FEBS Letters 587 (2013) 2055–2059

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Review The guanylate signaling system in zebrafish photoreceptors ⇑ Karl-Wilhelm Koch

Biochemistry Group, Faculty VI, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany Research Center Neurosensory Science, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany Center of Interface Science, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany article info abstract

Article history: Zebrafish express in the retina a large variety of three different membrane-bound guanylate cyclas- Received 24 April 2013 es and six different -activating (zGCAPs) belonging to the family of neu- Revised 25 April 2013 ronal calcium sensor proteins. Although these proteins are predominantly localized in rod and Accepted 26 April 2013 cone photoreceptor cells of the retina, they differ in their spatial–temporal expression profiles. Fur- Available online 7 May 2013 ther, each zGCAP has a different affinity for Ca2+ and displays different Ca2+-sensitivities of guanylate 2+ Edited by Alexander Gabibov, Vladimir cyclase activation. Thus, zGCAPs operate as cytoplasmic Ca -sensors that sense incremental changes 2+ Skulachev, Felix Wieland and Wilhelm Just of cytoplasmic Ca -concentration in rod and cone cells and control the activity of their target guan- ylate in a Ca2+-relay mode fashion. Keywords: Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Photoreceptor Guanylate cyclase Calcium signaling Guanylate cyclase-activating Zebrafish

1. Introduction the dark state of the cell and increase their activities, when the cytoplasmic [Ca2+] decreases after illumination. Although this gen- 1.1. General eral concept is well accepted and confirmed [1–8], several open questions remain. For example, what are the molecular mecha- Excitation and adaptation of vertebrate photoreceptor cells is nisms of protein–protein interaction during GC activation by mediated by two second messengers, guanosine-30,50-cyclic mono- GCAPs? Further, to what extents contribute other Ca2+-dependent phosphate (cGMP) and Ca2+ [1–4]. Light absorption of rhodopsin or Ca2+-independent mechanisms to the regulation of phototrans- triggers the hydrolysis of cGMP by activation of a well-known G- duction and the control of light adaptation [9]? Finally, zebrafish protein coupled pathway leading also with a short delay to a fall has attained increasing interest in vision research due to its fast in cytoplasmic Ca2+-concentration ([Ca2+]). The mutual depen- development, amenability to genetic manipulation and to the fact dence of cytoplasmic concentrations in cGMP and Ca2+ critically that it is equipped with a set of cone photoreceptor cells, which are controls the responsiveness of the under differ- sensitive to the whole visible spectrum and to UV-light. The zebra- ent illumination conditions and a change in cytoplasmic [Ca2+]is fish retina expresses three isoforms of sensory GCs and six forms of considered to be a critical step mediating light adaptation [1–4]. GCAPs raising the question, whether this apparently redundant Key proteins involved in the interplay of cGMP and Ca2+ are mem- expression is of physiological meaning [10,11]. The present mini- brane-bound guanylate cyclases (GCs) [4,5] and their Ca2+-sensi- review will discuss this topic, in particular by reflecting on the re- tive regulators, named guanylate cyclase-activating proteins cently suggested Ca2+-relay model that accounts for differential (GCAP) [6–8]. A combination of biochemical, genetic and physio- properties of GCAP forms [4,12,13]. logical studies has established that photoreceptor GCs have a low basal cyclase activity at high intracellular [Ca2+] corresponding to 1.2. Sensory GCs in teleost fishes

Teleost DNA data bases harbour sequence information of sev- Abbreviations: GC, guanylate cyclase; GCAP, guanylate cyclase-activating pro- eral putative membrane bound GCs. These include four different tein; z, in front of GC or GCAP denotes proteins expressed in zebrafish; ROS, rod sensory GCs denoted as OlGC3, OlGC4, OlGC5 and OlGC-R2 in me- outer segment; NCS, neuronal calcium sensor ⇑ Address: Biochemistry Group, Faculty VI, Carl von Ossietzky University Olden- daka fish (Oryzias latipes) and four orthologous forms in pufferfish burg, D-26111 Oldenburg, Germany. Fax: +49 441 193640. (Fugu rubripes) and three in carp (Cyprinus carpio) [10,14–19]. E-mail address: [email protected] Transcripts of the three sensory GCs in the zebrafish (Danio rerio)

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.04.023 2056 K.-W. Koch / FEBS Letters 587 (2013) 2055–2059 were detected in the photoreceptor layer of larval and adult fish conformational changes, GCAP-target interaction and activation denoted zGC1, zGC2, zGC3 [11,20]. profiles of GCs. These biochemical and biophysical approaches to Amino acid sequence alignments and a subsequent dot blot ma- understand Ca2+-sensor function have recently been reviewed trix analysis [21] between medaka and zebrafish GCs revealed high and will not be repeated here [33]. Furthermore, experiments on in the cytoplasmic part and significant regions transgenic mice with manipulated GCAP expression demonstrated of conserved amino acids in the extracellular part confirming the that mammalian GCAP1 and GCAP2 critically shape the photore- conclusion that these GCs belong to the group of sensory GCs ceptor light response [34–37]. Both, physiological recordings and [22]. Sensory GCs operating in rod and cone cells of the vertebrate in vitro data are consistent with a step-by-step signaling mode of retina have been investigated in the past by numerous studies, GCAPs, which allows the cell to respond to increment changes in which show collectively that these membrane bound GCs exist as cytoplasmic Ca2+. This signaling mode of a Ca2+-relay model has dimers in photoreceptor membranes, form complexes with GCAPs also recently been discussed and reviewed in detail [13]. A more and are regulated by GCAPs in a Ca2+-dependent manner (for re- complex operation of the GC/GCAP-complex seems to be realized view see [3,4]. So far, knowledge about the biochemical properties in the zebrafish retina with its abundant expression of six zGCAP of zGCs is very limited and the cDNAs have not been heterolo- isoforms. Thus, in the following I will focus this mini-review on gously expressed for functional studies. In situ hybridization GCAP function in zebrafish retina. experiments, however, showed that zGC1 and zGC2 were detected in rods and cones and no signals were seen in rods with zGC3 RNA 2. GCAP regulatory modes in zebrafish rods and cones probes. Instead, cones were labeled by zGC3 probes [11]. Screening of randomly mutagenized zebrafishes resulted in sev- 2.1. Ca2+-binding and Ca2+-induced conformational changes eral behavioral mutants with mutations in photoreceptor specific including one mutant named zatoichi, which has a defect All zGCAPs have successfully been purified as recombinant pro- in the coding for zGC3 [23]. The zatoichi mutant larva shows teins from Escherichia coli allowing detailed investigations of their no optokinetic response (OKR) and no optomotor response (OMR) biochemical properties [38–40]. A non-radioactive chelator assay measured at 6 days post fertilization (dpf), which points to the cru- developed by Linse and co-workers [41–43] was used to investi- cial role of GC expression for larval visual function. Further, in an- gate Ca2+-binding affinities. All zGCAPs had at least three binding other study the gene gucy2f coding for the GC zGC1 (s. above) was sites for Ca2+ with apparent affinity constants of high, medium overexpressed in zebrafish larvae in a gain-of-function approach and low affinity. However, these values differed among all investi- yielding larvae with multiple defects including a loss of forebrain gated zGCAPs and are unique for every isoform [38]. neurons [24]. Knocking down the expression of gucy2f by the mor- Binding of Ca2+ triggers conformational changes in GCAPs and pholino antisense-oligonucleotide approach led to impaired visual had been investigated thoroughly for the mammalian GCAP1 and function and shortening of photoreceptor cells in 6 day old larvae GCAP2. Common spectroscopic methods include in particular [25]. While these studies highlight the importance of genes coding intrinsic Tryptophan (Trp) fluorescence and circular dichroism for membrane bound GCs for normal visual performance they (CD), which had been applied in studying the Ca2+-induced confor- leave open questions that are related to regulatory features. mational changes of GCAPs and another retina specific NCS protein, recoverin [31,41–47]. Mammalian GCAP1 typically shows a bipha- 1.3. Expression of GCAPs and functional role in signaling sic pattern in Trp emission during a Ca2+-titration with decreasing emission from 0 to 1 lMCa2+ and increasing emission above 1 lM GCAPs belong to the family of neuronal calcium sensor (NCS) Ca2+. The Trp emission studies therefore demonstrated that GCAPs proteins that are mainly expressed in neurons, where they mediate undergo Ca2+-induced conformational changes in the physiological as Ca2+-sensors a wide range of physiological responses [26]. range of cytoplasmic [Ca2+]. Interestingly, although all zGCAPs GCAPs are specifically expressed in photoreceptor cells and have change their Trp emission on Ca2+-binding or Ca2+-dissociation, been identified in different vertebrates like human, bovine, mon- no zGCAP isoform followed the same pattern as GCAP1, which key, mice, chicken, fish and amphibians [6–8,10–12,27–29]. They demonstrates significant differences in Ca2+-sensing [38]. are small compact proteins of approximately 200 amino acid length containing four EF-hand Ca2+-binding motifs, of which the 2.2. Activation profiles of GC signaling first EF-hand is non-functional and probably involved in protein– protein interaction [30,31]. GCAPs can bind Mg2+, when Ca2+ disso- One remarkable feature of zGCAPs is that each form has a dis- ciates from its binding sites during light-induced changes of the tinct activation profile. Reconstitution of purified zGCAPs with cytoplasmic Ca2+-concentration, which transforms GCAPs into membrane bound GCs using preparations of bovine rod outer seg- their cyclase-activating conformation [32]. Mammalian rod and ment (ROS) membranes as assay system showed that all zGCAPs cone cells express two or three GCAP isoforms, but eight isoforms could activate GCs with apparent affinities (EC50-values) in the were predicted in pufferfish (Fugu rubripes) [10] and six isoforms submicromolar or lower micromolar range [38], which is very sim- were cloned from zebrafish (Danio rerio) [10,11] and carp (Cyprinus ilar to mammalian GCAPs that also have EC50 values around 1 lM. carpio) retinal cDNA libraries [19]. Prominent transcription and/ or Most significant differences are however observed, when maximal expression are observed for all GCAPs in the outer vertebrate ret- GC activities are compared and calculated as x-fold activation from ina, in particular in the outer and inner segments of photoreceptor the definition: GCmax GCmin/GCmin (GCmax: GC activity at maxi- cells. In addition, GCAP specific staining was also observed in cone mum; GCmin: GC activity at minimum). Maximal activities of somata, cell bodies, axons, axon terminals and synaptic pedicles zGCAP1, 5 and 7 are rather low (x-fold activation 1.5–3-fold), but [7,8,28,29], but their functional role in these cell compartments zGCAP2, 3 and 4 are strong activators having an x-fold activation is not understood, in contrast to their well-known function as between 6 and 13 [38]. When these features are compared with Ca2+-sensitive regulators of GC activity. GCAPs interact with their the Ca2+-sensitive regulatory properties it becomes apparent that main targets, membrane bound sensory GCs, and thereby activate zGCAPs can be classified into two groups having IC50 values either 2+ 2+ GCs, when they are Ca -free and inhibit GCs, when they are Ca - around 30 nM or around 400 nM [38–40]. The IC50 value is equiv- loaded. For a quantitative assessment of GCAP function in a phys- alent to the Ca2+-concentration at which GC activation is half-max- iological context it is necessary to examine crucial biochemical imal. Each of these groups contains further a combination of strong properties as Ca2+-binding and Ca2+-affinity, Ca2+-induced and weak GC activators (Fig. 1). If we look at the distribution of K.-W. Koch / FEBS Letters 587 (2013) 2055–2059 2057

Fig. 1. Ca2+-dependent activation profile of the photoreceptor guanylate cyclase. Different GCAP forms are active at different Ca2+-concentrations. The GC activities are normalized to 100% and are based on data of Ref. [39]. Curves are drawn to highlight the observation that zGCAP1–3 and zGCAP4, 5 and 7 each form a cluster of similar Ca2+-sensitivities. Isoforms of zGCAPs written in bold letters exhibit a high level of x-fold activation, those written in normal black show a low level of x-fold Fig. 2. Summary of zGCAP expression level in zebrafish rod and cone cells. activation. The black-to-gray bar in the background symbolizes the range of Drawings of rods (gray), double cones (long-wavelength sensitive, red and green), 2+ cytoplasmic Ca change during a light response. long single cones (short-wavelength sensitive, blue) and short single cones (UV- sensitive, violet) are shown in the upper part. Only zGCAP1 and 2 are expressed in rods. Expression in cones is different for each zGCAP and is indicated by conic zGCAPs in different photoreceptor cells, each cell type harbors shaped bars symbolizing from top to bottom high and low expression rates. Ca2+- strong and weak GC activators and all cone types express also a dependent regulation of GC by isoforms of zGCAPs is characterized by the IC50 value, which is around 30 nM free [Ca2+] for zGCAP1–3 (yellow background) and combination of zGCAPs having different IC50 values (Fig. 2). Double around 400 nM [Ca2+] for zGCAP4, 5 and 7 (blue background). cones and long single cones have two strong activators that both are also expressed at high rates (Fig. 2). Interestingly, short single cones have a high expression rate only for the zGCAP group with 2+ low IC50 values. The only zGCAP with a high IC50 value is zGCAP5, to shift the Ca -sensitivity into the physiological range. In addi- but this Ca2+-sensor shows the lowest degree of x-fold activation tion, the affinity of the GCAP1–ROS–GC1 interaction depends on among all tested GCAPs [38] and probably does not contribute the myristoyl modification [54–56]. In contrast, bovine GCAP2 much to the overall regulation and activation of GCs. It is unclear shows an almost identical activation pattern in its myristoylated at the moment, whether this special case of UV-cones reflects a dif- or non-myristoylated form, however, the myristoyl group in ferent Ca2+-homeostasis. Direct measurements of cytoplasmic GCAP2 seems to be more flexible and is involved in stabilizing Ca2+-concentration in the dark have been performed so far only the Ca2+-bound conformation [57,58]. These findings raised the fol- for UV-cones and yielded a value of 400 nM, which is suggested lowing questions, (a) whether zGCAPs are expressed in myristoy- to decrease after illumination [48]. It will be a major challenge to lated or non-myristoylated forms; (b) and if so, whether these further define exactly the changing pattern of cytoplasmic Ca2+ in forms differ in their key properties. Heterologous expression and zebrafish rod and cone cells. In any case, zGCAPs are operating as purification from bacteria revealed that zGCAP3 and zGCAP4 could activating GC regulators in a specific time frame depending on be myristoylated by a co-expressed N-myristoyl- from the illumination state of the cell. Thus, zGCAPs exhibiting a lower yeast, although a point mutant of zGCAP4 was necessary [39,40]. Ca2+-affinity or -sensitivity (zGCAP4, 5 and 7) would first be turned Probing larval and adult stages of zebrafish showed also that into an activating status followed by zGCAP 1, 2 or 3 that will be- zGCAP3 is myristoylated in native retinae [40], but a similar study come active at Ca2+-concentrations that are approx. one order of has not been conducted for zGCAP4 so far. In order to identify a magnitude lower. This gradual step-by-step mode of action repre- possible influence of the myristoyl group on the functional proper- sents a Ca2+-relay mechanism of GCAP action. ties of zGCAPs, zGCAP3 and 4 were purified as myristoylated and non-myristoylated recombinant proteins and investigated accord- 2.3. Comparison of zGCAP3 and zGCAP4 ing to several criteria yielding the following results: they do not undergo a classical Ca2+-myristoyl-switch, although membrane Two cone-specific Ca2+-sensors, zGCAP3 and 4, have been inves- binding of zGCAP3 is slightly improved by the myristoyl group tigated in more detail [39,40]. Each of them represents a strong [39,40]. Activation profiles of zGCAPs are nearly identical with sim- activator of GCs with high expression rate in cone cells, but with ilar IC50 and EC50 values for GC activation indicating no significant different operation profiles and Ca2+-sensitivities (Figs. 1 and 2). influence on Ca2+-sensor function. However, in zGCAP3 the myri- Most NCS proteins contain a consensus sequence for the attach- stoyl group has several other effects. It enhances the Ca2+sensitiv- ment of a myristoyl group at the amino-terminus [26] and in par- ity of conformational changes, stabilizes the protein conformation ticular recoverin and GCAPs from mammalian (ROS) exhibit in general and has a modulating effect on kinetic parameters of the differences in their functional properties, whether they are myri- GC target [40]. Interestingly, zebrafish larvae first express non- stoylated or not. For example, recoverin undergoes a classical myristoylated zGCAP3 at 3.25 dpf, but myristoylated zGCAP3 was Ca2+-myristoyl switch, it binds to membranes, when it is saturated not detected before 7 dpf [40]. Apparently, the impact of the myr- with Ca2+ and the myristoyl group is exposed. In its Ca2+-free form istoyl group on zGCAP3 functions seems less important in the lar- the myristoyl group is buried in a hydrophobic protein pocket val than in the adult stage. making the protein less hydrophobic [49], which in turn leads to In summary, GCAPs are NCS proteins that operate like their the release of recoverin into the cytoplasm. In contrast to recover- mammalian counterparts in a relay mode allowing rod and cone in, GCAP1 and 2 do not undergo a classical Ca2+-myristoyl switch cells to detect incremental changes in cytoplasmic Ca2+. The bio- [50–53], but presence of the myristoyl group in GCAP1 is essential chemical complexity of zGCAPs reflects a fine-tuned system of 2058 K.-W. Koch / FEBS Letters 587 (2013) 2055–2059

Ca2+-sensing and Ca2+-mediating proteins playing key roles in pho- [20] Brockerhoff, S.E., Rieke, F., Matthews, H.R., Taylor, M.R., Kennedy, B., toreceptor excitation and adaptation. However, many unresolved Ankoudinova, I., Niemi, G.A., Tucker, C.L., Xiao, M., Cilluffo, M.C., Fain, G.L. and Hurley, J.B. (2003) Light stimulates a transducin-independent increase of questions remain as for example to examine, whether the action cytoplasmic Ca2+ and suppression of current in cones from the zebrafish mode of each zGCAP changes under varying illumination condi- mutant nof. J. Neurosci. 23, 470–480. tions. Furthermore, which other Ca2+-dependent control mecha- [21] Schwartz, R.M., Dayhoff, M.O. (1978) Atlas of protein sequence and structure. Nat. Biomed. Res. Found. (Washington, DC, USA) 5(Suppl. 3), 353–358. nisms targeting different or proteins are involved in [22] Rätscho, N., Scholten, A. and Koch, K.-W. (2010) Diversity of guanylate adjusting the photoreceptor’s light adapting properties and form- cyclases in teleost fishes. Mol. Cell. Biochem. 334, 207–214. ing the molecular basis for the dynamic range extension in cone [23] Muto, A., Orger, M.B., Wehmann, A.M., Smear, M.C., Kay, J.N., Page-McCaw, P.S., Gathan, E., Xiao, T., Nevin, L.M., Gosse, N.J., Staub, W., Finger-Baier, K. and adaptation of the fish retina. Baier, H. (2005) Forward genetic analysis of visual behavior in zebrafish. PLoS Genet. 1 (5), e66. Acknowledgement [24] Maddison, L., Lu, J., Victoroff, T., Scott, E., Baier, H. and Chen, W. (2009) A gain- of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration. Mol. Genet. Genomics 281, 551–563. Experimental work done in the laboratory of the author is [25] Stiebel-Kalish, H., Reich, E., Rainy, N., Vaatine, G., Nisgav, Y., Tovar, A., Gothilf, funded by several Grants of the Deutsche Forschungsgemeinschaft Y. and Bach, M. (2012) Gucy2f zebrafish knockdown – a model for Gucy2d- (DFG). related leber congenital amaurosis. Eur. J. Hum. Genet. 20, 884–889. [26] Burgoyne, R.D. (2007) Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signaling. Nat. Rev. Neurosci. 8, 182–193. References [27] Subbaraya, I., Ruiz, C.C., Helekar, B.S., Zhao, X., Gorczyca, W.A., Pettenati, M.J., Rao, P.N., Palczewski, K. and Baehr, W. (1994) Molecular characterization of human [1] Pugh Jr., E.N. and Lamb, T.D. (2000) Phototransduction in vertebrate rods and and mouse photoreceptor guanylate cyclase-activating protein (GCAP) and cones: molecular mechanisms of amplification, recovery and light adaptation chromosomal localization of the human gene. J. Biol. Chem. 269, 31080–31089. in: Handbook of Biological Physics (Stavenga, D.G., DeGrip, W.J. and Pugh, E.N. [28] Howes, K., Bronson, J.D., Dang, Y.L., Li, N., Zhang, K., Ruiz, C., Helekar, B., Lee, Jr.Jr., Eds.), pp. 183–255, Elsevier Science BV 3. M., Subbaraya, I., Kolb, H., Chen, J. and Baehr, W. (1998) Gene array and [2] Luo, D.G., Xue, T. and Yau, K.-W. (2008) How vision begins: an odyssey. Proc. expression of mouse retina guanylate cyclase activating proteins 1 and 2. Natl. Acad. Sci. USA 105, 9855–9862. Invest. Ophthalmol. Vis. Sci. 39, 867–875. [3] Stephen, R., Filipek, S., Palczewski, K. and and, Sousa, M.C., and (2008) Ca2+- [29] Cuenca, N., Lopez, S., Howes, K. and Kolb, H. (1998) The localization of dependent regulation of phototransduction. Photochem. Photobiol. 84, 903– guanylyl cyclase-activating proteins in the mammalian retina. Invest. 910. Ophthalmol. Vis. Sci. 39, 1243–1250. [4] Koch, K.-W., Duda, T. and and, Sharma, R.K., and (2010) Ca2+-modulated [30] Ermilov, A.N., Olshevskaya, E.V. and Dizhoor, A.M. (2001) Instead of binding vision-linked ROS-GC guanylate cyclase transduction machinery. Mol. Cell. calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 Biochem. 334, 105–115. is required for targeting photoreceptor guanylyl cyclase. J. Biol. Chem. 276, [5] Baehr, W., Karan, S., Maeda, T., Luo, D.G., Li, S., Bronson, J.D., Watt, C.B., Yau, 48143–48148. K.W., Frederick, J.M. and Palczewski, K. (2007) The function of guanylate [31] Hwang, J.Y., Schlesinger, R. and Koch, K.-W. (2004) Irregular dimerization of cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors. J. Biol. guanylate cyclase-activating protein 1 mutants causes loss of target Chem. 282, 8837–8847. activation. Eur. J. Biochem. 271, 3785–3793. 2+ 2+ [6] Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, [32] Dizhoor, A.M., Olshevskaya, E.V. and Peshenko, I.V. (2010) Mg /Ca cation H., Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., binding cycle of guanylyl cyclase activating proteins (GCAPs): role in Detwiler, P.B. and Baehr, W. (1994) Molecular cloning and characterization of regulation of photoreceptor guanylyl cyclase. Mol. Cell. Biochem. 334, 117– retinal photoreceptor guanylyl cyclase-activating protein. Neuron 13, 395– 124. 404. [33] Koch, K.-W. (2012) Biophysical investigation of retinal calcium sensor [7] Dizhoor, A.M., Olshevskaya, E.V., Henzel, W.J., Wong, S., Stults, J.T., function. Biochim. Biophys. Acta 1820, 1228–1233. Ankoudinova, I. and Hurley, J.B. (1995) Cloning, sequencing, and expression [34] Mendez, A., Burns, M.E., Sokal, I., Dizhoor, A.M., Baehr, W., Palczewski, K., of a 24-kDa Ca2+-binding protein activating photoreceptor guanylyl cyclase. J. Baylor, D. and Chen, J. (2001) Role of guanylate cyclase-activating proteins Biol. Chem. 270, 25200–25206. (GCAPs) in setting the flash sensitivity of rod photoreceptors. Proc. Natl. Acad. [8] Frins, S., Bönigk, W., Müller, F., Kellner, R. and Koch, K.-W. (1996) Functional Sci. USA 98, 9948–9953. characterization of a guanylyl cyclase-activating protein from vertebrate rods. [35] Howes, K.A., Pennesi, M.E., Sokal, I., Church-Kopish, J., Schmidt, B., Margolis, D., J. Biol. Chem. 271, 8022–8027. Frederick, J.M., Rieke, F., Palczewski, K., Wu, S.M., Detwiler, P.B. and Baehr, W. [9] Fain, G.L. (2011) Adaptation of mammalian photoreceptors to background (2002) GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout light: putative role for direct modulation of phosphodiesterase. Mol. mice. EMBO J. 21, 1545–1554. Neurobiol. 44, 374–382. [36] Makino, C.L., Peshenko, I.V., Wen, X.H., Olshevskaya, E.V., Barrett, R. and [10] Imanishi, Y., Yang, L., Sokal, I., Filipek, S., Palczewski, K. and Baehr, W. (2004) Dizhoor, A.M. (2008) A role for GCAP2 in regulating the photoresponse. J. Biol. Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: Chem. 283, 29135–29143. characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish [37] Makino, C.L., Wen, X.H., Olshevskaya, E.V., Peshenko, I.V., Savchenko, A.B. and (Danio rerio) and prediction of eight GCAPs (GCAP1–8) in pufferfish (fugu Dizhoor, A.M. (2012) Enzymatic relay mechanism stimulates cyclic GMP rubripes). J. Mol. Evol. 59, 204–217. synthesis in rod photoresponse: biochemical and physiological study in [11] Rätscho, N., Scholten, A. and Koch, K.-W. (2009) Expression profiles of three guanyly cyclase activating protein 1 knockout mice. PLoS ONE 7, e47637. novel sensory guanylate cyclases and guanylate cyclase-activating proteins in [38] Scholten, A. and Koch, K.-W. (2011) Differential calcium signaling by cone the zebrafish retina. Biochim. Biopyhs. Acta 1793, 1110–1114. specific guanylate cyclase-activating proteins from the zebrafish retina. PLoS [12] Koch, K.-W. (2006) GCAPs, the classical neuronal calcium sensors in the retina ONE 6, e23117. –aCa2+-relay model of guanylate cyclase activation. Calcium Binding Proteins [39] Behnen, P., Scholten, A., Rätscho, N. and Koch, K.-W. (2009) The cone-specific 1, 3–6. calcium sensor guanylate cyclase activating protein 4 from the zebrafish [13] Koch, K.-W., Dell’Orco, D. (in press) A calcium – relay mechanism in vertebrate retina. J. Biol. Inorg. Chem. 14, 89–99. phototransduction. ACS Chem. Neurosci. http://dx.doi.org/10.1021/ [40] Fries, R., Scholten, A., Säftel, W. and Koch, K.-W. (2012) Operation profile of cn400027z. zebrafish guanylate cyclase-activating protein 3. J. Neurochem. 121, 54–65. 2+ [14] Seimiya, M., Kusakabe, T. and Suzuki, N. (1997) Primary structure and [41] Andre, I. and Linse, S. (2002) Measurement of Ca -binding constants of differential gene expression of three membrane forms of guanylyl cyclase proteins and presentation of the CaLigator software. Anal. Biochem. 305, 195– found in the eye of the teleost Oryzias latipes. J. Biol. Chem. 272, 23407–23417. 205. [15] Hisatome, O., Honkawa, H., Imanishsi, Y., Satoh, T. and Tokunaga, F. (1999) [42] Linse, S. (2009) Calcium binding to proteins studied via competition with Three kinds of guanylate cyclase expressed in medaka photoreceptor cells in chromophoric chelators in: Calcium-Binding Protein Protocols: Volume 2: both retina and pineal organ. Biochem. Biophys. Res. Commun. 255, 216–220. Methods and Techniques (Vogel, H.J., Ed.), pp. 15–24, Humana Press. [16] Kusakabe, T. and Suzuke, N. (2000) Photoreceptors and olfactory cells express [43] Linse, S., Johansson, C., Brodin, P., Grundstrom, T., Drakenberg, T. and Forsen, 2+ the same retinal guanyly cyclase isoform in medaka: visualization by promote T.S. (1991) Electrostatic contribution to the binding of Ca in calbindin D9k. transgenics. FEBS Lett. 483, 143–148. Biochemistry 30, 154–162. [17] Harumi, T., Watanabe, T., Yamamoto, T., Tanabe, Y. and Suzuki, N. (2003) [44] Sokal, I., Otto-Bruc, A.E., Surgucheva, I., Verlinde, C.L., Wang, C.K., Baehr, W. Expression of membrane-bound and soluble guanylyl-cyclase mRNAs in and Palczewski, K. (1999) Conformational changes in guanylyl cyclase- embryonic and adult retina of the Medaka fish Oryzias latipes. Zool. Sci. 20, activating protein 1 (GCAP1) and its tryptophan mutants as a function of 133–140. calcium concentration. J. Biol. Chem. 274, 19829–19837. 2+ [18] Yamagami, S. and Suzuki, N. (2005) Diverse forms of guanylyl cyclases in [45] Hughes, R.E., Brzovic, P.S., Dizhoor, A.M., Klevit, R.E. and Hurley, J.B. (1998) Ca - Medaka fish – their genomic structure and phylogenetic relationships to those dependent conformational changes in bovine GCAP-2. Protein Sci. 7, 2675–2680. in vertebrates and invertebrates. Zool. Sci. 22, 819–835. [46] Gensch, T., Komolov, K.E., Senin, I.I., Philippov, P.P. and Koch, K.-W. (2007) 2+ 2+ [19] Takemoto, N., Tachibanaki, S. and Kawamura, S. (2009) High cGMP synthetic Ca -dependent conformational changes in the neuronal Ca -sensor activity in carp cones. Proc. Natl. Acad. Sci. USA 106, 11788–11793. recoverin probed by the fluorescent dye Alexa647. Proteins 65, 492–499. K.-W. Koch / FEBS Letters 587 (2013) 2055–2059 2059

[47] Dell’Orco, D., Behnen, P., Linse, S. and Koch, K.-W. (2010) Calcium binding, protein 1 induced by Ca2+ and N-terminal fatty acid acylation. Structure 18, structural stability and guanylate cyclase activation in GCAP1 variants 116–126. associated with human cone dystrophy. Cell. Mol. Life Sci. 67, 973–984. [54] Hwang, J.Y., Lange, C., Helten, A., Höppner-Heitmann, D., Duda, T., Sharma, R.K. [48] Cilluffo, M.C., Matthews, H.R., Brockerhoff, S.E. and Fain, G.L. (2004) Light- and Koch, K.-W. (2003) Regulatory modes of rod outer segment membrane induced Ca2+-release in the visible cones of the zebrafish. Vis. Neurosci. 21, guanylate cyclase differ in catalytic efficiency and Ca2+-sensitivity. Eur. J. 599–609. Biochem. 270, 3814–3821. [49] Ames, J.B., Ishima, R., Tanaka, T., Gordon, J.I., Stryer, L. and Ikura, M. (1997) [55] Peshenko, I.V., Olshevskaya, E.V. and Dizhoor, A.M. () Interaction of GCAP1 Molecular mechanics of calcium-myristoyl switches. Nature 389, 198–202. with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation. Front. [50] Olshevskaya, E.V., Hughes, R.E., Hurley, J.B. and Dizhoor, A.M. (1997) Calcium Mol. Neurosci. 5, 19, http://dx.doi.org/10.3389/fnmol.2012.00019. binding, but not a calcium-myristoyl switch, controls the ability of guanylyl [56] Peshenko, I.V., Olshevskaya, E.V., Lim, S., Ames, J.B. and Dizhoor, A.M. (2012) cyclase-activating protein GCAP-2 to regulate photoreceptor guanylyl cyclase. Calcium-myristoyl tug is a new mechanisms for intramolecular tuning of J. Biol. Chem. 272, 1432714333. calcium sensitivity and target interaction for guanylyl cyclase- [51] Hwang, J.Y. and Koch, K.-W. (2002) Calcium- and myristoyl-dependent activating protein 1. J. Biol. Chem. 287, 13972–13984. properties of guanylate cyclase-activating protein-1 and protein-2. [57] Schröder, T., Lilie, H. and Lange, C. (2011) The myristoylation of guanylate Biochemistry 41, 13021–13028. cyclase-activating protein-2 causes an increase in thermodynamic stability in [52] Stephen, R., Bereta, G., Golczak, M., Palczewski, K. and Sousa, M.C. (2007) the presence but not in the absence of Ca2+. Protein Sci. 20, 1155–1165. Stabilizing function for myristoyl group revealed by the crystal structure of a [58] Kollmann, H., Becker, S.F., Shirdel, J., Scholten, A., Ostendorp, A., Lienau, C. and neuronal calcium sensor, guanylate cyclase-activating protein 1. Structure 15, Koch, K.-W. (2012) Probing the Ca2+ switch of the neuronal Ca2+ sensor GCAP2 1392–1402. by time-resolved fluorescence spectroscopy. ACS Chem. Biol. 7, 1006–1014. [53] Orban, T., Bereta, G., Miyagi, M., Wang, B., Chance, M.R., Sousa, M.C. and Palczewski, K. (2010) Conformation changes in guanylate cyclase-activating