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The guanylate cyclase signaling system in zebrafish photoreceptors
FEBS Letters 587 (2013) 2055–2059
journal homepage: www.FEBSLetters.org
Review
The guanylate cyclase 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
a r t i c l e
i n f o
Article history: Received 24 April 2013 Revised 25 April 2013 Accepted 26 April 2013 Available online 7 May 2013 Edited by Alexander Gabibov, Vladimir Skulachev, Felix Wieland and Wilhelm Just Keywords: Photoreceptor Guanylate cyclase Calcium signaling Guanylate cyclase-activating protein Zebrafish
a b s t r a c t Zebrafish express in the retina a large variety of three different membrane-bound guanylate cyclases and six different guanylate cyclase-activating proteins (zGCAPs) belonging to the family of neuronal calcium sensor proteins. Although these proteins are predominantly localized in rod and cone photoreceptor cells of the retina, they differ in their spatial–temporal expression profiles. Further, each zGCAP has a different affinity for Ca2+ and displays different Ca2+-sensitivities of guanylate cyclase activation. Thus, zGCAPs operate as cytoplasmic Ca2+-sensors that sense incremental changes of cytoplasmic Ca2+-concentration in rod and cone cells and control the activity of their target guanylate cyclases in a Ca2+-relay mode fashion. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction 1.1. General Excitation and adaptation of vertebrate photoreceptor cells is mediated by two second messengers, guanosine-30 ,50 -cyclic monophosphate (cGMP) and Ca2+ [1–4]. Light absorption of rhodopsin triggers the hydrolysis of cGMP by activation of a well-known Gprotein coupled pathway leading also with a short delay to a fall in cytoplasmic Ca2+-concentration ([Ca2+]). The mutual dependence of cytoplasmic concentrations in cGMP and Ca2+ critically controls the responsiveness of the photoreceptor cell under different illumination conditions and a change in cytoplasmic [Ca2+] is considered to be a critical step mediating light adaptation [1–4]. Key proteins involved in the interplay of cGMP and Ca2+ are membrane-bound guanylate cyclases (GCs) [4,5] and their Ca2+-sensitive regulators, named guanylate cyclase-activating proteins (GCAP) [6–8]. A combination of biochemical, genetic and physiological studies has established that photoreceptor GCs have a low basal cyclase activity at high intracellular [Ca2+] corresponding to
Abbreviations: GC, guanylate cyclase; GCAP, guanylate cyclase-activating protein; z, in front of GC or GCAP denotes proteins expressed in zebrafish; ROS, rod outer segment; NCS, neuronal calcium sensor ⇑ Address: Biochemistry Group, Faculty VI, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany. Fax: +49 441 193640. E-mail address: [email protected]
the dark state of the cell and increase their activities, when the cytoplasmic [Ca2+] decreases after illumination. Although this general concept is well accepted and confirmed [1–8], several open questions remain. For example, what are the molecular mechanisms of protein–protein interaction during GC activation by GCAPs? Further, to what extents contribute other Ca2+-dependent or Ca2+-independent mechanisms to the regulation of phototransduction and the control of light adaptation [9]? Finally, zebrafish has attained increasing interest in vision research due to its fast development, amenability to genetic manipulation and to the fact that it is equipped with a set of cone photoreceptor cells, which are sensitive to the whole visible spectrum and to UV-light. The zebrafish retina expresses three isoforms of sensory GCs and six forms of GCAPs raising the question, whether this apparently redundant expression is of physiological meaning [10,11]. The present minireview will discuss this topic, in particular by reflecting on the recently suggested Ca2+-relay model that accounts for differential properties of GCAP forms [4,12,13]. 1.2. Sensory GCs in teleost fishes Teleost DNA data bases harbour sequence information of several putative membrane bound GCs. These include four different sensory GCs denoted as OlGC3, OlGC4, OlGC5 and OlGC-R2 in medaka fish (Oryzias latipes) and four orthologous forms in pufferfish (Fugu rubripes) and three in carp (Cyprinus carpio) [10,14–19]. 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
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were detected in the photoreceptor layer of larval and adult fish denoted zGC1, zGC2, zGC3 [11,20]. Amino acid sequence alignments and a subsequent dot blot matrix analysis [21] between medaka and zebrafish GCs revealed high sequence homology in the cytoplasmic part and significant regions of conserved amino acids in the extracellular part confirming the conclusion that these GCs belong to the group of sensory GCs [22]. Sensory GCs operating in rod and cone cells of the vertebrate retina have been investigated in the past by numerous studies, which show collectively that these membrane bound GCs exist as dimers in photoreceptor membranes, form complexes with GCAPs and are regulated by GCAPs in a Ca2+-dependent manner (for review see [3,4]. So far, knowledge about the biochemical properties of zGCs is very limited and the cDNAs have not been heterologously expressed for functional studies. In situ hybridization experiments, however, showed that zGC1 and zGC2 were detected in rods and cones and no signals were seen in rods with zGC3 RNA probes. Instead, cones were labeled by zGC3 probes [11]. Screening of randomly mutagenized zebrafishes resulted in several behavioral mutants with mutations in photoreceptor specific genes including one mutant named zatoichi, which has a defect in the gene coding for zGC3 [23]. The zatoichi mutant larva shows no optokinetic response (OKR) and no optomotor response (OMR) measured at 6 days post fertilization (dpf), which points to the crucial role of GC expression for larval visual function. Further, in another study the gene gucy2f coding for the GC zGC1 (s. above) was overexpressed in zebrafish larvae in a gain-of-function approach yielding larvae with multiple defects including a loss of forebrain neurons [24]. Knocking down the expression of gucy2f by the morpholino antisense-oligonucleotide approach led to impaired visual function and shortening of photoreceptor cells in 6 day old larvae [25]. While these studies highlight the importance of genes coding for membrane bound GCs for normal visual performance they leave open questions that are related to regulatory features. 1.3. Expression of GCAPs and functional role in signaling GCAPs belong to the family of neuronal calcium sensor (NCS) proteins that are mainly expressed in neurons, where they mediate as Ca2+-sensors a wide range of physiological responses [26]. GCAPs are specifically expressed in photoreceptor cells and have been identified in different vertebrates like human, bovine, monkey, mice, chicken, fish and amphibians [6–8,10–12,27–29]. They are small compact proteins of approximately 200 amino acid length containing four EF-hand Ca2+-binding motifs, of which the first EF-hand is non-functional and probably involved in protein– protein interaction [30,31]. GCAPs can bind Mg2+, when Ca2+ dissociates from its binding sites during light-induced changes of the cytoplasmic Ca2+-concentration, which transforms GCAPs into their cyclase-activating conformation [32]. Mammalian rod and cone cells express two or three GCAP isoforms, but eight isoforms were predicted in pufferfish (Fugu rubripes) [10] and six isoforms were cloned from zebrafish (Danio rerio) [10,11] and carp (Cyprinus carpio) retinal cDNA libraries [19]. Prominent transcription and/ or expression are observed for all GCAPs in the outer vertebrate retina, in particular in the outer and inner segments of photoreceptor cells. In addition, GCAP specific staining was also observed in cone somata, cell bodies, axons, axon terminals and synaptic pedicles [7,8,28,29], but their functional role in these cell compartments is not understood, in contrast to their well-known function as Ca2+-sensitive regulators of GC activity. GCAPs interact with their main targets, membrane bound sensory GCs, and thereby activate GCs, when they are Ca2+-free and inhibit GCs, when they are Ca2+loaded. For a quantitative assessment of GCAP function in a physiological context it is necessary to examine crucial biochemical properties as Ca2+-binding and Ca2+-affinity, Ca2+-induced
conformational changes, GCAP-target interaction and activation profiles of GCs. These biochemical and biophysical approaches to understand Ca2+-sensor function have recently been reviewed and will not be repeated here [33]. Furthermore, experiments on transgenic mice with manipulated GCAP expression demonstrated that mammalian GCAP1 and GCAP2 critically shape the photoreceptor light response [34–37]. Both, physiological recordings and in vitro data are consistent with a step-by-step signaling mode of GCAPs, which allows the cell to respond to increment changes in cytoplasmic Ca2+. This signaling mode of a Ca2+-relay model has also recently been discussed and reviewed in detail [13]. A more complex operation of the GC/GCAP-complex seems to be realized in the zebrafish retina with its abundant expression of six zGCAP isoforms. Thus, in the following I will focus this mini-review on GCAP function in zebrafish retina. 2. GCAP regulatory modes in zebrafish rods and cones 2.1. Ca2+-binding and Ca2+-induced conformational changes All zGCAPs have successfully been purified as recombinant proteins from Escherichia coli allowing detailed investigations of their biochemical properties [38–40]. A non-radioactive chelator assay developed by Linse and co-workers [41–43] was used to investigate Ca2+-binding affinities. All zGCAPs had at least three binding sites for Ca2+ with apparent affinity constants of high, medium and low affinity. However, these values differed among all investigated zGCAPs and are unique for every isoform [38]. Binding of Ca2+ triggers conformational changes in GCAPs and had been investigated thoroughly for the mammalian GCAP1 and GCAP2. Common spectroscopic methods include in particular intrinsic Tryptophan (Trp) fluorescence and circular dichroism (CD), which had been applied in studying the Ca2+-induced conformational changes of GCAPs and another retina specific NCS protein, recoverin [31,41–47]. Mammalian GCAP1 typically shows a biphasic pattern in Trp emission during a Ca2+-titration with decreasing emission from 0 to 1 lM Ca2+ and increasing emission above 1 lM Ca2+. The Trp emission studies therefore demonstrated that GCAPs undergo Ca2+-induced conformational changes in the physiological range of cytoplasmic [Ca2+]. Interestingly, although all zGCAPs change their Trp emission on Ca2+-binding or Ca2+-dissociation, no zGCAP isoform followed the same pattern as GCAP1, which demonstrates significant differences in Ca2+-sensing [38]. 2.2. Activation profiles of GC signaling One remarkable feature of zGCAPs is that each form has a distinct activation profile. Reconstitution of purified zGCAPs with membrane bound GCs using preparations of bovine rod outer segment (ROS) membranes as assay system showed that all zGCAPs could activate GCs with apparent affinities (EC50-values) in the submicromolar or lower micromolar range [38], which is very similar to mammalian GCAPs that also have EC50 values around 1 lM. Most significant differences are however observed, when maximal GC activities are compared and calculated as x-fold activation from the definition: GCmax GCmin/GCmin (GCmax: GC activity at maximum; GCmin: GC activity at minimum). Maximal activities of zGCAP1, 5 and 7 are rather low (x-fold activation 1.5–3-fold), but zGCAP2, 3 and 4 are strong activators having an x-fold activation between 6 and 13 [38]. When these features are compared with the Ca2+-sensitive regulatory properties it becomes apparent that zGCAPs can be classified into two groups having IC50 values either around 30 nM or around 400 nM [38–40]. The IC50 value is equivalent to the Ca2+-concentration at which GC activation is half-maximal. Each of these groups contains further a combination of strong and weak GC activators (Fig. 1). If we look at the distribution of
K.-W. Koch / FEBS Letters 587 (2013) 2055–2059
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 activation. The black-to-gray bar in the background symbolizes the range of cytoplasmic Ca2+ change during a light response.
zGCAPs in different photoreceptor cells, each cell type harbors strong and weak GC activators and all cone types express also a combination of zGCAPs having different IC50 values (Fig. 2). Double 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 low IC50 values. The only zGCAP with a high IC50 value is zGCAP5, but this Ca2+-sensor shows the lowest degree of x-fold activation among all tested GCAPs [38] and probably does not contribute much to the overall regulation and activation of GCs. It is unclear at the moment, whether this special case of UV-cones reflects a different Ca2+-homeostasis. Direct measurements of cytoplasmic Ca2+-concentration in the dark have been performed so far only for UV-cones and yielded a value of 400 nM, which is suggested to decrease after illumination [48]. It will be a major challenge to further define exactly the changing pattern of cytoplasmic Ca2+ in zebrafish rod and cone cells. In any case, zGCAPs are operating as activating GC regulators in a specific time frame depending on the illumination state of the cell. Thus, zGCAPs exhibiting a lower Ca2+-affinity or -sensitivity (zGCAP4, 5 and 7) would first be turned into an activating status followed by zGCAP 1, 2 or 3 that will become active at Ca2+-concentrations that are approx. one order of magnitude lower. This gradual step-by-step mode of action represents a Ca2+-relay mechanism of GCAP action. 2.3. Comparison of zGCAP3 and zGCAP4 Two cone-specific Ca2+-sensors, zGCAP3 and 4, have been investigated in more detail [39,40]. Each of them represents a strong activator of GCs with high expression rate in cone cells, but with different operation profiles and Ca2+-sensitivities (Figs. 1 and 2). Most NCS proteins contain a consensus sequence for the attachment of a myristoyl group at the amino-terminus [26] and in particular recoverin and GCAPs from mammalian (ROS) exhibit differences in their functional properties, whether they are myristoylated or not. For example, recoverin undergoes a classical Ca2+-myristoyl switch, it binds to membranes, when it is saturated with Ca2+ and the myristoyl group is exposed. In its Ca2+-free form the myristoyl group is buried in a hydrophobic protein pocket making the protein less hydrophobic [49], which in turn leads to the release of recoverin into the cytoplasm. In contrast to recoverin, GCAP1 and 2 do not undergo a classical Ca2+-myristoyl switch [50–53], but presence of the myristoyl group in GCAP1 is essential
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Fig. 2. Summary of zGCAP expression level in zebrafish rod and cone cells. Drawings of rods (gray), double cones (long-wavelength sensitive, red and green), long single cones (short-wavelength sensitive, blue) and short single cones (UVsensitive, 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 shaped bars symbolizing from top to bottom high and low expression rates. Ca2+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 around 400 nM [Ca2+] for zGCAP4, 5 and 7 (blue background).
to shift the Ca2+-sensitivity into the physiological range. In addition, the affinity of the GCAP1–ROS–GC1 interaction depends on the myristoyl modification [54–56]. In contrast, bovine GCAP2 shows an almost identical activation pattern in its myristoylated or non-myristoylated form, however, the myristoyl group in GCAP2 seems to be more flexible and is involved in stabilizing the Ca2+-bound conformation [57,58]. These findings raised the following questions, (a) whether zGCAPs are expressed in myristoylated or non-myristoylated forms; (b) and if so, whether these forms differ in their key properties. Heterologous expression and purification from bacteria revealed that zGCAP3 and zGCAP4 could be myristoylated by a co-expressed N-myristoyl-transferase from yeast, although a point mutant of zGCAP4 was necessary [39,40]. Probing larval and adult stages of zebrafish showed also that zGCAP3 is myristoylated in native retinae [40], but a similar study has not been conducted for zGCAP4 so far. In order to identify a possible influence of the myristoyl group on the functional properties of zGCAPs, zGCAP3 and 4 were purified as myristoylated and non-myristoylated recombinant proteins and investigated according to several criteria yielding the following results: they do not undergo a classical Ca2+-myristoyl-switch, although membrane binding of zGCAP3 is slightly improved by the myristoyl group [39,40]. Activation profiles of zGCAPs are nearly identical with similar IC50 and EC50 values for GC activation indicating no significant influence on Ca2+-sensor function. However, in zGCAP3 the myristoyl group has several other effects. It enhances the Ca2+sensitivity of conformational changes, stabilizes the protein conformation in general and has a modulating effect on kinetic parameters of the GC target [40]. Interestingly, zebrafish larvae first express nonmyristoylated zGCAP3 at 3.25 dpf, but myristoylated zGCAP3 was not detected before 7 dpf [40]. Apparently, the impact of the myristoyl group on zGCAP3 functions seems less important in the larval than in the adult stage. In summary, GCAPs are NCS proteins that operate like their mammalian counterparts in a relay mode allowing rod and cone cells to detect incremental changes in cytoplasmic Ca2+. The biochemical complexity of zGCAPs reflects a fine-tuned system of
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Ca2+-sensing and Ca2+-mediating proteins playing key roles in photoreceptor excitation and adaptation. However, many unresolved questions remain as for example to examine, whether the action mode of each zGCAP changes under varying illumination conditions. Furthermore, which other Ca2+-dependent control mechanisms targeting different enzymes or proteins are involved in adjusting the photoreceptor’s light adapting properties and forming the molecular basis for the dynamic range extension in cone adaptation of the fish retina. Acknowledgement Experimental work done in the laboratory of the author is funded by several Grants of the Deutsche Forschungsgemeinschaft (DFG). References [1] Pugh Jr., E.N. and Lamb, T.D. (2000) Phototransduction in vertebrate rods and cones: molecular mechanisms of amplification, recovery and light adaptation in: Handbook of Biological Physics (Stavenga, D.G., DeGrip, W.J. and Pugh, E.N. Jr.Jr., Eds.), pp. 183–255, Elsevier Science BV 3. [2] Luo, D.G., Xue, T. and Yau, K.-W. (2008) How vision begins: an odyssey. Proc. Natl. Acad. Sci. USA 105, 9855–9862. [3] Stephen, R., Filipek, S., Palczewski, K. and and, Sousa, M.C., and (2008) Ca2+dependent regulation of phototransduction. Photochem. Photobiol. 84, 903– 910. [4] Koch, K.-W., Duda, T. and and, Sharma, R.K., and (2010) Ca2+-modulated vision-linked ROS-GC guanylate cyclase transduction machinery. Mol. Cell. Biochem. 334, 105–115. [5] Baehr, W., Karan, S., Maeda, T., Luo, D.G., Li, S., Bronson, J.D., Watt, C.B., Yau, K.W., Frederick, J.M. and Palczewski, K. (2007) The function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors. J. Biol. Chem. 282, 8837–8847. [6] Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H., Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B. and Baehr, W. (1994) Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein. Neuron 13, 395– 404. [7] Dizhoor, A.M., Olshevskaya, E.V., Henzel, W.J., Wong, S., Stults, J.T., Ankoudinova, I. and Hurley, J.B. (1995) Cloning, sequencing, and expression of a 24-kDa Ca2+-binding protein activating photoreceptor guanylyl cyclase. J. Biol. Chem. 270, 25200–25206. [8] Frins, S., Bönigk, W., Müller, F., Kellner, R. and Koch, K.-W. (1996) Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. J. Biol. Chem. 271, 8022–8027. [9] Fain, G.L. (2011) Adaptation of mammalian photoreceptors to background light: putative role for direct modulation of phosphodiesterase. Mol. Neurobiol. 44, 374–382. [10] Imanishi, Y., Yang, L., Sokal, I., Filipek, S., Palczewski, K. and Baehr, W. (2004) Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1–8) in pufferfish (fugu rubripes). J. Mol. Evol. 59, 204–217. [11] Rätscho, N., Scholten, A. and Koch, K.-W. (2009) Expression profiles of three novel sensory guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish retina. Biochim. Biopyhs. Acta 1793, 1110–1114. [12] Koch, K.-W. (2006) GCAPs, the classical neuronal calcium sensors in the retina – a Ca2+-relay model of guanylate cyclase activation. Calcium Binding Proteins 1, 3–6. [13] Koch, K.-W., Dell’Orco, D. (in press) A calcium – relay mechanism in vertebrate phototransduction. ACS Chem. Neurosci. http://dx.doi.org/10.1021/ cn400027z. [14] Seimiya, M., Kusakabe, T. and Suzuki, N. (1997) 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. 272, 23407–23417. [15] Hisatome, O., Honkawa, H., Imanishsi, Y., Satoh, T. and Tokunaga, F. (1999) Three kinds of guanylate cyclase expressed in medaka photoreceptor cells in both retina and pineal organ. Biochem. Biophys. Res. Commun. 255, 216–220. [16] Kusakabe, T. and Suzuke, N. (2000) Photoreceptors and olfactory cells express the same retinal guanyly cyclase isoform in medaka: visualization by promote transgenics. FEBS Lett. 483, 143–148. [17] Harumi, T., Watanabe, T., Yamamoto, T., Tanabe, Y. and Suzuki, N. (2003) Expression of membrane-bound and soluble guanylyl-cyclase mRNAs in embryonic and adult retina of the Medaka fish Oryzias latipes. Zool. Sci. 20, 133–140. [18] Yamagami, S. and Suzuki, N. (2005) Diverse forms of guanylyl cyclases in Medaka fish – their genomic structure and phylogenetic relationships to those in vertebrates and invertebrates. Zool. Sci. 22, 819–835. [19] Takemoto, N., Tachibanaki, S. and Kawamura, S. (2009) High cGMP synthetic activity in carp cones. Proc. Natl. Acad. Sci. USA 106, 11788–11793.
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K.-W. Koch / FEBS Letters 587 (2013) 2055–2059 [47] Dell’Orco, D., Behnen, P., Linse, S. and Koch, K.-W. (2010) Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy. Cell. Mol. Life Sci. 67, 973–984. [48] Cilluffo, M.C., Matthews, H.R., Brockerhoff, S.E. and Fain, G.L. (2004) Lightinduced Ca2+-release in the visible cones of the zebrafish. Vis. Neurosci. 21, 599–609. [49] Ames, J.B., Ishima, R., Tanaka, T., Gordon, J.I., Stryer, L. and Ikura, M. (1997) Molecular mechanics of calcium-myristoyl switches. Nature 389, 198–202. [50] Olshevskaya, E.V., Hughes, R.E., Hurley, J.B. and Dizhoor, A.M. (1997) Calcium binding, but not a calcium-myristoyl switch, controls the ability of guanylyl cyclase-activating protein GCAP-2 to regulate photoreceptor guanylyl cyclase. J. Biol. Chem. 272, 1432714333. [51] Hwang, J.Y. and Koch, K.-W. (2002) Calcium- and myristoyl-dependent properties of guanylate cyclase-activating protein-1 and protein-2. Biochemistry 41, 13021–13028. [52] Stephen, R., Bereta, G., Golczak, M., Palczewski, K. and Sousa, M.C. (2007) Stabilizing function for myristoyl group revealed by the crystal structure of a neuronal calcium sensor, guanylate cyclase-activating protein 1. Structure 15, 1392–1402. [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
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journal homepage: www.FEBSLetters.org
Review
The guanylate cyclase 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
a r t i c l e
i n f o
Article history: Received 24 April 2013 Revised 25 April 2013 Accepted 26 April 2013 Available online 7 May 2013 Edited by Alexander Gabibov, Vladimir Skulachev, Felix Wieland and Wilhelm Just Keywords: Photoreceptor Guanylate cyclase Calcium signaling Guanylate cyclase-activating protein Zebrafish
a b s t r a c t Zebrafish express in the retina a large variety of three different membrane-bound guanylate cyclases and six different guanylate cyclase-activating proteins (zGCAPs) belonging to the family of neuronal calcium sensor proteins. Although these proteins are predominantly localized in rod and cone photoreceptor cells of the retina, they differ in their spatial–temporal expression profiles. Further, each zGCAP has a different affinity for Ca2+ and displays different Ca2+-sensitivities of guanylate cyclase activation. Thus, zGCAPs operate as cytoplasmic Ca2+-sensors that sense incremental changes of cytoplasmic Ca2+-concentration in rod and cone cells and control the activity of their target guanylate cyclases in a Ca2+-relay mode fashion. Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction 1.1. General Excitation and adaptation of vertebrate photoreceptor cells is mediated by two second messengers, guanosine-30 ,50 -cyclic monophosphate (cGMP) and Ca2+ [1–4]. Light absorption of rhodopsin triggers the hydrolysis of cGMP by activation of a well-known Gprotein coupled pathway leading also with a short delay to a fall in cytoplasmic Ca2+-concentration ([Ca2+]). The mutual dependence of cytoplasmic concentrations in cGMP and Ca2+ critically controls the responsiveness of the photoreceptor cell under different illumination conditions and a change in cytoplasmic [Ca2+] is considered to be a critical step mediating light adaptation [1–4]. Key proteins involved in the interplay of cGMP and Ca2+ are membrane-bound guanylate cyclases (GCs) [4,5] and their Ca2+-sensitive regulators, named guanylate cyclase-activating proteins (GCAP) [6–8]. A combination of biochemical, genetic and physiological studies has established that photoreceptor GCs have a low basal cyclase activity at high intracellular [Ca2+] corresponding to
Abbreviations: GC, guanylate cyclase; GCAP, guanylate cyclase-activating protein; z, in front of GC or GCAP denotes proteins expressed in zebrafish; ROS, rod outer segment; NCS, neuronal calcium sensor ⇑ Address: Biochemistry Group, Faculty VI, Carl von Ossietzky University Oldenburg, D-26111 Oldenburg, Germany. Fax: +49 441 193640. E-mail address: [email protected]
the dark state of the cell and increase their activities, when the cytoplasmic [Ca2+] decreases after illumination. Although this general concept is well accepted and confirmed [1–8], several open questions remain. For example, what are the molecular mechanisms of protein–protein interaction during GC activation by GCAPs? Further, to what extents contribute other Ca2+-dependent or Ca2+-independent mechanisms to the regulation of phototransduction and the control of light adaptation [9]? Finally, zebrafish has attained increasing interest in vision research due to its fast development, amenability to genetic manipulation and to the fact that it is equipped with a set of cone photoreceptor cells, which are sensitive to the whole visible spectrum and to UV-light. The zebrafish retina expresses three isoforms of sensory GCs and six forms of GCAPs raising the question, whether this apparently redundant expression is of physiological meaning [10,11]. The present minireview will discuss this topic, in particular by reflecting on the recently suggested Ca2+-relay model that accounts for differential properties of GCAP forms [4,12,13]. 1.2. Sensory GCs in teleost fishes Teleost DNA data bases harbour sequence information of several putative membrane bound GCs. These include four different sensory GCs denoted as OlGC3, OlGC4, OlGC5 and OlGC-R2 in medaka fish (Oryzias latipes) and four orthologous forms in pufferfish (Fugu rubripes) and three in carp (Cyprinus carpio) [10,14–19]. 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
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were detected in the photoreceptor layer of larval and adult fish denoted zGC1, zGC2, zGC3 [11,20]. Amino acid sequence alignments and a subsequent dot blot matrix analysis [21] between medaka and zebrafish GCs revealed high sequence homology in the cytoplasmic part and significant regions of conserved amino acids in the extracellular part confirming the conclusion that these GCs belong to the group of sensory GCs [22]. Sensory GCs operating in rod and cone cells of the vertebrate retina have been investigated in the past by numerous studies, which show collectively that these membrane bound GCs exist as dimers in photoreceptor membranes, form complexes with GCAPs and are regulated by GCAPs in a Ca2+-dependent manner (for review see [3,4]. So far, knowledge about the biochemical properties of zGCs is very limited and the cDNAs have not been heterologously expressed for functional studies. In situ hybridization experiments, however, showed that zGC1 and zGC2 were detected in rods and cones and no signals were seen in rods with zGC3 RNA probes. Instead, cones were labeled by zGC3 probes [11]. Screening of randomly mutagenized zebrafishes resulted in several behavioral mutants with mutations in photoreceptor specific genes including one mutant named zatoichi, which has a defect in the gene coding for zGC3 [23]. The zatoichi mutant larva shows no optokinetic response (OKR) and no optomotor response (OMR) measured at 6 days post fertilization (dpf), which points to the crucial role of GC expression for larval visual function. Further, in another study the gene gucy2f coding for the GC zGC1 (s. above) was overexpressed in zebrafish larvae in a gain-of-function approach yielding larvae with multiple defects including a loss of forebrain neurons [24]. Knocking down the expression of gucy2f by the morpholino antisense-oligonucleotide approach led to impaired visual function and shortening of photoreceptor cells in 6 day old larvae [25]. While these studies highlight the importance of genes coding for membrane bound GCs for normal visual performance they leave open questions that are related to regulatory features. 1.3. Expression of GCAPs and functional role in signaling GCAPs belong to the family of neuronal calcium sensor (NCS) proteins that are mainly expressed in neurons, where they mediate as Ca2+-sensors a wide range of physiological responses [26]. GCAPs are specifically expressed in photoreceptor cells and have been identified in different vertebrates like human, bovine, monkey, mice, chicken, fish and amphibians [6–8,10–12,27–29]. They are small compact proteins of approximately 200 amino acid length containing four EF-hand Ca2+-binding motifs, of which the first EF-hand is non-functional and probably involved in protein– protein interaction [30,31]. GCAPs can bind Mg2+, when Ca2+ dissociates from its binding sites during light-induced changes of the cytoplasmic Ca2+-concentration, which transforms GCAPs into their cyclase-activating conformation [32]. Mammalian rod and cone cells express two or three GCAP isoforms, but eight isoforms were predicted in pufferfish (Fugu rubripes) [10] and six isoforms were cloned from zebrafish (Danio rerio) [10,11] and carp (Cyprinus carpio) retinal cDNA libraries [19]. Prominent transcription and/ or expression are observed for all GCAPs in the outer vertebrate retina, in particular in the outer and inner segments of photoreceptor cells. In addition, GCAP specific staining was also observed in cone somata, cell bodies, axons, axon terminals and synaptic pedicles [7,8,28,29], but their functional role in these cell compartments is not understood, in contrast to their well-known function as Ca2+-sensitive regulators of GC activity. GCAPs interact with their main targets, membrane bound sensory GCs, and thereby activate GCs, when they are Ca2+-free and inhibit GCs, when they are Ca2+loaded. For a quantitative assessment of GCAP function in a physiological context it is necessary to examine crucial biochemical properties as Ca2+-binding and Ca2+-affinity, Ca2+-induced
conformational changes, GCAP-target interaction and activation profiles of GCs. These biochemical and biophysical approaches to understand Ca2+-sensor function have recently been reviewed and will not be repeated here [33]. Furthermore, experiments on transgenic mice with manipulated GCAP expression demonstrated that mammalian GCAP1 and GCAP2 critically shape the photoreceptor light response [34–37]. Both, physiological recordings and in vitro data are consistent with a step-by-step signaling mode of GCAPs, which allows the cell to respond to increment changes in cytoplasmic Ca2+. This signaling mode of a Ca2+-relay model has also recently been discussed and reviewed in detail [13]. A more complex operation of the GC/GCAP-complex seems to be realized in the zebrafish retina with its abundant expression of six zGCAP isoforms. Thus, in the following I will focus this mini-review on GCAP function in zebrafish retina. 2. GCAP regulatory modes in zebrafish rods and cones 2.1. Ca2+-binding and Ca2+-induced conformational changes All zGCAPs have successfully been purified as recombinant proteins from Escherichia coli allowing detailed investigations of their biochemical properties [38–40]. A non-radioactive chelator assay developed by Linse and co-workers [41–43] was used to investigate Ca2+-binding affinities. All zGCAPs had at least three binding sites for Ca2+ with apparent affinity constants of high, medium and low affinity. However, these values differed among all investigated zGCAPs and are unique for every isoform [38]. Binding of Ca2+ triggers conformational changes in GCAPs and had been investigated thoroughly for the mammalian GCAP1 and GCAP2. Common spectroscopic methods include in particular intrinsic Tryptophan (Trp) fluorescence and circular dichroism (CD), which had been applied in studying the Ca2+-induced conformational changes of GCAPs and another retina specific NCS protein, recoverin [31,41–47]. Mammalian GCAP1 typically shows a biphasic pattern in Trp emission during a Ca2+-titration with decreasing emission from 0 to 1 lM Ca2+ and increasing emission above 1 lM Ca2+. The Trp emission studies therefore demonstrated that GCAPs undergo Ca2+-induced conformational changes in the physiological range of cytoplasmic [Ca2+]. Interestingly, although all zGCAPs change their Trp emission on Ca2+-binding or Ca2+-dissociation, no zGCAP isoform followed the same pattern as GCAP1, which demonstrates significant differences in Ca2+-sensing [38]. 2.2. Activation profiles of GC signaling One remarkable feature of zGCAPs is that each form has a distinct activation profile. Reconstitution of purified zGCAPs with membrane bound GCs using preparations of bovine rod outer segment (ROS) membranes as assay system showed that all zGCAPs could activate GCs with apparent affinities (EC50-values) in the submicromolar or lower micromolar range [38], which is very similar to mammalian GCAPs that also have EC50 values around 1 lM. Most significant differences are however observed, when maximal GC activities are compared and calculated as x-fold activation from the definition: GCmax GCmin/GCmin (GCmax: GC activity at maximum; GCmin: GC activity at minimum). Maximal activities of zGCAP1, 5 and 7 are rather low (x-fold activation 1.5–3-fold), but zGCAP2, 3 and 4 are strong activators having an x-fold activation between 6 and 13 [38]. When these features are compared with the Ca2+-sensitive regulatory properties it becomes apparent that zGCAPs can be classified into two groups having IC50 values either around 30 nM or around 400 nM [38–40]. The IC50 value is equivalent to the Ca2+-concentration at which GC activation is half-maximal. Each of these groups contains further a combination of strong and weak GC activators (Fig. 1). If we look at the distribution of
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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 activation. The black-to-gray bar in the background symbolizes the range of cytoplasmic Ca2+ change during a light response.
zGCAPs in different photoreceptor cells, each cell type harbors strong and weak GC activators and all cone types express also a combination of zGCAPs having different IC50 values (Fig. 2). Double 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 low IC50 values. The only zGCAP with a high IC50 value is zGCAP5, but this Ca2+-sensor shows the lowest degree of x-fold activation among all tested GCAPs [38] and probably does not contribute much to the overall regulation and activation of GCs. It is unclear at the moment, whether this special case of UV-cones reflects a different Ca2+-homeostasis. Direct measurements of cytoplasmic Ca2+-concentration in the dark have been performed so far only for UV-cones and yielded a value of 400 nM, which is suggested to decrease after illumination [48]. It will be a major challenge to further define exactly the changing pattern of cytoplasmic Ca2+ in zebrafish rod and cone cells. In any case, zGCAPs are operating as activating GC regulators in a specific time frame depending on the illumination state of the cell. Thus, zGCAPs exhibiting a lower Ca2+-affinity or -sensitivity (zGCAP4, 5 and 7) would first be turned into an activating status followed by zGCAP 1, 2 or 3 that will become active at Ca2+-concentrations that are approx. one order of magnitude lower. This gradual step-by-step mode of action represents a Ca2+-relay mechanism of GCAP action. 2.3. Comparison of zGCAP3 and zGCAP4 Two cone-specific Ca2+-sensors, zGCAP3 and 4, have been investigated in more detail [39,40]. Each of them represents a strong activator of GCs with high expression rate in cone cells, but with different operation profiles and Ca2+-sensitivities (Figs. 1 and 2). Most NCS proteins contain a consensus sequence for the attachment of a myristoyl group at the amino-terminus [26] and in particular recoverin and GCAPs from mammalian (ROS) exhibit differences in their functional properties, whether they are myristoylated or not. For example, recoverin undergoes a classical Ca2+-myristoyl switch, it binds to membranes, when it is saturated with Ca2+ and the myristoyl group is exposed. In its Ca2+-free form the myristoyl group is buried in a hydrophobic protein pocket making the protein less hydrophobic [49], which in turn leads to the release of recoverin into the cytoplasm. In contrast to recoverin, GCAP1 and 2 do not undergo a classical Ca2+-myristoyl switch [50–53], but presence of the myristoyl group in GCAP1 is essential
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Fig. 2. Summary of zGCAP expression level in zebrafish rod and cone cells. Drawings of rods (gray), double cones (long-wavelength sensitive, red and green), long single cones (short-wavelength sensitive, blue) and short single cones (UVsensitive, 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 shaped bars symbolizing from top to bottom high and low expression rates. Ca2+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 around 400 nM [Ca2+] for zGCAP4, 5 and 7 (blue background).
to shift the Ca2+-sensitivity into the physiological range. In addition, the affinity of the GCAP1–ROS–GC1 interaction depends on the myristoyl modification [54–56]. In contrast, bovine GCAP2 shows an almost identical activation pattern in its myristoylated or non-myristoylated form, however, the myristoyl group in GCAP2 seems to be more flexible and is involved in stabilizing the Ca2+-bound conformation [57,58]. These findings raised the following questions, (a) whether zGCAPs are expressed in myristoylated or non-myristoylated forms; (b) and if so, whether these forms differ in their key properties. Heterologous expression and purification from bacteria revealed that zGCAP3 and zGCAP4 could be myristoylated by a co-expressed N-myristoyl-transferase from yeast, although a point mutant of zGCAP4 was necessary [39,40]. Probing larval and adult stages of zebrafish showed also that zGCAP3 is myristoylated in native retinae [40], but a similar study has not been conducted for zGCAP4 so far. In order to identify a possible influence of the myristoyl group on the functional properties of zGCAPs, zGCAP3 and 4 were purified as myristoylated and non-myristoylated recombinant proteins and investigated according to several criteria yielding the following results: they do not undergo a classical Ca2+-myristoyl-switch, although membrane binding of zGCAP3 is slightly improved by the myristoyl group [39,40]. Activation profiles of zGCAPs are nearly identical with similar IC50 and EC50 values for GC activation indicating no significant influence on Ca2+-sensor function. However, in zGCAP3 the myristoyl group has several other effects. It enhances the Ca2+sensitivity of conformational changes, stabilizes the protein conformation in general and has a modulating effect on kinetic parameters of the GC target [40]. Interestingly, zebrafish larvae first express nonmyristoylated zGCAP3 at 3.25 dpf, but myristoylated zGCAP3 was not detected before 7 dpf [40]. Apparently, the impact of the myristoyl group on zGCAP3 functions seems less important in the larval than in the adult stage. In summary, GCAPs are NCS proteins that operate like their mammalian counterparts in a relay mode allowing rod and cone cells to detect incremental changes in cytoplasmic Ca2+. The biochemical complexity of zGCAPs reflects a fine-tuned system of
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Ca2+-sensing and Ca2+-mediating proteins playing key roles in photoreceptor excitation and adaptation. However, many unresolved questions remain as for example to examine, whether the action mode of each zGCAP changes under varying illumination conditions. Furthermore, which other Ca2+-dependent control mechanisms targeting different enzymes or proteins are involved in adjusting the photoreceptor’s light adapting properties and forming the molecular basis for the dynamic range extension in cone adaptation of the fish retina. Acknowledgement Experimental work done in the laboratory of the author is funded by several Grants of the Deutsche Forschungsgemeinschaft (DFG). References [1] Pugh Jr., E.N. and Lamb, T.D. (2000) Phototransduction in vertebrate rods and cones: molecular mechanisms of amplification, recovery and light adaptation in: Handbook of Biological Physics (Stavenga, D.G., DeGrip, W.J. and Pugh, E.N. Jr.Jr., Eds.), pp. 183–255, Elsevier Science BV 3. [2] Luo, D.G., Xue, T. and Yau, K.-W. (2008) How vision begins: an odyssey. Proc. Natl. Acad. Sci. USA 105, 9855–9862. [3] Stephen, R., Filipek, S., Palczewski, K. and and, Sousa, M.C., and (2008) Ca2+dependent regulation of phototransduction. Photochem. Photobiol. 84, 903– 910. [4] Koch, K.-W., Duda, T. and and, Sharma, R.K., and (2010) Ca2+-modulated vision-linked ROS-GC guanylate cyclase transduction machinery. Mol. Cell. Biochem. 334, 105–115. [5] Baehr, W., Karan, S., Maeda, T., Luo, D.G., Li, S., Bronson, J.D., Watt, C.B., Yau, K.W., Frederick, J.M. and Palczewski, K. (2007) The function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors. J. Biol. Chem. 282, 8837–8847. [6] Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H., Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B. and Baehr, W. (1994) Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein. Neuron 13, 395– 404. [7] Dizhoor, A.M., Olshevskaya, E.V., Henzel, W.J., Wong, S., Stults, J.T., Ankoudinova, I. and Hurley, J.B. (1995) Cloning, sequencing, and expression of a 24-kDa Ca2+-binding protein activating photoreceptor guanylyl cyclase. J. Biol. Chem. 270, 25200–25206. [8] Frins, S., Bönigk, W., Müller, F., Kellner, R. and Koch, K.-W. (1996) Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. J. Biol. Chem. 271, 8022–8027. [9] Fain, G.L. (2011) Adaptation of mammalian photoreceptors to background light: putative role for direct modulation of phosphodiesterase. Mol. Neurobiol. 44, 374–382. [10] Imanishi, Y., Yang, L., Sokal, I., Filipek, S., Palczewski, K. and Baehr, W. (2004) Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1–8) in pufferfish (fugu rubripes). J. Mol. Evol. 59, 204–217. [11] Rätscho, N., Scholten, A. and Koch, K.-W. (2009) Expression profiles of three novel sensory guanylate cyclases and guanylate cyclase-activating proteins in the zebrafish retina. Biochim. Biopyhs. Acta 1793, 1110–1114. [12] Koch, K.-W. (2006) GCAPs, the classical neuronal calcium sensors in the retina – a Ca2+-relay model of guanylate cyclase activation. Calcium Binding Proteins 1, 3–6. [13] Koch, K.-W., Dell’Orco, D. (in press) A calcium – relay mechanism in vertebrate phototransduction. ACS Chem. Neurosci. http://dx.doi.org/10.1021/ cn400027z. [14] Seimiya, M., Kusakabe, T. and Suzuki, N. (1997) 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. 272, 23407–23417. [15] Hisatome, O., Honkawa, H., Imanishsi, Y., Satoh, T. and Tokunaga, F. (1999) Three kinds of guanylate cyclase expressed in medaka photoreceptor cells in both retina and pineal organ. Biochem. Biophys. Res. Commun. 255, 216–220. [16] Kusakabe, T. and Suzuke, N. (2000) Photoreceptors and olfactory cells express the same retinal guanyly cyclase isoform in medaka: visualization by promote transgenics. FEBS Lett. 483, 143–148. [17] Harumi, T., Watanabe, T., Yamamoto, T., Tanabe, Y. and Suzuki, N. (2003) Expression of membrane-bound and soluble guanylyl-cyclase mRNAs in embryonic and adult retina of the Medaka fish Oryzias latipes. Zool. Sci. 20, 133–140. [18] Yamagami, S. and Suzuki, N. (2005) Diverse forms of guanylyl cyclases in Medaka fish – their genomic structure and phylogenetic relationships to those in vertebrates and invertebrates. Zool. Sci. 22, 819–835. [19] Takemoto, N., Tachibanaki, S. and Kawamura, S. (2009) High cGMP synthetic activity in carp cones. Proc. Natl. Acad. Sci. USA 106, 11788–11793.
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