodopsin kinase (RK), binds a 48-kDa protein, arrestin (Arr), and thus becomes inactive (see Refs. [1 ± 3] for review). Interaction with the inhibitory subunit of PDE (PDEg) and RGS-9/Gb5 complex [92,93] accelerates the intrinsic GTPase activity of transducin and returns it into the inactive GDP-bound state (T-GDP). The PDEg released by T-GDP recombines with the PDE* and inhibits cGMP hydrolysis. RetGC, stimulated by Ca2 + -free sensor protein (GCAP), restores cGMP concentration, and the cGMP-gated channels re-open.

photoreceptors, Ca2 + extruded from the photoreceptor outer segment by a Na + /K + , Ca2 + exchanger re-enters the outer segment through the open cGMP-gated channels, so that the average free intracellular Ca2 + level lies between 500 and 600 nM [10]. In the light, when cGMP is hydrolyzed and the channels are closed, the exchanger continues to extrude Ca2 + ions from the photoreceptor outer segment. Therefore, the intracellular Ca2 + concentrations in illuminated rods and cones can decrease below 50 nM [10,11]. The decrease in the intracellular Ca2 + concentrations caused by light contributes to photoreceptor recovery and adaptation through the activation of guanylyl cyclase [12], stimulation of cGMP binding to the cGMP-gated channels [13,14] and, most likely, inactivation of photoexcited rhodopsin [15]. Among the ways in which Ca2 + feedback contributes to the recovery and light adaptation, regulation of photoreceptor membrane guanylyl cyclase is most significant (reviewed in Refs. [6± 8]). Along with the fact that synthesis of cGMP in photoreceptors increases when the free Ca2 + concentrations drop below 100 nM [12], clamping the free Ca2 + concentrations at higher levels typical for dark-adapted photoreceptors makes the cells incapable of light adaptation [16,17]. Mechanisms of cGMP synthesis in preparations of photoreceptor membranes have been studied in vitro by

using several different techniques recently reviewed elsewhere [18,19]. Earlier observations by Lolley and Racz [20] implicated Ca2 + as a potential regulator of the cGMP synthesis in photoreceptors. A major milestone in the early studies of the cyclase regulation was the work by Koch and Stryer [12] who found that Ca2 + regulated guanylyl cyclase in photoreceptor membranes in a cooperative manner, within the physiologically relevant range of the free Ca2 + concentrations and via a soluble Ca2 + sensor protein. Photoreceptor membranes washed at low ionic strength lack the soluble Ca2 + -sensor activator and have relatively low basal cyclase activity (near 3 ± 5 nmol of cGMP min ÿ 1 mg ÿ 1 of rhodopsin, Refs. [21 ± 23]), which also becomes insensitive to Ca2 + . In the presence of the Ca2 + sensor activator proteins in vitro, the guanylyl cyclase activity accelerates typically four- to five-fold at the [Ca2 + ]free near or below 50 nM [21 ±27]. Further increase in the [Ca2 + ]free inhibits the cyclase in a cooperative manner, with an EC50 Ca of 200 ± 300 nM, and a Hill coefficient of  1.7 ±2 [21 ± 27]. Following the original observation by Koch and Stryer [12], two isozymes of the retinal membrane guanylyl cyclase (RetGC) were found in photoreceptor membranes, and at least two of their soluble Ca2 + -sensor proteins, guanylyl cyclase activator proteins (GCAPs), were identified throughout vertebrate species (reviewed in Refs. [18,28]).

Fig. 2. Ca2 + feedback in photoreceptors. (A) In the dark, Ca2 + removed from the outer segment cytoplasm by a Na + /Ca2 + , K + exchanger re-enters the cell through the open cGMP-gated Na + /Ca2 + channels. (B) While causing the hyperpolarization of the membrane, cGMP hydrolysis by photoactivated PDE* stops the influx of Ca2 + and causes its intracellular concentration to drop. (C) Low Ca2 + concentrations stimulate Rh* phosphorylation by RK (likely by negating the inhibitory effect of recoverin or S-modulin, Ref. [15]), activate cGMP synthesis by RetGC [12] and increase the affinity of the channels to cGMP [13,14].

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2. Photoreceptor guanylyl cyclases. The unusual members of the membrane guanylyl cyclase family The first cDNA clone coding for RetGC-1 (GC-E) was found by Shyjan et al. [29]. The second isozyme, RetGC-2 (GC-F), was later cloned from human and rat retinal cDNA libraries by two independent groups [30,31]. Shortly after that, homologues of RetGC-1 and RetGC-2 have been found in other vertebrate species [32,33]. Heterologously expressed recombinant RetGC-1 and RetGC-2 have been identified as Ca2 + /GCAP-regulated cyclases based on in vitro assays of their activity [21,31]. The expression of RetGC-1 and RetGC-2 in photoreceptors has been confirmed by in situ hybridization using specific oligonucleotide probes [29,31], and both proteins have been detected in membranes of the inner and outer segments of rods and cones using immunofluorescence and immunogold staining [21,34,35]. However, while the antibodies against RetGC-1 produce a strong signal in cones, they give only a weak signal in rods [21,34]. Disruption of a mouse RetGC-1 gene results in degeneration of cones but does not significantly affect viability or electrophysiology of rods [36], which is also consistent with the RetGC-1 being a cone-specific isoform. The detailed immunolocalization of RetGC-2 has not yet been reported, but our preliminary observations suggest that RetGC-2 is strongly expressed in rods and rather weakly in cones. RetGC-1 and RetGC-2 are not only closely related to each other, but also very much resemble other membrane guanylyl cyclases, such as natriuretic peptide receptor cyclases (GC-A, GC-B), guanylin- and enterotoxin-activated intestinal cyclase (GC-C), peptide receptors in sea urchin sperm cells (GC-D) and olfactory guanylyl cyclase (GC-G) (reviewed in Refs. [37,38]). RetGC-1 and -2 have three major homology regions common to transmembrane guanylyl cyclases (Fig. 3). In every receptor guanylyl cyclase, the extracellular domain (ECD) forms a binding center for the extracellular activator (i.e. peptide ligand). The homology between RetGC-1, RetGC-2, and other members of the GC family is rather weak within this domain compared to other regions of the molecule [30,31]. In RetGC-1, after the

Fig. 3. A schematic representation of structural domains in RetGC molecule. Explanations are in the text.

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removal of its N-terminal leader peptide, the ECD undergoes post-translational glycosylation [39]. Together with other members of this family, RetGC is a transmembrane protein, and its ECD is connected to the cytoplasmic portion of the molecule by a single hydrophobic transmembrane region. Similar to other membrane guanylyl cyclases, the intracellular part of RetGC includes a region homologous to protein kinases [usually called kinase homology domain (KHD)] [37,38]. RetGC, isolated from the retina by several steps of chromatography, has been reported to undergo phosphorylation in vitro [40] by autophosphorylation. Although in natriuretic peptide receptors, phosphorylation of the intracellular domain stimulates the cyclase activity (see Ref. [41] for review); the physiological significance of phosphorylation in the case of RetGC remains unclear. Moreover, the protein kinase activity detected in preparations of RetGC-1 purified from the retina may possibly reflect a residual contamination from the retinal protein kinases. The actual consensus sequence implicated in protein kinase activity has not been found in KHD of membrane guanylyl cyclases, and there are serious doubts that KHD of RetGC can even posses such activity [38]. Additional experimental evidence will be required in order to prove or to exclude the presence of the intrinsic protein kinase activity in RetGC. ATP stabilizes RetGC activity in photoreceptor membranes in vitro [42,43] by slowing its spontaneous inactivation [44]. In this case, RetGC apparently requires direct ATP binding rather than ATP-dependent phosphorylation, since the non-hydrolyzable ATP analogs produce similar effect [43,44]. The attempts to identify the ATP-binding center in RetGCs have not yet been successful, especially because a consensus motif, GXGXXXG, implicated in ATP binding in natriuretic receptor guanylyl cyclases [45], is absent from the corresponding part of the KHD domain. However, there are indications that more than one such center may exist in RetGC-1 [46]. A short region between the KHD and the catalytic domain in membrane GC is required for the ability of the cyclase to form a homodimer. Consequently, it has been referred to as ``dimerization domain'' [38,41]. Although there is no direct experimental evidence that it actually creates a direct contact between two RetGC polypeptides, the corresponding putative dimerization domain in RetGC apparently plays a very important functional role, because several mutations in this short region of the molecule have been linked to genetic disorders of vision [47], as described in Section 6 of this review. The catalytic domain of RetGC located within the Cterminal third of the protein converts its substrate, MgGTP (or MnGTP in vitro), into cGMP and inorganic pyrophosphate (PPi). Evidently, the hydrolysis of the PPi by inorganic pyrophosphatase in photoreceptors is required in order to prevent inhibition of the cGMP synthesis by pyrophosphate [48].

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The catalytic domain of RetGC closely resembles other guanylyl cyclases and a Type II adenylyl cyclase [49]. High selectivity of the cyclase for the MgGTP as a substrate, compared to MgATP, is determined by only a few amino acid residues, so that just two amino acid substitutions, E925K together with C995D, within the catalytic domain of RetGC-1, converts it into an adenylyl cyclase [49]. Although both isoforms of RetGC are highly homologous to GC-A and GC-B and other members of the membrane guanylyl cyclases family, they demonstrate a very unusual property that clearly separates them from the rest of the family. First, RetGCs do not respond to the extracellular peptide ligands that activate other membrane GCs [29 ± 31]. Due to the lack of any obvious extracellular ligand, they are also regarded as ``orphan'' membrane guanylyl cyclases [38]. Second, there is strong evidence that RetGC-1 and RetGC-2 do not require the extracellular peptide(s) for their activation. Instead, both RetGC isozymes are regulated by Ca2 + -binding proteins, GCAPs that interact with the cytoplasmic portion of the cyclase molecule [21,31,43,50]. 3. GCAPs: Ca2+ sensors for photoreceptor guanylyl cyclases Two soluble Ca 2 + -binding proteins, GCAP-1 and GCAP-2, which impart Ca2 + sensitivity to RetGC, were isolated in 1994 [21,25]. GCAP-1 was purified from the retinal fraction containing photoreceptor outer segment membranes based on its ability to stimulate RetGC activity in washed photoreceptor outer segment membranes [25]. GCAP-2 was isolated from a heat-stable fraction of soluble retinal proteins and it was purified to homogeneity by the same criterion but using a different purification scheme [21]. Amino acid sequences of GCAP-1 and GCAP-2 are over 40% identical and include three distinct EF-hand Ca2 + binding domains [51] and the signal for the N-terminal fatty acylation [22,52]. Both proteins are highly acidic, and the average isotopic molecular masses of fatty-acylated GCAPs are close to 24 kDa [52,53]. Unlike GCAP-1, GCAP-2 tends to bind irreversibly to various chromatographic media, especially, anion exchangers, and is difficult to purify from photoreceptor outer segment membranes using the same scheme of purification as for GCAP-1 [27,54]. On the other hand, GCAP-2 is thermostable in the presence of Ca2 + and partially retains its activity after reverse-phase chromatography in the presence of acetonitrile [21]. GCAP-1 and GCAP-2 are both present in photoreceptors in vivo [22,54 ± 57]. The specific anti-GCAP-1 antibodies detect expression of GCAP-1 almost exclusively in cones [55,57], while GCAP-2 has been detected primarily in outer and inner segments of rods and at a lower level in cones [22,55,57]. The signal for GCAP-1 and GCAP-2 within the intracellular compartments of rods and cones may partially depend on the strength of a particular antibody and on how

different methods of fixation affect their epitopes exposure in various specimens [55,57]. However, additional evidence to support that GCAP-1 is more specific for cones comes from the fact that a mutation, Y99C, in GCAP-1 in humans, results in cone degeneration, but has virtually no effect on the viability or electrophysiological behavior of rods [58]. More recently, a cDNA for the third homologue, GCAP3, was cloned from a human retinal cDNA library. Although mRNA for GCAP-3 is present in human retina, this protein has not been isolated from the retinal tissue, nor was it found in retinal cDNA libraries from several other animal species [59]. Unlike GCAP-1 and GCAP-2 whose genes are arranged in a tail-to-tail orientation within the same chromosomal locus, 6p21.1 [60], the GCAP-3 gene is in the locus 3q13.1 [59]. The role of GCAP-3 in recovery and adaptation is not immediately apparent, since the disruption of both GCAP-1 and GCAP-2 genes in transgenic mice seems to be sufficient to decelerate the recovery in rods in a manner consistent with the lack of the Ca2 + -feedback regulation of the cGMP synthesis [61]. However, it cannot be excluded that GCAP-3 may actually function in a certain population of cones. There are also non-photoreceptor proteins that can activate RetGC in vitro under different than GCAPs conditions. A small 10-kDa EF-hand protein, S100b, originally referred to as CDGCAP, can stimulate RetGC activity at rather high free Ca2 + concentrations ( > 5 mM) [62]. It also has been reported that neurocalcin can weakly activate recombinant RetGC at the free Ca2 + concentrations above 5 mM [63]. However, neither S100b nor neurocalcin have measurable effect on cyclase activity within the physiologically relevant range of the free Ca2 + concentrations in photoreceptors [62 ±64]. Hence, it does not seem feasible that either S100b or neurocalcin can contribute to regulation of RetGC activity in vivo. Yet, the ability to interact with RetGC at high Ca2 + concentrations in vitro may reflect their potential involvement in regulation of cGMP synthesis in some other cells with a different, presently unknown, target cyclase. 4. Structure and function of GCAPs GCAPs belong to a family of the recoverin-like proteins that are distantly homologous to calmodulins, but constitute a well-defined separate group within the EF-hand [51] superfamily. Unlike the ubiquitous calmodulins, recoverinlike proteins are N-fatty-acylated and are predominantly localized to neurons (see Ref. [65] for review). Similarly to calmodulin, the recoverin-like proteins have four EF-hand motifs (usually referred to as EF-1 through EF-4), but only three (or sometimes just two) of them form true Ca2 + -binding domains. The N-terminal EF-handrelated motif, EF-1, deviates from the consensus sequence and lacks several key side-chain residues required for coordinating Ca2 + ions [51,66]. GCAP-1 and GCAP-2 have three functional EF-hands (Fig. 4), and inactivation

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Fig. 4. Three-dimensional model of the Ca2 + -bound GCAP-2 established by Ames et al. [68]. Calcium positions are represented by the black spheres within the Ca2 + -binding loops of the EF-hands (EF-2 through EF-4). The first EF-hand-related structure (EF-1) does not bind Ca2 + . The regions that contain GCAP-2-specific amino acid sequences required for the RetGC regulation [64] are highlighted.

of these EF-hands by point mutations renders GCAPs in a constitutively active form that efficiently stimulates RetGC both at low and high Ca2 + concentrations [23,67]. According to direct measurement of Ca2 + binding using equilibrium dialysis, GCAP-2 binds three Ca2 + ions with a cooperativity factor of  2 and an apparent dissociation constant of 300 nM [68]. These values closely resemble the cooperativity and the EC50 values for the Ca2 + effects on RetGC regulation by GCAP-2 [21,23,53,64]. Upon binding of Ca2 + , GCAPs undergo an ``activator-to-inhibitor'' transition, so that fully Ca2 + -loaded GCAPs ([Ca2 + ]free  1 ± 10 mM) inhibit basal activity of RetGC in photoreceptor membranes [23,67]. However, at the free Ca2 + concentrations typical for the dark-adapted photoreceptors, the ratio between the Ca2 + -free (activator) and the Ca2 + -bound (inhibitor) forms of GCAP-2 is such that the activity of RetGC is fairly close to its basal activity measured in the absence of GCAPs [23]. The relative contribution of each EF-hand to the overall Ca2 + sensitivity of the cyclase regulation is slightly different between GCAP-1 and GCAP-2. Inactivation of EF-3 or EF-4 by point mutations in GCAP-1 produces a strong decrease in Ca2 + sensitivity of the cyclase activation, while inactivation of the EF-2 has only a small effect. Contrary to this, inactivation of the third Ca2 + -binding loop, EF-3, in GCAP-2 has relatively small effect on Ca2 + sensitivity of RetGC compared to EF-2 and EF-4. Inactivation of any pair of EF-hands is sufficient to shift the Ca2 + sensitivity of RetGC regulation outside the physiological submicromolar range, and inactivation of all three EF-hands completely eliminates Ca2 + sensitivity of GCAPs and turns them into constitutive activators of RetGC [23,67]. The solution structure of the Ca2 + -bound GCAP-2 recently established by Ames et al. [68] is drastically different from calmodulin and closely resembles recoverin and neurocalcin. The GCAP-2 molecule consists of two globular halves connected by a flexible ``hinge'' region

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between the exiting helix of the EF-2 and the entering helix of the EF-3 (Fig. 4). Each half of the molecule is formed by a pair of the EF-hands. The two pairs of EF-hands form a well-defined core of the molecule, while its N- and Ctermini are rather flexible, and their signals on the NMR spectra have poor resolution [68]. The patterns of proteolytic cleavage and tryptophan fluorescence spectra of GCAPs demonstrate that the binding and the release of Ca2 + substantially affect GCAP conformation [67,69,70]. Yet, the actual three-dimensional structure of the Ca2 + -free GCAP-2 remains undetermined. The three-dimensional structure of GCAP-1 (Ca2 + -free or Ca2 + -bound) has not been determined either. Similar to other recoverin-like proteins, the N-terminal glycine in GCAP-1 and GCAP-2 is fatty-acylated by the Nmyristoyl transferase [22,52]. This modification is often referred to as ``myristoylation'', although in photoreceptors the N-acyl groups have heterogeneous (both saturated and unsaturated) C14 and C12 fatty chains [71,72]. GCAP-1 and GCAP-2 can be produced in myristoylated form in Escherichia coli by co-expression with the N-myristoyl transferase [53,73]. The presence of the N-fatty acyl group significantly increases activity of GCAP-1 [74], but makes no major contribution to the activity of GCAP-2 [53]. In recoverin, the N-fatty acyl moiety constrained within the protein structure is released by Ca2 + binding. This conformational transition is known as ``calcium ± myristoyl switch'' (see Ref. [75] for review). No evidence for the existence of such switch has been found in GCAP-2, and its N-terminal fatty acyl group is likely to be constrained neither in the Ca2 + bound nor in the Ca2 + -free state [69]. It is well-documented that GCAP-2 can activate both recombinant RetGC-1 and RetGC-2 in vitro [21,31,59,64]. The matter of selectivity between the two RetGC isozymes in the case of GCAP-1 remains unclear. Some authors observed only activation of recombinant RetGC-1 by GCAP-1, expressed from baculoviral vectors in cultured insect cells [59]. According to other group, fully myristoylated GCAP-1 produced in E. coli can activate recombinant RetGC-2 in vitro [76]. This discrepancy may possibly reflect variations in a degree of the fatty acylation of GCAP-1 produced in different expression systems. In addition to that, the apparent affinity of GCAPs toward the heterologously expressed RetGC isozymes (for example, see Refs. [59,77]) is 1 ± 2 orders of magnitude lower compared to the cyclase present in photoreceptor membranes [78], meaning that the heterologously expressed RetGC isozymes do not necessarily reflect all the properties of the native enzyme. 5. Mechanism of RetGC regulation by GCAP. The ``dimer ±adapter'' hypothesis Despite that the actual three-dimensional structure of the RetGC/GCAP complex is unknown, recent observations

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indicate that such complex likely consists of a dimer of the cyclase and a dimer of GCAP. Both RetGC-1 and RetGC-2 have a putative ``dimerization domain'' typical for peptide receptor guanylyl cyclases (Fig. 3) (for review see also Refs. [37,38,41]). Although coexpression of RetGC-1 and RetGC-2 in transfected cells can result in a complex that contains both isozymes, they are likely to form only homodimers in vivo [35]. RetGC in photoreceptor outer segment membranes can be chemically cross-linked into a covalent dimer, and the yield of the specific cross-linked product increases in the presence of the Ca2 + -free GCAP-1 and GCAP-2, while Ca2 + -loaded GCAPs inhibit this reaction [79]. Also, the catalytic domain of RetGC is closely related to the adenylyl cyclase II in which the active center of the enzyme is formed by its dimerized catalytic domains [49]. Therefore, it is tempting to suggest that the functional role of GCAP is to stimulate closer interactions between the catalytic domains within the RetGC homodimer. Both GCAP-1 and GCAP-2 can also form functional homodimers. The structure of each dimer is affected by Ca2 + binding, and in the case of GCAP-2, its Ca2 + -loaded form completely dissociates to monomers during gel-chromatography [78]. Moreover, the ability of GCAP-2 and its chimerical mutants to activate RetGC at low free Ca2 + concentrations correlates with their ability to dimerize in the absence of Ca2 + . Thus, not only GCAP:RetGC, but GCAP:GCAP interactions, as well, are important for the cyclase regulation [78]. GCAPs themselves can bind to the membrane [53,54], therefore, their presence in a membrane fraction does not necessarily directly reflect their binding to the target enzyme [53]. Yet, there are additional evidences that GCAP tightly binds to the cyclase and that GCAP dimer mediates RetGC:RetGC interaction, likely within a tetrameric complex, [(RetGC)2(GCAP)2]. In contrast to the Ca2 + -free GCAPs that activate RetGC, the Ca2 + -loaded GCAPs inhibit the intrinsic basal activity of the cyclase in photoreceptor membranes in vitro [23,67,76,78]. This clearly indicates that GCAPs remains bound to RetGC both when in its Ca2 + -free and the Ca2 + -bound forms. Consistent with this, GCAP-2 partially protects the intracellular fragment of RetGC from proteolysis both in the presence and in the absence of Ca2 + [44]. A putative mechanism (Fig. 5) that we referred to as a ``dimer ±adapter'' model [78] can fit with the following observations: (1) GCAP-2 both forms dimers and activates RetGC in low Ca2 + ; (2) While binding of Ca2 + promotes dissociation of the GCAP-2 dimer, it still remains bound to the cyclase; and (3) Ca2 + -free, but not Ca2 + -bound GCAP2 stimulates interactions between the RetGC molecules. According to this model, GCAP monomers bound to each molecule of the cyclase form a GCAP:GCAP dimer that mediates interactions between the catalytic domains of the cyclase subunits. The Ca 2 + -loaded GCAP molecules attached to the individual RetGC subunits may interfere

Fig. 5. A putative ``dimer ± adapter'' mechanism for the RetGC activation by GCAP-2 [78]. (Left) The Ca2 + -bound GCAP molecules attached to the RetGC subunits interfere with dimerization of the cyclase catalytic domains and inhibit RetGC activity. (Right) The release of Ca2 + results in a conformational change in GCAP-2 accompanied by tight association between the two GCAP-2 molecules. The tight (GCAP-2)2 dimer brings RetGC subunits into a closer contact between their catalytic domains that form the active center of the enzyme.

with dimerization of the cyclase catalytic domains, and therefore inhibit the function of the active center of the enzyme. The dissociation of Ca2 + in the light changes the GCAP dimer conformation and also increases the affinity between the two GCAP-2 molecules. ``Pulling'' together the GCAP molecules while they are tightly bound to the cyclase subunits can bring the RetGC subunits (either directly or by causing a secondary conformational change in RetGC) into a proper contact between their catalytic domains and thus activate the catalytic center in a RetGC homodimer. To further evaluate this model, it would require to determine the exact binding sites in GCAPs and RetGC, as well as the ratio between GCAP and RetGC subunits within such complex in vivo. The places where the contacts between RetGCs and GCAPs occur, have not yet been clearly determined. One of the putative sites for the binding of GCAP-1 and GCAP-2 reportedly includes a peptide sequence, GTFRMRHMPEVPVRIRIG, within the cyclase catalytic domain [80]. However, there may be additional or even alternative sites in RetGC that interact with GCAPs. Based on the study of the chimerical RetGC-1 and RetGC-2, their affinity for GCAPs may be determined by the KHD rather than the cyclase catalytic domain [77]. Also, in a bovine homologue of RetGC-1, two different fragments, M445-L456 and L503I522, have been implicated as possible sites for the interaction with GCAP [81]. Compared to other recoverin-like proteins, there are several regions in GCAP-1 and GCAP-2 that determine their specificity as the cyclase activators. The positions of these regulatory domains are only slightly different in GCAP-1 and GCAP-2, and in both cases, they include a region engulfing the EF-1 loop, a ``hinge'' region that bridges EF-hands 2 and 3, and a part of the C-terminal

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region, immediately adjacent to the EF-4 (Fig. 4) [64,76]. The region between EF-2 and EF-3 determines that the Ca2 + -free, not a Ca2 + -bound form of the GCAP-2, activates the cyclase [76]. In contrast to the earlier beliefs [52], the very N-terminus of GCAP-1 is unlikely to participate in cyclase activation [76,82]. It now remains to be determined which part(s) of the GCAP molecule directly binds to the cyclase, and where the GCAP:GCAP contacts may occur to promote its dimerization. 6. Mutations in RetGC and GCAP associated with congenital retinal diseases Severe cases of Leber congenital amaurosis (photoreceptor degeneration in early childhood) have been linked to frame shift and missense mutations in RetGC-1 [83]. The missense mutation, F514S, decreases stimulation of RetGC1 by GCAP in vitro [84]. Defects in the RetGC-1 gene were also found in patients with dominant cone ± rod dystrophy, a disease that causes degeneration of cones followed by the death of rods. These defects include two single missense mutations: E837D and R838C; and a triple mutation: E837D, R838C and T839M. In all three cases, the substitutions are in the putative dimerization domain of RetGC1 [85,86]. The R838C substitution reduces the overall catalytic activity of RetGC-1 in vitro and drastically suppresses stimulation by GCAP-2 [47,87]. The R838C substitution also alters the Ca2 + sensitivity of the GCAP-1 response in vitro, thus allowing the mutant cyclase to be stimulated by GCAP-1 at higher Ca2 + concentrations than wild type [47]. A case of congenital dominant cone degeneration has been linked to a mutation, Y99C, found within the third EFhand of GCAP-1 [58]. This mutation causes slow degeneration of cones and results in a complete loss of color perception and daylight vision, yet rods are unaffected and their responses to dim light remain normal. Although the Y99C GCAP-1 activates RetGC in vitro to the same extent as the wild type GCAP-1, in the presence of the Y99C mutant, the cyclase remains active even at the free Ca2 + concentrations above 1 mM [73,88]. The Y99C GCAP-1 efficiently competes with the Ca2 + -loaded wild type GCAP-1 and GCAP-2, and that may account for the dominant nature of this mutation in humans [73]. High intracellular Ca2 + concentrations can alter the permeability of rod mitochondria and promote apoptosis in photoreceptors [89,90]. Since activation of RetGC in vitro by the Y99C GCAP-1 is not fully suppressed even at  10 mM free Ca2 + , it is very tempting to speculate that in live photoreceptors, it also cannot be properly suppressed when the free Ca2 + concentrations reach their normal ``dark'' level. In normal photoreceptors, the ``dark'' free concentrations of the circulating Ca2 + are at their  500 nM steady-state level because the low rate of cGMP synthesis matched by the low PDE activity maintains the free

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cGMP concentrations near 2 ± 4 mM [91]. Only several percent of the total cGMP-gated channels are open in the dark and provide the influx of Na + and Ca2 + [91]. The increased rate of cGMP synthesis by the Y99C GCAP-1, if not matched by a corresponding increase in the basal PDE activity, would lead to the opening of higher number of the cGMP-gated channels in cones. That would increase the influx of Na + /Ca2 + until the intracellular Ca2 + concentrations reach abnormally high levels (>10 mM, based on Ref. [73]) that are sufficient to prevent the mutant GCAP-1 from activating the cyclase. Concentrations of Ca2 + , in that case, may be also sufficient to provoke the apoptosis in cones. A similar scenario may also take place in the case of the R838C mutation in RetGC-1 linked to cone ± rod dystrophy [47] discussed earlier. However, it is important to emphasize that at present, there is no direct experimental evidence that the Y99C GCAP-1 increases the free Ca2 + level in cones in vivo. Even if it were the case, it is also difficult to explain why the cone death in patients that carry such mutation occurs only around their mid-life ages [58]. There could be additional functions of GCAP-1 that are affected by this mutation, or a combination of several different factors may contribute to the onset and/or acceleration of the cone death. Transgenic models should help to verify the actual biochemical links between the mutant GCAP-1 and the photoreceptor degeneration.

Acknowledgments The financial support for A.M.D. has been provided in part by the National Institutes of Health (grant EY11522) and Research to Prevent Blindness.

References [1] Pugh EN, Lamb TD. Biochim Biophys Acta: Bio-Energ 1993; 1141:111 ± 49. [2] Baylor D. Proc Natl Acad Sci USA 1996;93:560 ± 5. [3] Molday RS. Invest Ophthalmol Visual Sci 1998;39:2491 ± 513. [4] Leskov IB, Klenchin VA, Handy JW, Whitlock GG, Govardovskii VI, Bownds MD, Lamb TD, Pugh EN, Arshavsky VY. Neuron 2000; 27:525 ± 53. [5] Baylor DA, Lamb TD, Yau K-W. J Physiol (London) 1979; 288:613 ± 34. [6] Pugh EN, Nikonov S, Lamb TD. Curr Opin Neurobiol 1999;9:410 ± 8. [7] Koutalos Y, Yau KW. Trends Neurosci 1996;19:73 ± 81. [8] Pugh EN, Duda T, Sitaramayya A, Sharma RK. Biosci Rep 1998; 17:429 ± 73. [9] Miller JL, Picones A, Korenbrot JI. Curr Opin Neurobiol 1994; 4:488 ± 95. [10] Gray-Keller MP, Detwiler PB. Neuron 1994;13:849 ± 61. [11] Sampath AP, Matthews HR, Cornwall MC, Bandarchi J, Fain GL. J Gen Physiol 1999;113:267 ± 77. [12] Koch K-W, Stryer L. Nature 1988;334:64 ± 71. [13] Hsu YT, Molday RS. Nature 1993;361:76 ± 9. [14] Hackos DH, Korenbrot JI. J Gen Physiol 1997;110:515 ± 28.

718

A.M. Dizhoor / Cellular Signalling 12 (2000) 711±719

[15] Kawamura S. Nature 1993;362:855 ± 7. [16] Matthews HR, Murphy RLW, Fain GL, Lamb TD. Nature 1988; 334:67 ± 9. [17] Nakatani K, Yau K-W. Nature 1988;334:69 ± 71. [18] Dizhoor AM, Hurley JB. Methods 1999;19:521 ± 31. [19] Hurley JB, Dizhoor AM. Methods Enzymol 2000;315:708 ± 17. [20] Lolley RN, Racz E. Vision Res 1982;22:1481 ± 6. [21] Dizhoor AM, Lowe DG, Olshevskaya EV, Laura RP, Hurley JB. Neuron 1994;12:1345 ± 52. [22] Dizhoor AM, Olshevskaya EV, Henzel WJ, Wong SC, Stults JT, Ankoudinova I, Hurley JB. J Biol Chem 1995;270:25200 ± 6. [23] Dizhoor AM, Hurley JB. J Biol Chem 1996;271:19346 ± 50. [24] Wolbring G, Schnetkamp PP. Biochemistry 1996;35:11013 ± 8. [25] Gorczyca WA, Gray-Keller MP, Detwiler PB, Palczewski K. Proc Natl Acad Sci USA 1994;91:4014 ± 8. [26] Lambrecht HG, Koch KW. EMBO J 1991;10:793 ± 8. [27] Frins S, Bonigk W, Muller F, Kellner R, Koch KW. J Biol Chem 1996;271:8022 ± 7. [28] Palczewski K, Polans AS, Baehr W, Ames JB. BioEssays 2000; 22:337 ± 50. [29] Shyjan AW, de Sauvage FJ, Gillett NA, Goeddel DV, Lowe DG. Neuron 1992;9:727 ± 37. [30] Yang RB, Foster DC, Garbers DL, Fulle HJ. Proc Natl Acad Sci USA 1995;92:602 ± 6. [31] Lowe DG, Dizhoor AM, Liu K, Gu Q, Spencer M, Laura R, Lu L, Hurley JB. Proc Natl Acad Sci USA 1995;92:5535 ± 9. [32] Goraczniak R, Duda T, Sharma RK. Biochem Biophys Res Commun 1997;234:666 ± 70. [33] Semple-Rowland SL, Lee NR, Van Hooser JP, Palczewski K, Baehr W. Proc Natl Acad Sci USA 1998;95:1271 ± 6. [34] Liu X, Seno K, Nishizawa Y, Hayashi F, Yamazaki A, Matsumoto H, Wakabayashi T, Usukura J. Exp Eye Res 1994;59:761 ± 8. [35] Yang RB, Garbers DL. J Biol Chem 1997;272:13738 ± 42. [36] Yang RB, Robinson SW, Xiong WH, Yau KW, Birch DG, Garbers DL. J Neurosci 1999;19:5889 ± 97. [37] Garbers DL, Lowe DG. J Biol Chem 1994;269:30741 ± 4. [38] Garbers DL. Methods 1999;19:477 ± 84. [39] Koch KW. J Biol Chem 1991;266:8634 ± 7. [40] Aparicio JG, Applebury ML. J Biol Chem 1996;271:27083 ± 9. [41] Foster DC, Wedel BJ, Robinson SW, Garbers DL. Rev Physiol, Biochem Pharmacol 1999;135:1 ± 39. [42] Gorczyca WA, Van Hooser JP, Palczewski K. Biochemistry 1994; 33:3217 ± 22. [43] Laura RP, Dizhoor AM, Hurley JB. J Biol Chem 1996;271:11646 ± 51. [44] Tucker CL, Laura RP, Hurley JB. Biochemistry 1997;36:11995 ± 2000. [45] Duda T, Goraczniak RM, Sharma RK. FEBS Lett 1993;335:309 ± 14. [46] Duda T, Goraczniak R, Sharma RK. Biochem J 1996;319:279 ± 83. [47] Tucker CL, Woodcock SC, Kelsell RE, Ramamurthy V, Hunt DM, Hurley JB. Proc Natl Acad Sci USA 1999;96:9039 ± 44. [48] Yang Z, Wensel TG. J Biol Chem 1992;267:24634 ± 40. [49] Tucker CL, Hurley JH, Miller TR, Hurley JB. Proc Natl Acad Sci USA 1998;95:5993 ± 7. [50] Duda T, Goraczniak R, Surgucheva I, Rudnicka-Nawrot M, Gorczyca WA, Palczewski K, Sitaramayya A, Baehr W, Sharma RK. Biochemistry 1996;35:8478 ± 82. [51] Persechini A, Moncrief ND, Kretsinger RH. Trends Neurosci 1989; 12:462 ± 7. [52] Palczewski K, Subbaraya I, Gorczyca WA, Helekar BS, Ruiz CC, Ohguro H, Huang J, Zhao X, Crabb JW, Johnson RS, Walsh KA, Gray-Keller MP, Detwiler PB, Baehr W. Neuron 1994;13:395 ± 404. [53] Olshevskaya EV, Hughes RE, Hurley JB, Dizhoor AM. J Biol Chem 1997;272:14327 ± 33. [54] Gorczyca WA, Polans AS, Surgucheva IG, Subbaraya I, Baehr W, Palczewski K. J Biol Chem 1995;270:22029 ± 36. [55] Howes K, Bronson JD, Dang YL, Li N, Zhang K, Ruiz C, Helekar B, Lee M, Subbaraya I, Kolb H, Chen J, Baehr W. Invest Ophthalmol Visual Sci 1998;39:867 ± 75.

[56] Otto-Bruc A, Fariss RN, Haeseleer F, Huang J, Buczylko J, Surgucheva I, Baehr W, Milam AH, Palczewski K. Proc Natl Acad USA 1997;94:4727 ± 32. [57] Kachi S, Nishizawa Y, Olshevskaya E, Yamazaki A, Miyake Y, Wakabayashi T, Dizhoor A, Usukura J. Exp Eye Res 1999; 68:465 ± 73. [58] Payne AM, Downes SM, Bessant DA, Taylor R, Holder GE, Warren MJ, Bird AC, Bhattacharya SS. Hum Mol Genet 1998; 7(2):273 ± 7. [59] Haeseleer F, Sokal I, Li N, Pettenati M, Rao N, Bronson D, Wechter R, Baehr W, Palczewski K. J Biol Chem 1999;274:6526 ± 35. [60] Surguchov A, Bronson JD, Banerjee P, Knowles JA, Ruiz C, Subbaraya I, Palczewski K, Baehr W. Genomics 1997;39:312 ± 22. [61] Mendez A, Burns ME, Sokal I, Baeh J, Palczewski K, Baylor D. Invest Ophthalmol Visual Sci 1999;40:2056. [62] Pozdnyakov N, Goraczniak R, Margulis A, Duda T, Sharma RK, Yoshida A, Sitaramayya A. Biochemistry 1997;36:14159 ± 66. [63] Kumar VD, Vijay-Kumar S, Krishnan A, Duda T, Sharma RK. Biochemistry 1999;38:12614 ± 20. [64] Olshevskaya EV, Boikov S, Ermilov A, Krylov D, Hurley JB, Dizhoor AM. J Biol Chem 1999;274:10823 ± 32. [65] Nef P. Guidebook to the calcum-binding proteins. In: Celio MR, Pauls T, Schwaller B, editors. New York: Oxford Univ. Press, 1996. pp. 94 ± 120. [66] Strynadka NC, James MN. Annu Rev Biochem 1989;58:951 ± 98. [67] Rudnicka-Nawrot M, Surgucheva I, Hulmes JD, Haeseleer F, Sokal I, Crabb JW, Baehr W, Palczewski K. Biochemistry 1998; 37:248 ± 57. [68] Ames JB, Dizhoor AM, Ikura M, Palczewski K, Stryer L. J Biol Chem 1999;274:19329 ± 37. [69] Hughes RE, Brzovic PS, Dizhoor AM, Klevit RE, Hurley JB. Protein Sci 1998;7:2675 ± 80. [70] Sokal I, Otto-Bruc AE, Surgucheva I, Verlinde CL, Wang CK, Baehr W, Palczewski K. J Biol Chem 1999;274:19829 ± 37. [71] Dizhoor AM, Ericsson LH, Johnson RS, Kumar S, Olshevskaya E, Zozulya S, Neubert TA, Stryer L, Hurley JB, Walsh KA. J Biol Chem 1992;267:16033 ± 6. [72] Johnson RS, Ohguro H, Palczewski K, Hurley JB, Walsh KA, Neubert TA. J Biol Chem 1994;269:21067 ± 71. [73] Dizhoor AM, Boikov SG, Olshevskaya EV. J Biol Chem 1998; 273:17311 ± 4. [74] Otto-Bruc A, Buczylko J, Surgucheva I, Subbaraya I, Rudnicka-Nawrot M, Crabb JW, Arendt A, Hargrave PA, Baehr W, Palczewski K. Biochemistry 1997;3:4295 ± 302. [75] Ames JB, Tanaka T, Stryer L, Ikura M. Curr Opin Struct Biol 1996; 6:432 ± 8. [76] Krylov DM, Niemi GA, Dizhoor AM, Hurley JB. J Biol Chem 1999;274:10833 ± 9. [77] Laura RP, Hurley JB. Biochemistry 1998;37:11264 ± 71. [78] Olshevskaya EV, Ermilov AN, Dizhoor AM. J Biol Chem 1999; 274:25583 ± 7. [79] Yu H, Olshevskaya E, Duda T, Seno K, Hayashi F, Sharma RK, Dizhoor AM, Yamazaki A. J Biol Chem 1999;274:15547 ± 55. [80] Sokal I, Haeseleer F, Arendt A, Adman ET, Hargrave PA, Palczewski K. Biochemistry 1999;38:1387 ± 93. [81] Lange C, Duda T, Beyermann M, Sharma RK, Koch KW. FEBS Lett 1999;460:27 ± 31. [82] Schrem A, Lange C, Beyermann M, Koch KW. J Biol Chem 1999; 274:6244 ± 9. [83] Perrault I, Rozet JM, Calvas P, Gerber S, Camuzat A, Dollfus H, Chatelin S, Souied E, Ghazi I, Leowski C, Bonnemaison M, Le Paslier D, Frezal J, Dufier JL, Pittler S, Munnich A, Kaplan J. Nat Genet 1996;14:461 ± 4. [84] Duda T, Venkataraman V, Goraczniak R, Lange C, Koch KW, Sharma RK. Biochemistry 1999;38:509 ± 15. [85] Kelsell RE, Gregory-Evans K, Payne AM, Perrault I, Kaplan J, Yang RB, Garbers DL, Bird AC, Moore AT, Hunt DM. Hum Mol Genet 1998;7:1179 ± 84.

A.M. Dizhoor / Cellular Signalling 12 (2000) 711±719 [86] Perrault I, Rozet JM, Gerber S, Kelsell RE, Souied E, Cabot A, Hunt DM, Munnich A, Kaplan J. Am J Hum Genet 1998;63: 651 ± 4. [87] Duda T, Krishnan A, Venkataraman V, Lange C, Koch KW, Sharma RK. Biochemistry 1999;38:13912 ± 9. [88] Sokal I, Li N, Surgucheva I, Warren MJ, Payne AM, Bhattacharya SS, Baehr W, Palczewski K. Mol Cell 1998;2:129 ± 33.

719

[89] He L, Poblenz AT, Medrano CJ, Fox DA. J Biol Chem 2000; 275:12175 ± 84. [90] Nicotera P, Orrenius S. Cell Calcium 1998;23:173 ± 80. [91] Pugh EN, Lamb TD. Vision Res 1990;30:1923 ± 48. [92] He W, Cowan CW, Wensel TG. Neuron 1998;20:95 ± 102. [93] Makino ER, Handy JW, Li T, Arshavsky VY. Proc Natl Acad Sci USA 1999;96:1947 ± 52.