Interaction of GCAP1 with Retinal Guanylyl Cyclase and Calcium: Sensitivity to Fatty Acylation

ORIGINAL RESEARCH ARTICLE published: 22 February 2012 MOLECULAR NEUROSCIENCE doi: 10.3389/fnmol.2012.00019 Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

Igor V. Peshenko , Elena V. Olshevskaya and Alexander M. Dizhoor*

Department of Basic Science and Pennsylvania College of Optometry, Salus University, Elkins Park, PA, USA

Edited by: Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins Michael R. Kreutz, Leibniz-Institute within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. for Neurobiology, Germany GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response Reviewed by: to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four Carlo Sala, CNR Institute of Neuroscience, Italy EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required Karl-Wilhelm Koch, Carl von for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting Ossietzky University Oldenburg, N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with Germany its target enzyme when heterologously expressed in HEK293 cells. We also compared *Correspondence: Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and Alexander M. Dizhoor, Department of Basic Science and Pennsylvania C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was College of Optometry, Salus able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less University, 8360 Old York Road, effectively stimulated cyclase activity in vitro.Ca2+ binding isotherm of the G2A GCAP1 Elkins Park, PA 19027, USA. was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity e-mail: [email protected] of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.

Keywords: photoreceptors, calcium, guanylyl cyclase, myristoylation

INTRODUCTION et al., 2011). Rods and cones maintain partially depolarized Retinal guanylyl cyclase activating proteins (GCAPs) form a sub- state in the dark by opening a fraction of cGMP-gated chan- + family within the neuronal calcium sensor (NCS) proteins group nels in the outer segment, thus allowing influx of Na and + of the EF-hand superfamily (reviewed in: Burgoyne, 2007). The Ca2 . In the light, when bleached photopigment activates pho- GCAPs are closely related to neurocalcin, hippocalcin, recoverin, totransduction cascade, cGMP becomes rapidly depleted by and other NCS proteins and share a number of typical features light-activated phosphodiesterase and the cGMP-gated chan- for the group: they consist of two pairs of calmodulin-type EF- nels become closed, thus causing hyperpolarization of the hands (Ames et al., 1999; Stephen et al., 2006, 2007), of which membrane—the first cellular step in visual signal detection the N-terminal EF-hand 1 does not bind divalent cation, and are (reviewed in: Fu and Yau, 2007). Photoreceptors can quickly modified at the N-terminus by fatty acyl group (Figure 1A). The recover from excitation and adapt to background illumination modification is commonly referred to as myristoylation, although by re-opening cGMP-gated channels, and GCAPs control one in mammalian retinal proteins, the N-terminal Gly group can be of the key biochemical pathways expediting the recovery and fatty acylated by one of four different C14 and C12 derivatives light adaptation. In response to illumination, GCAPs activate (Dizhoor et al., 1992; Johnson et al., 1994), all of which can be cGMP re-synthesis via negative calcium feedback mechanism found in GCAP (Palczewski et al., 1994). that trails the excitation state of the photoreceptor (reviewed + Among the NCS proteins, GCAPs arguably have the most in: Pugh et al., 1997, 1999). Ca2 is constantly extruded from clearly understood physiological function. The GCAPs regu- the outer segment via Na/Ca, K-exchanger, in the dark, when late activity of retinal membrane guanylyl cyclase (RetGC) in several percent of the cGMP-gated channels are open at any + photoreceptors in a Ca2+-sensitive manner (Koch and Stryer, given time, Ca2 can re-enter the outer segment through the 1988; Dizhoor et al., 1994, 1995; Gorczyca et al., 1994)and open channels and accumulates in the cytoplasm at submi- thus critically expedite recovery and light adaptation of rods cromolar concentrations. In mouse rods, the measured steady- and cones (Mendez et al., 2001; Burns et al., 2002; Sakurai state concentrations in the dark approach 250 nm (as high as 410–560 nm free Ca2+ were reported in lower vertebrates)

and rapidly fall nearly 10-fold in the light, when the cGMP- Abbreviations: EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N ,N - gated channels are shut down (Gray-Keller and Detwiler, 1994; tetraacetic acid; GCAP, guanylyl cyclase activating protein; RetGC, retinal membrane guanylyl cyclase; GFP, green ﬂuorescent protein; NCS proteins, Sampath et al., 1999; Woodruff et al., 2002). Therefore, GCAPs + neuronal calcium binding proteins. bind Ca2 and maintain their inhibitory conformation that

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FIGURE 1 | (A) Structure of myristoylated GCAP1 [Stephen et al. (2007)]. Myristoyl residue buried inside the EF-1/EF-2 pair of EF-hands is shown in red. (B) Dose-dependence of recombinant RetGC1 activation by myristoylated and G2A GCAP1 (mean ± SD). For details of the assay—see Materials and Methods. decelerates RetGC in the dark (Dizhoor and Hurley, 1996; MATERIALS AND METHODS + + Dizhoor et al., 1998). Mg2 can actively compete with Ca2 MUTAGENESIS binding in EF-hand domains of GCAPs (Peshenko and Dizhoor, Mutations were introduced in bovine GCAP1 cDNA by “splicing + 2004), and at the low free Ca2 levels in the light it converts by overlap extension” technique using PCR reactions catalyzed by + GCAPs into Mg2 -bound, RetGC activator state. This stimu- high-fidelity Phusion Flash polymerase (Finnzymes). The resul- lates cGMP synthesis and, therefore, allows the rod to timely tant products were ligated into the NcoI/BamHI sites of pET11d re-open the cGMP-gated channels and recover from the hyper- (Novagene) vector, sequenced, and transformed into expressing + polarization state. Following the completion of recovery, Ca2 cell lines as described previously in detail (Peshenko and Dizhoor, returned through the re-opened cGMP-gated channels converts 2006). RetGC1 tagged by mOrange was constructed by insert- + GCAPs back to their Ca2 -bound inhibitory form, thus com- ing mOrange (Clontech) cDNA into a modified human RetGC1 pleting the light-driven functional cycle of GCAP (reviewed in: cDNA-harboring pRCCMV plasmid (Laura et al., 1996)asfol- Dizhoor et al., 2010). lows. The XhoI-XhoI fragment of the vector was excised by XhoI GCAPs exist in several isoforms (Imanishi et al., 2004), of digest and self-ligation, then the coding region for the extracellu- which GCAP1 (Gorczyca et al., 1994; Palczewski et al., 1994) lar domain of RetGC1 was modified by ligating a linker fragment and GCAP2 (Dizhoor et al., 1994, 1995) are ubiquitous among into the HindIII/BsteII sites to introduce two new restriction all tested vertebrate species. They regulate RetGC with dis- sites, NheI and AgeI beginning after 33 base pairs downstream + tinctly different sensitivities to Ca2 (Hwang and Koch, 2002; from the leader peptide-coding fragment. The mOrange cDNA, Hwang et al., 2003; Peshenko et al., 2004, 2011) and, therefore, PCR-amplified with the NheI and AgeI sites at the 5-and3- sequentially activate RetGC at different phases of rod photore- end, respectively, was ligated into the corresponding restriction sponse (Makino et al., 2008). The two GCAPs also differ in their sites of the modified pRetGC1-RCCMV plasmid. The resultant requirement of the N-fatty acylation for their function. While construct encoded 238 a.a. mOrange protein sequence down- N-myristoylation has relatively modest impact on the regulatory stream from the 51 a.a. leader peptide, replacing a short fragment, activity of GCAP2 in vitro (Olshevskaya et al., 1997), it is much Ala63–Phe68, of the RetGC1 extracellular domain. more critically needed for the activity of GCAP1 (Otto-Bruc et al., + 1997; Hwang and Koch, 2002). The best-known Ca2 -dependent GCAP1 PURIFICATION conformational change described for NCS proteins is a “calcium- Myristoylated bovine D6S GCAP1 was produced in BLR(DE3) + myristoyl switch”—Ca2 -dependent release of the myristoylated E. coli strains harboring yeast N-myristoyl transferase (NMT), N-terminus from the cavity created by alpha-helical structures extracted from inclusion bodies and purified to ∼95% elec- ofEF-hands1and2(Zozulya and Stryer, 1992; Dizhoor et al., trophoretic purity using Ca2+ precipitation, butyl-Sepharose 1993; Ames et al., 1995, 1997; Lim et al., 2011). In contrast, NMR chromatography, and high-resolution gel-filtration as previously + data argue that myristoyl chain does not undergo Ca2 -myristoyl described in detail (Peshenko and Dizhoor, 2006). The express- switch in GCAP1 and GCAP2 (Hughes et al., 1998; Lim et al., ing strain for non-myristoylated G2A GCAP1 lacked the NMT 2009), and it remains buried inside the protein in the X-ray crys- plasmid. tal structure of GCAP1 (Stephen et al., 2007)(Figure 1A). In this study, we addressed functional effects of N-fatty acylation in Ca2+/EGTA BUFFERS bovine GCAP1 on its interaction with the target enzyme and the Ca2+/EGTA mixtures were prepared according to Tsien and + ability to “sense” Ca2 . Pozzan (1989), protocol and verified with Ca2+ fluorescent

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indicator dyes as described previously (Peshenko and Dizhoor, E. coli in comparison with the myristoylated GCAP1 (Figure 1B). 2006). The free metal concentrations in assays containg 2 mM Both myristoylated and non-myristoylated protein were pro- Ca2+/EGTAbufferwerecalculatedusingBoundandDetermined duced using D6S variant of GCAP1, required for high-efficiency and MaxChelator software with proper corrections for pH, salt myristoylation in BLR(DE3) E. coli strain harboring yeast myris- and nucleotide concentrations, and temperature. toyl transferase (Dizhoor et al., 1998; Krylov et al., 1999). To ensurethecompletelackofmyristoylation,theN-terminalGly2 + Ca2 BINDING ASSAY (Figure 1A) replaced with Ala prevents recognition of GCAP1 by + Ca2 binding isotherms were obtained using previously described NMT (Otto-Bruc et al., 1997). Molecular masses of the purified modification of a fluorescent indicator dye titration approach non-myristoylated G2A GCAP1 and myristoylated GCAP1 (Peshenko and Dizhoor, 2006). Briefly, each GCAP1 was diluted verified by ESI-QTOF mass-spectrometry matched, within one from 300–350 μm stock solution to 20–40 μm final concen- mass unit accuracy, their predicted molecular masses (23,365 and tration in 0.6 ml of 100 mm MOPS/KOH (pH 7.2), 40 mM 23,561, respectively). Consistently with the previously reported KCl, 1 mM dithiothreitol, and 0.5 μM BAPTA 2 (Molecular observations using native photoreceptor membranes (Otto-Bruc Probes/Invitrogen). The mixture assembled in a plastic cuvette et al., 1997; Hwang and Koch, 2002), the G2A GCAP1 stimulated ◦ was titrated at 23 C with addition of 3 μlaliquotsofcalibrated the activity of the recombinant RetGC1 expressed in HEK293 CaCl2 solution. cells much less efficiently (Figure 1B). The dose-dependence of the RetGC activation suggests that the GUANYLYL CYLASE ASSAYS binding of the G2A GCAP1 to its target enzyme is impeded. It RetGC activity was assayed as described previously (Peshenko needs to be emphasized that GCAP binding to RetGC cannot be and Dizhoor, 2007; Peshenko et al., 2011). Briefly, the assay mix- measured directly, because detergents required for extraction of ◦ ture (25 μl) incubated at 30 Ccontained30mMMOPS–KOH RetGC from the membranes inactivate GCAP/RetGC interaction + (pH 7.2), 60 mM KCl, 4 mM NaCl, 1mM DTT, 2 mM Ca2 /EGTA (Koch, 1991; Lambrecht and Koch, 1992). We, therefore, tested + buffer, 1 mM free Mg2 , 0.3 mM ATP, 4 mM cGMP, 10 mM crea- GCAP/RetGC complex formation using previously described tine phosphate, 0.5 unit of creatine phosphokinase, 1 mM GTP, semi-quantitative analysis of GCAP1-GFP co-localization with 1 μCi of [α-32P]GTP, 0.1 μCi of [8–3H]cGMP (Perkin Elmer), RetGC in HEK293 cells (Peshenko et al., 2008)(Figure 2). PDE6 inhibitors zaprinast, and dipyridamole. The resultant GCAP1-GFP expressed in the absence of the target enzyme [32P]cGMP product and the [3H]cGMP internal standard was (Figure 2A) distributes in a diffuse pattern throughout the cell, analyzed by TLC using fluorescently backed polyethyleneimine with similar intensity of the fluorescence in the cytoplasm and cellulose plates (Merck) developed in 0.2 M LiCl. the nucleus (with the exception of the nucleoli and vacuoles) (Peshenko et al., 2008). The first EF-hand in GCAP1 and GCAP2 EXPRESSION OF RetGC1 IN HEK293 CELLS does not bind Ca2+, but has been implicated in target recogni- ◦ HEK293 cells grown at 37 C, 5% CO2, in high-glucose Dulbecco’s tion/binding instead (Ermilov et al., 2001; Hwang et al., 2004). modified Eagle medium (DMEM, Invitrogen) supplemented with Therefore, a mutation replacing the conserved CysPro in the 10% fetal bovine serum (Invitrogen) were transfected using the EF-hand 1 loop with Gly prevents RetGC stimulation (Hwang + Ca2 -phosphate method (a Promega Profection protocol) with et al., 2004). This mutation results in a diffuse pattern of GCAP1- 40 μg per 100 μm culture dish of pRCCMV plasmid coding for GFP, even when it is co-expressed with RetGC1 (Figure 2B). human RetGC1, and the membranes containing recombinant In a sharp contrast, wild type GCAP1-GFP is co-localizes to RetGC1 were isolated as previously described in detail (Peshenko the membranes containing co-expressed RetGC1 (Figure 2C), et al., 2004). thus producing a “tennis racquet” membrane localization pattern specific for GCAP1/RetGC1 binding, as was previously demon- CO-EXPRESSION OF RetGC1 AND GCAP1 IN HEK 293 CELLS AND strated (Peshenko et al., 2008, 2011). The G2A GCAP1-GFP CONFOCAL LASER SCANNING MICROSCOPY co-localization with RetGC1 was generally similar to that of wild Fluorescently tagged GCAP1 was co-expressed in HEK293 cells type GCAP1, although not as sharply defined as for the wild with human RetGC1 as previously described (Peshenko et al., type (Figure 2D). This indicates that the lack of myristoylation 2008, 2010).Cellsgrownin2cm2 cover slip chambers were per se does not block RetGC/GCAP interaction, consistently with typically transfected with a mixture of 3 μg pRCCMV plas- the activity assay in Figure 1 and the earlier observations by mid harboring RetGC1 cDNA and 0.02 μg of the GCAP1-GFP Hwang and Koch (2002)andDell’Orco et al. (2010). However, a pQBI25fN3 plasmid. In 24 h the cells were either viewed directly semi-quantitative analysis performed on a large number of cells or fixed with freshly prepared 4% paraformaldehyde at room reveals that the efficiency of the complex formation has been temperature for subsequent immunostaining. The images were reduced (Figures 2E,F). The quantification analysis is based on collected using FV1000 Spectral laser confocal system and ana- thedecreaseofthefluorescenceintensityinthenucleusrelativeto lyzed using Olympus FluoView FV10-ASW software as previously the cell membranes, caused by absorption of the tagged GCAP1 described (Peshenko et al., 2008). by RetGC1 expressed in the membranes (Peshenko et al., 2008, 2011). In the absence of RetGC, the ratio of the GCAP1-GFP RESULTS AND DISCUSSION compartmentalization between the cytoplasm and the nucleus We tested the RetGC1—stimulating activity of a non- (excluding nucleoli) for average cell is close to 1, but in the pres- myristoylated recombinant bovine G2A GCAP1 produced in ence of RetGC1 it drastically, almost 14-fold (Figure 2), increases

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FIGURE 2 | The effect of myristoylation on co-localization of GCAP1 with GCAP1-GFP, b—anti-RetGC1 immunofluorescence of AlexaFluor 568, RetGC in HEK293 cells. (A) GCAP1-GFP expressed in HEK293 cells without c—GCAP1 GFP (green) and anti-RetGC1 (red) fluorescence profile RetGC1 produces diffuse pattern spreading over the cytoplasm and the recorded across the cell along the white line shown in “b”; the nuclei in nucleus (Peshenko et al., 2008); a—fluorescence of GCAP1-GFP, b—same, “c” were counterstained with TO-PRO3 (pseudo-blue). (D) Same as C, but superimposed on DIC image of the cells, c—GCAP1 GFP fluorescence but using G2A GCAP1-GFP mutant. (E) Distribution of GCAP1-GFP profile recorded across the cell along the black line in “b.” (B) GCAP1 GFP fluorescence between the membranes and the nucleus quantified as mutant, in which a conserved CysPro pair in EF-hand 1 loop required for described in (Peshenko et al., 2008); each data point corresponds to an interaction with RetGC is replaced by Gly (Hwangetal.,2004), was individual cell; ◦—GCAP1-GFP expressed alone, •—GCAP1-GFP co-expressed with RetGC1 tagged at the N-terminus with mOrange co-expressed with RetGC1, •—G2A GCAP1-GFP co-expressed variant of red fluorescent protein using protocol described in (Peshenko et al., with RetGC1. (F) The GCAP1-GFP fluorescence distribution ratio (mean ± 2008, 2011); notice that the diffuse pattern of GCAP1-GFP persists despite SEM) averaged from panel E demonstrates that the G2A GCAP1 mutant the presence of RetGC1. (C) Membrane localization of wild type compartmentalizes with the RetGC1, although less efficiently than the GCAP1-GFP (green) co-expressed with RetGC1 (red); a—fluorescence of wild type.

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in favor of the membrane localization (Peshenko et al., 2008). microcalorimetric pattern of heat release by GCAP1 in response Same analysis for the G2A GCAP1 yields more modest average to Ca2+ binding (Lim et al., 2009). However, using the Ca2+ value of 6-fold. Therefore, although myristoylation is not essential binding isotherm (such as in Peshenko and Dizhoor (2006) for the RetGC/GCAP binding, it evidently improves the efficiency and this study) is a more reliable approach to determine the ofthecomplexformation.Atthesametime,thelowerlevel actual binding constants, because, unlike microcalorimetry, it of activation of the cyclase in Figure 1B as compared with the is not affected by a complex relationship between heat release less prominently decreased co-localization efficiency in Figure 2F and absorption resulting from Ca2+ binding and conformational implies that there is additional effect of myristoylation—it has changes in the GCAP1 molecule (Lim et al., 2009). to be involved in the creation of the conformation required To verify that our analysis did not simply fail to reveal a for the optimal cyclase stimulation within the GCAP/RetGC major difference between the two forms of GCAP1 due to an complex. It also needs to be noted that the attachment of insufficient resolution of the Ca2+ binding assay, we also tested the C-terminal GFP tag per se does not affect the regulatory Ca2+ binding by non-acylated GCAP1 mutants in which we properties of GCAP1 as the Ca2+ sensor of guanylyl cyclase inactivated one (EF-4) or two (EF-3 and EF-4) EF-hands using the (Peshenko et al., 2008). D144N/D148G (EF4−) and the D100N/D102G/D144N/D148G The effect of N-myristoylation in GCAP1 on the cyclase bind- (EF3,4−) substitutions, respectively, (Peshenko and Dizhoor, ing and activation could aprioriresult from stabilizing effect of 2006). In both cases, we clearly observed the corresponding + the fatty chain buried inside the protein structure (Stephen et al., decrease of Ca2 binding by one (Figure 3C)ortwo(Figure 3D) + 2007), such that in the absence of myristoylation GCAP1 becomes mol Ca2 per mol GCAP1, respectively. So we find no indications more easily misfolded. Previous analysis of the non-acylated that any of the three metal binding EF-hands in the non-acylated GCAP1 by circular dichroism spectra (Dell’Orco et al., 2010) GCAP1 undergo possible misfolding severely affecting binding of + argues that the metal-bound non-myristoylated protein is fairly Ca2 . Therefore, the general fold of GCAP1 is highly unlikely stable, at least at the level of the secondary structure. On the other to be critically compromised by the absence of the myristoyl hand, the Ca2+ binding isotherms (Dell’Orco et al., 2010)also group. It is much more likely that the fatty acyl group changes suggested that at least one of the EF-hands in non-myristoylated GCAP1 structure in a fairly subtle manner, only slightly affecting + + GCAP1 became unable to bind Ca2+ within submicromolar Ca2+ Ca2 binding within the physiological free Ca2 range. + range. Hence, one could not rule out the possibility of partial mis- ThereasonforthedifferenceintheCa2 binding affinities folding of GCAP1 three-dimensional structure. However, the data in our study and that by Dell’Orco et al. (2010) is not immedi- in Figure 3 argue that under the conditions of our experiments ately apparent. The trace amount of EDTA (∼1:20 molar ratio + the G2A GCAP1 is affected in a very subtle manner that merely to GCAP1, or 1:60 per mol of Ca2 binding sites) was neg- results in a slight change of its Ca2+ sensitivity, rather than critical ligible in our assays and could not skew the results by more loss of Ca2+ binding affinity in any of the three metal-binding EF- than few percent [which also would be toward lower, contrary hands. The fluorescent indicator dye titration analysis (Peshenko to the higher than in Dell’Orco et al. (2010), average affinity in and Dizhoor, 2006) shows that the G2A GCAP1 has a normal sto- our experiments (Figure 3B)].Inthemethodweuse(Peshenko ichiometry of Ca2+ binding—three per molecule, saturating at and Dizhoor, 2006), the fluorescent chelator indicator was also low micromolar range of free Ca2+ (Figure 3B). present at a very low (∼1:60–1:40) molar ratio to GCAP1 (as low + The overall apparent affinity of the G2A GCAP1 for Ca2+ as 1:180–1:120 per mol of Ca2 binding sites), quite negligible in our experiments (Figure 3B) was markedly better than we in comparison with the calcium buffer capacity of GCAP1 itself could expect for the non-myristoylated GCAP1 based on previ- and ∼40–60 times lower than in the method used by Dell’Orco ous reports. The respective macroscopic equilibrium constants, et al. (2010). This may have prevented some potential effect of the K1, K2,andK3, derived from the three-center binding model chelator dye itself on the non-myristoylated protein. Alternatively, (Figure 3B)were2.0 × 107,1.7 × 107,and3.2 × 106 M−1 for the the difference could be related to the use of the protein purifica- myristoylated and 1.5 × 107,1.0 × 107,and2.2 × 106 M−1 for tion protocol that more efficiently removes poorly folded fraction the non-acylated G2A GCAP1. These values substantially devi- of low-affinity GCAP1 in our study. ate from those observed by Dell’Orco et al. (2010)—2.5 × 108, Despite the substantial quantitative difference, we would 3.2 × 107,and7.9 × 107 M−1 for myristoylated versus 6.3 × 107, nonetheless emphasize that we have a qualitative agreement + 5.0 × 106,and2.0 × 103 M−1 for non-myristoylated GCAP1. with the findings by Dell’Orco et al. (2010)—Ca2 -sensitivity The binding isotherm in our study (solid line) looks substantially of the G2A GCAP1 is reduced compared to the myristoylated different from the isotherm “reconstituted” using the previously form (Figure 3A). Even though the binding ratio at saturation reported (Dell’Orco et al., 2010)constants(dashed line)—both remained three Ca2+ ions per GCAP1, there was a slight shift of reach the same binding stoichiometry, but in very different free the curve toward higher micromolar free Ca2+ range for the non- Ca2+ range. Based on our results, we can positively state that not myristoylated GCAP1. Evidently, it is the lack of myristoylation only the stoichiometry of Ca2+ binding does not change in the what alters the properties of GCAP1, not the G2A mutation per se non-myristoylated GCAP1, but it also reaches saturation within (Figure 3A). nearly the same low micromolar range as myristoylated protein, From the overall shift in Ca2+ binding affinity in non-acylated with only a slight right-shift of the binding curve (Figures 3A,B). GCAP1 (Figure 3A), the result shown in Figure 3D appears most It needs to be mentioned that indirect evaluation of Ca2+ bind- intriguing. The affinity of EF-hand 2, the only metal-binding site ing affinities was also performed previously using a complex left in the non-acylated D100N/D102G/D144N/D148G GCAP1

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FIGURE 3 | Ca2+ sensitivity of GCAP1 is affected by myristoylation. constants; panels (C,D)—by simplified hyperbolic saturation function, 2+ (A–D),Ca binding isotherms obtained using fluorescent indicator dye (Cabound/GCAP) = Bmax× Cafree/(Cafree + Kd ), where Cabound is the 2+ 2+ BAPTA-2 titration protocol (Peshenko and Dizhoor, 2006). (A) Ca concentration of Ca bound to GCAP1, calculated as Cabound = Catotal – 2+ binding by myristoylated D6S GCAP1 (◦), non-myristoylated Cafree, Bmax is mol of Ca bound per mol of GCAP1 at saturation, WT GCAP1 (•), and non-myristoylated G2A GCAP1 (). (B) Comparison of Kd is the apparent dissociation constant. The data shown are representative the experimental data for Ca2+ binding by G2A GCAP1 () with the from 3 to 5 independent experiments producing virtually identical results. theoretical curve for three-center binding model calculated using previously (E) Normalized activity of the recombinant RetGC1 expressed in HEK293 reported macroscopic association constants, 6.3 × 107,5.0 × 106,and cells reconstituted with 10 μM purified myristoylated GCAP1 (◦)orG2A 2.0 × 103 M−1 (Dell’Orco et al., 2010) (- - -); the corresponding dissociation GCAP1 (•) at different free Ca2+ concentrations and 1 mm free Mg2+.The constants are shown next to each trace. (C,D) Change of the binding activities in each series were normalized by the maximal activity in the stoichiometry in non-myristoylated GCAP1 with one (D144N/D148G, C)or corresponding series. The data were fitted by the equation, n two (D100N/D102G/D144N/D148G, D) EF-hands inactivated. The data were A = Amax + (Amax − Amin)/(1 + (Cafree/Ca1/2) ), where A is RetGC activity, 2+ fitted using two different models: panels (A,B)—by three-center binding Ca1/2 is the free Ca concentration producing 50% effect and n is the = ( + 2 + 3)/( + + model, Cabound/GCAP K1Caf 2K1K2Caf 3K1K2K3Caf 1 K1Caf Hill coefficient. For other conditions of the assay see Materials and 2 + 3 , K1K2Caf K1K2K3Caf ), where K1 K2,andK3 are macroscopic equilibrium Methods.

mutant (Figure 3D), remains high (Kd ∼ 0.04 μm ± 0.01), vir- In our previous studies (Peshenko and Dizhoor, 2007; tually the same as in the myristoylated D100N/D102G/D144N/ Peshenko et al., 2008), we identified the EF-hand 3/EF-hand D148G mutant [0.03 ± 0.01 μm, Peshenko and Dizhoor (2006)]. 4 portion of the molecule as critical for the Ca2+-dependent In other words, myristoylation appears to have a surprisingly activator-to-inhibitor transition of GCAP1. In a good qualita- little effect on the high-affinity Ca2+ binding in the EF-hand 2 tive agreement with the earlier observations of Hwang and Koch proximal to the fatty acyl group in the three-dimensional struc- (2002) and the shift in the Ca2+-binding isotherm to the higher ture of GCAP1 (Figure 1). Instead of affecting metal binding in Ca2+ range (Figure 3A), the G2A GCAP1 regulated recombi- 2+ the neighboring EF-hand 2, the myristoyl moiety, in a rather nant RetGC1 with a noticeably lower sensitivity to Ca [(Ca)1/2 paradoxical manner, seemingly influences the efficiency of Ca2+ increased from 260 nM in wild type to 520 nM in the G2A mutant binding in a more distal portion of the molecule (EF-hand 3 or 4). (Figure 3E)]. Since the high-affinity binding in EF-hand 2 does

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not seem to be critically affected by myristoylation (Figure 3D), Hwang et al., 2004), but also inﬂuences Ca2+ binding efﬁcacy in + the observed changes in Ca2 sensitivity of RetGC1 (Figure 3E) a more distal part of the molecule through a presently unknown are, again, likely attributable to the change in the function of EF- and somewhat paradoxical mechanism. The possibility that the hand 3 and/or 4. It also needs to be pointed that even though the intramolecular interactions exist between the myristoyl moiety + decrease in Ca2 sensitivity of RetGC regulation by GCAP1 is rel- and the remote portions of the GCAP1 structure also suggests atively modest, ∼2-fold, a shift like this can be large enough to that such interaction can be affected by the environment of the + adversely affect the levels of free cGMP and Ca2 in the dark and myristoyl group and deserves closer in-depth study. This study provoke photoreceptor death in transgenic mice (Olshevskaya is currently in progress and could provide a new insight into et al., 2004; Woodruff et al., 2007). Therefore, the role of myris- the structure-function relationships underlying the Ca2+ sensor + toylation in maintaining normal Ca2 sensitivity of GCAP1 as a action of GCAP1 as a member of the NCS protein group. Ca2+ sensor should be rather important for its normal physiological function. ACKNOWLEDGMENTS Our results indicate that myristoyl residue inside the GCAP1 This work was supported by the NIH grant EY11522 from NEI. molecule affects proximal structural element(s) involved in the Alexander M. Dizhoor is the Martin and Florence Hafter Chair target enzyme recognition, such as EF-1 (see Ermilov et al., 2001; Professor of Pharmacology.

REFERENCES terminusofrecoverininCa(2+)- required for targeting photorecep- of three novel GCAPs (GCAP4, Ames, J. B., Dizhoor, A. M., Ikura, dependent membrane interaction. tor guanylyl cyclase. J. Biol. Chem. GCAP5, GCAP7) from zebrafish M., Palczewski, K., and Stryer, L. Science 259, 829–832. 276, 48143–48148. (Danio rerio) and prediction of (1999). Three-dimensional struc- Dizhoor, A. M., Ericsson, L. H., Fu, Y., and Yau, K. W. (2007). eight GCAPs (GCAP1–8) in puffer- ture of guanylyl cyclase activat- Johnson, R. S., Kumar, S., Phototransduction in mouse fish (Fugu rubripes). J. Mol. Evol. 59, ing protein-2, a calcium-sensitive Olshevskaya, E. V., Zozulya, S., rods and cones. Pflugers Arch. 454, 204–217. modulator of photoreceptor guany- Neubert, T. A., Stryer, L., Hurley, 805–819. Johnson, R. S., Ohguro, H., Palczewski, lyl cyclases. J. Biol. Chem. 274, J. B., and Walsh, K. A. (1992). The Gorczyca,W.A.,Gray-Keller,M.P., K.,Hurley,J.B.,Walsh,K.A., 19329–19337. NH2 terminus of retinal recoverin Detwiler, P. B., and Palczewski, K. and Neubert, T. A. (1994). Ames, J. B., Ishimam, R., Tanaka, T., is acylated by a small family of (1994). Purification and physiolog- Heterogeneous N-acylation is a Gordon, J. I., Stryer, L., and Ikura, fatty acids. J. Biol. Chem. 267, ical evaluation of a guanylate cyclase tissue- and species-specific post- M. (1997). Molecular mechanics of 16033–16036. activating protein from retinal rods. translational modification. J. Biol. calcium-myristoyl switches. Nature Dizhoor, A. M., and Hurley, J. B. Proc. Natl. Acad. Sci. U.S.A. 91, Chem. 269, 21067–21071. 389, 198–202. (1996). Inactivation of EF-hands 4014–4018. Koch, K. W. (1991). Purification and Ames,J.B.,Tanaka,T.,Ikura,M.,and makes GCAP-2 (p24) a constitutive Gray-Keller, M. P., and Detwiler, P. identification of photoreceptor Stryer, L. (1995). Nuclear magnetic activator of photoreceptor guanylyl B. (1994). The calcium feedback guanylate cyclase. J. Biol. Chem. resonance evidence for Ca(2+)- cyclase by preventing a Ca2+- signal in the phototransduction 266, 8634–8637. induced extrusion of the myristoyl induced “activator-to-inhibitor” cascade of vertebrate rods. Neuron Koch, K. W., and Stryer, L. (1988). group of recoverin. J. Biol. Chem. transition. J. Biol. Chem. 271, 13, 849–861. Highly cooperative feedback con- 270, 30909–30913. 19346–19350. Hughes, R. E., Brzovic, P. S., Dizhoor, trol of retinal rod guanylate cyclase Burgoyne, R. D. (2007). Neuronal Dizhoor, A. M., Lowe, D. G., A.M.,Klevit,R.E.,andHurley,J.B. by calcium ions. Nature 334, calcium sensor proteins, generat- Olshevskaya, E. V., Laura, R. P., (1998). Ca2+-dependent conforma- 64–66. ing diversity in neuronal Ca2+ and Hurley, J. B. (1994). The tional changes in bovine GCAP-2. Krylov, D. M., Niemi, G. A., Dizhoor, signalling. Nat. Rev. Neurosci. 8, human photoreceptor membrane Protein Sci. 7, 2675–2680. A. M., and Hurley, J. B. (1999). 182–193. guanylyl cyclase, RetGC, is present Hwang, J. Y., and Koch, K. W. (2002). Mapping sites in guanylyl cyclase Burns,M.E.,Mendez,A.,Chen,J.,and in outer segments and is regulated Calcium- and myristoyl-dependent activating protein-1 required for Baylor, D. A. (2002). Dynamics of by calcium and a soluble activator. properties of guanylate cyclase- regulation of photoreceptor mem- cyclic GMP synthesis in retinal rods. Neuron 12, 1345–1352. activating protein-1 and protein-2. brane guanylyl cyclases. J. Biol. Neuron 36, 81–91. Dizhoor, A. M., Olshevskaya, E. V., Biochemistry 41, 13021–13028. Chem. 274, 10833–10839. Dell’Orco, D., Behnen, P., Linse, S., and Henzel,W.J.,Wong,S.C.,Stults, Hwang, J. Y., Lange, C., Helten, A., Laura, R. P., Dizhoor, A. M., and Koch, K. W. (2010). Calcium bind- J. T., Ankoudinova, I., and Hurley, Hoppner-Heitmann, D., Duda, T., Hurley, J. B. (1996). The membrane ing, structural stability and guany- J. B. (1995). Cloning, sequenc- Sharma, R. K., and Koch, K. W. guanylyl cyclase, retinal guanylyl late cyclase activation in GCAP1 ing, and expression of a 24-kDa (2003). Regulatory modes of rod cyclase-1, is activated through its variants associated with human Ca(2+)-binding protein activating outer segment membrane guany- intracellular domain. J. Biol. Chem. cone dystrophy. Cell. Mol. Life Sci. photoreceptor guanylyl cyclase. late cyclase differ in catalytic effi- 271, 11646–11651. 67, 973–984. J. Biol. Chem. 270, 25200–25206. ciency and Ca2+-sensitivity. Eur. J. Lim, S., Peshenko, I., Dizhoor, A., Dizhoor, A. M., Boikov, S. G., Dizhoor, A. M., Olshevskaya, E. V., and Biochem. 270, 3814–3821. and Ames, J. B. (2009). Effects and Olshevskaya, E. V. (1998). Peshenko, I. V. (2010). Mg2+/Ca2+ Hwang, J. Y., Schlesinger, R., and Koch, of Ca2+, Mg2+, and myristoyla- Constitutive activation of pho- cation binding cycle of guanylyl K. W. (2004). Irregular dimeriza- tion on guanylyl cyclase activat- toreceptor guanylate cyclase by cyclase activating proteins (GCAPs), tion of guanylate cyclase-activating ing protein 1 structure and stability. Y99C mutant of GCAP-1. Possible role in regulation of photoreceptor protein1mutantscauseslossoftar- Biochemistry 48, 850–862. role in causing human autosomal guanylyl cyclase. Mol. Cell. Biochem. get activation. Eur. J. Biochem. 271, Lim,S.,Strahl,T.,Thorner,J.,and dominant cone degeneration. J. 334, 117–124. 3785–3793. Ames, J. B. (2011). Structure of Biol. Chem. 273, 17311–17314. Ermilov, A. N., Olshevskaya, E. V., Imanishi, Y., Yang, L., Sokal, I., Filipek, a Ca2+-myristoyl switch pro- Dizhoor, A. M., Chen, C. K., and Dizhoor, A. M. (2001). Instead S., Palczewski, K., and Baehr, W. tein that controls activation of a Olshevskaya, E. V., Sinelnikova, of binding calcium, one of the (2004). Diversity of guanylate phosphatidylinositol 4-kinase in V., Phillipov, P., and Hurley, J. B. EF-hand structures in guanylyl cyclase-activating proteins (GCAPs) fission yeast. J. Biol. Chem. 286, (1993). Role of the acylated amino cyclase activating protein-2 is in teleost fish, characterization 12565–12577.

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Lambrecht, H. G., and Koch, K. W. Palczewski, K., Subbaraya, I., Gorczyca, EF-hands. J. Biol. Chem. 283, Tsien, R., and Pozzan, T. (1989). (1992). Recoverin, a novel calcium- W. A., Helekar, B. S., Ruiz, C. 21747–21757. Measurement of cytosolic free Ca2+ binding protein from vertebrate C.,Ohguro,H.,Huang,J.,Zhao, Peshenko, I. V., Olshevskaya, E. with quin2. Methods Enzymol. 172, photoreceptors. Biochim. Biophys. X.,Crabb,J.W.,Johnson,R.S., V.,Savchenko,A.B.,Karan,S., 230–262. Acta 1160, 63–66. Walsh, K. A., Gray-Keller, M. Palczewski,K.,Baehr,W.,and Woodruff, M. L., Olshevskaya, E. V., Makino,C.L.,Peshenko,I.V.,Wen, P., Detwiller, P. B., and Baehr, Dizhoor, A. M. (2011). Enzymatic Savchenko, A. B., Peshenko, I. V., X.-H., Elena, V., Olshevskaya, E. W. (1994). Molecular cloning properties and regulation of the Barrett, R., Bush, R. A., Sieving, P. V., Barrett, R., and Dizhoor, A. and characterization of retinal native isozymes of retinal mem- A., Fain, G. L., and Dizhoor, A. M. (2008). A role for GCAP2 photoreceptor guanylyl cyclase- brane guanylyl cyclase (RetGC) M. (2007). Constitutive excitation in regulating the photoresponse: activating protein. Neuron 13, from mouse photoreceptors. by Gly90Asp rhodopsin rescues rods guanylyl cyclase activation and 395–404. Biochemistry 50, 5590–5600. from degeneration caused by ele- rod electrophysiology in GUCA1B Peshenko, I. V., and Dizhoor, A. M. Peshenko, I. V., Olshevskaya, E. V., Yao, vated production of cGMP in the knockout mice. J. Biol. Chem. 283, (2004). Guanylyl cyclase-activating S.,Ezzeldin,H.H.,Pittler,S.J.,and dark. J. Neurosci. 27, 8805–8815. 29135–29143. proteins (GCAPs) are Ca2+/Mg2+ Dizhoor, A. M. (2010). Activation of Woodruff,M.L.,Sampath,A.P., Mendez, A., Burns, M. E., Sokal, sensors: implications for photore- retinal guanylyl cyclase RetGC1 by Matthews, H. R., Krasnoperova, N. I., Dizhoor, A. M., Baehr, W., ceptor guanylyl cyclase (RetGC) GCAP1: stoichiometry of binding V., Lem, J., and Fain, G. L. (2002). Palczewski,K.,Baylor,D.A.,and regulation in mammalian pho- and effect of new LCA-related muta- Measurement of cytoplasmic cal- Chen, J. (2001). Role of guanylate toreceptors. J. Biol. Chem. 279, tions. Biochemistry 49, 709–717. cium concentration in the rods of cyclase-activating proteins (GCAPs) 16903–16906. Pugh,E.N.Jr.,Duda,T.,Sitaramayya, wild-type and transducin knock- in setting the flash sensitivity of rod Peshenko, I. V., and Dizhoor, A. M. A., and Sharma, R. K. (1997). out mice. J. Physiol. 542, 843–854. photoreceptors. Proc. Natl. Acad. (2006). Ca2+ and Mg2+ binding Photoreceptor guanylate cyclases: a Zozulya, S., and Stryer, L. (1992). Sci. U.S.A. 98, 9948–9953. properties of GCAP-1. Evidence that review. Biosci. Rep. 17, 429–473. Calcium-myristoyl protein switch. Olshevskaya, E. V., Calvert, P. D., Mg2+-bound form is the physio- Pugh,E.N.Jr.,Nikonov,S.,andLamb, Proc. Natl. Acad. Sci. U.S.A. 89, Woodruff, M. L., Peshenko, I. V., logical activator of photoreceptor T. D. (1999). Molecular mechanisms 11569–11573. Savchenko, A. B., Makino, C. L., Ho, guanylyl cyclase. J. Biol. Chem. 281, of vertebrate photoreceptor light Y. S., Fain, G. L., and Dizhoor, A. 23830–23841. adaptation. Curr. Opin. Neurobiol. 9, M. (2004). The Y99C mutation in Peshenko, I. V., and Dizhoor, A. M. 410–418. Conflict of Interest Statement: The guanylyl cyclase-activating protein (2007). Activation and inhibition Sakurai, K., Chen, J., and Kefalov, V. authors declare that the research 1 increases intracellular Ca2+ and of photoreceptor guanylyl cyclase J. (2011). Role of guanylyl cyclase was conducted in the absence of any causes photoreceptor degeneration by guanylyl cyclase activating pro- modulation in mouse cone pho- commercial or financial relationships in transgenic mice. J. Neurosci. 24, tein 1 (GCAP-1): the functional totransduction. J. Neurosci. 31, that could be construed as a potential 6078–6085. role of Mg2+/Ca2+ exchange in 7991–8000. conflict of interest. Olshevskaya, E. V., Hughes, R. E., EF-hand domains. J. Biol. Chem. Sampath, A. P., Matthews, H. R., Hurley, J. B., and Dizhoor, A. 282, 21645–21652. Epub 22007 Jun Cornwall, M. C., Bandarchi, J., and M. (1997). Calcium binding, but 21641. Fain, G. L. (1999). Light-dependent Received: 09 January 2012; accepted: not a calcium-myristoyl switch, Peshenko, I. V., Moiseyev, G. P., changes in outer segment free-Ca2+ 09 February 2012; published online: 22 controls the ability of guanylyl Olshevskaya, E. V., and Dizhoor, A. concentration in salamander cone February 2012. cyclase-activating protein GCAP-2 M. (2004). Factors that determine photoreceptors. J. Gen. Physiol. 113, Citation: Peshenko IV, Olshevskaya EV to regulate photoreceptor guany- Ca2+ sensitivity of photoreceptor 267–277. and Dizhoor AM (2012) Interaction of lyl cyclase. J. Biol. Chem. 272, guanylyl cyclase. Kinetic analysis Stephen, R., Bereta, G., Golczak, M., GCAP1 with retinal guanylyl cyclase and 14327–14333. of the interaction between the Palczewski,K.,andSousa,M.C. calcium: sensitivity to fatty acylation. Otto-Bruc, A., Buczylko, J., Ca2+-bound and the Ca2+-free (2007). Stabilizing function for Front. Mol. Neurosci. 5:19. doi: 10.3389/ Surgucheva, I., Subbaraya, I., guanylyl cyclase activating pro- myristoyl group revealed by the crys- fnmol.2012.00019 Rudnicka-Nawrot, M., Crabb, J. teins (GCAPs) and recombinant tal structure of a neuronal calcium Copyright © 2012 Peshenko, W.,Arendt,A.,Hargrave,P.A., photoreceptor guanylyl cyclase sensor, guanylate cyclase-activating Olshevskaya and Dizhoor. This is Baehr, W., and Palczewski, K. 1(RetGC-1).Biochemistry 43, protein 1. Structure 15, 1392–1402. an open-access article distributed under (1997). Functional reconstitution 13796–13804. Stephen, R., Palczewski, K., and Sousa, the terms of the Creative Commons of photoreceptor guanylate cyclase Peshenko, I. V., Olshevskaya, E. V., and M. C. (2006). The crystal struc- Attribution Non Commercial License, with native and mutant forms Dizhoor, A. M. (2008). Binding of ture of GCAP3 suggests molecular which permits non-commercial use, of guanylate cyclase-activating guanylyl cyclase activating protein 1 mechanism of GCAP-linked cone distribution, and reproduction in other protein 1. Biochemistry 36, (GCAP1) to retinal guanylyl cyclase dystrophies. J. Mol. Biol. 359, forums, provided the original authors 4295–4302. (RetGC1). The role of individual 266–275. and source are credited.

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