Regulation of the methylation status of G protein-coupled receptor kinase 1 (rhodopsin kinase)

Cellular Signalling 24 (2012) 2259–2267

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Regulation of the methylation status of G protein-coupled receptor kinase 1 (rhodopsin kinase) Mikhail A. Kutuzov a, b,⁎, Alexandra V. Andreeva c,⁎, Nelly Bennett a, 1 a b c

Laboratoire de Biophysique Moléculaire & Cellulaire, URA CNRS N°520, Département de Biologie Moléculaire et Structurale, C.E.A.—Grenoble, 38054 Grenoble Cedex 9, France. Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow, Russia School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom

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Article history: Received 30 May 2012 Received in revised form 9 July 2012 Accepted 24 July 2012 Available online 28 July 2012 Keywords: CAAX moif Calcium signalling Light adaptation Carboxymethylation Neuronal calcium sensors Visual phototransduction

a b s t r a c t Rhodopsin kinase (GRK1) is a member of G protein-coupled receptor kinase family and a key enzyme in the quenching of photolysed rhodopsin activity and desensitisation of the rod photoreceptor neurons. Like some other rod proteins involved in phototransduction, GRK1 is posttranslationally modified at the C terminus by isoprenylation (farnesylation), endoproteolysis and α-carboxymethylation. In this study, we examined the potential mechanisms of regulation of GRK1 methylation status, which have remained unexplored so far. We found that considerable fraction of GRK1 is endogenously methylated. In isolated rod outer segments, its methylation is inhibited and demethylation stimulated by low-affinity nucleotide binding. This effect is not specific for ATP and was observed in the presence of a non-hydrolysable ATP analogue AMP-PNP, GTP and other nucleotides, and thus may involve a site distinct from the active site of the kinase. GRK1 demethylation is inhibited in the presence of Ca2+ by recoverin. This inhibition requires recoverin myristoylation and the presence of the membranes, and may be due to changes in GRK1 availability for processing enzymes upon its redistribution to the membranes induced by recoverin/Ca 2+. We hypothesise that increased GRK1 methylation in dark-adapted rods due to elevated cytoplasmic Ca2+ levels would further increase its association with the membranes and recoverin, providing a positive feedback to efficiently suppress spurious phosphorylation of non-activated rhodopsin molecules and thus maximise senstivity of the photoreceptor. This study provides the first evidence for dynamic regulation of GRK1 α-carboxymethylation, which might play a role in the regulation of light sensitivity and adaptation in the rod photoreceptors. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Rod photoreceptor cells are highly specialised neurons that express, at relatively high levels, proteins participating in photoreception. The abundance of these proteins has allowed a detailed investigation of signalling events in vertebrate phototransduction, which has become a textbook example of G protein‐coupled signalling [1–4]. Within rod outer segments (ROS), light-activated G protein‐coupled receptor rhodopsin activates a heterotrimeric G protein transducin, which in turn stimulates cGMP-specific phosphodiesterase PDE6. cGMP hydrolysis results in the closure of cGMP-gated cation channels, resulting in

Abbreviations: GRK, G protein‐coupled receptor kinase; ICME, isoprenylcysteine carboxyl methyltesterase; ICMT, isoprenylcysteine carboxyl methyltransferase; SAM, S-adenosyl-L-methionine. ⁎ Corresponding authors at: Department of Medicine, Northwestern University, 240 E. Huron St., M-300, Chicago, IL 60611, USA. Tel.: +1 312 545 5510; fax: +1 312 908 4650. E-mail addresses: [email protected] (M.A. Kutuzov), [email protected] (A.V. Andreeva). 1 Deceased. 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2012.07.020

hyperpolarisation. The activity of photoexcited rhodopsin is terminated by its phosphorylation by rhodopsin kinase (GRK1), a member of the family of G protein‐coupled receptor kinases (GRK) [5]. Phosphorylated rhodopsin is recognised by arrestin, which binds to rhodopsin and impedes further transducin activation. Closure of cation channels and continuing activity of the Na+/Ca2+ exchanger in the plasma membrane results in a decrease in cytoplasmic Ca2+ concentrations. This drop in Ca2+ is sensed by several mechanisms that regulate the speed of signal termination and light sensitivity of the cell [6–8]. One of these mechanisms relies on Ca 2+-dependent inhibition of GRK1 by an EF-hand Ca2+-binding protein recoverin [9–11]. In the dark, GRK1 is inhibited by the Ca2+-bound form of recoverin via direct interaction of the two proteins [12–14]; this inhibition is released when the Ca 2 + levels drop as a result of illumination. Several proteins directly involved in phototransduction have a CAAX motif at their C-termini (where A are aliphatic amino acid residues) and undergo a specific posttranslational modification, which includes consecutive isoprenylation (farnesylation or geranylgeranylation) of the Cys residue in the CAAX motif, proteolytic removal of the 3 C-terminal amino acids (AAX), followed by α-carboxymethylation of the

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isoprenylated Cys (reviewed by [15,16]). These proteins include the γ-subunit of transducin [17,18], α- and β-subunits of PDE6 [19–21], and GRK1 [22]. Human genome encodes between 120 and 280 proteins that may undergo similar modifications [23]. The role of isoprenylation/ α-carboxymethylation is thought to consist in enhancing protein association with the membranes, in mediating protein–protein interactions, or regulating protein sorting and degradation [23–26]. The effect of α-carboxymethylation on hydrophobicity and the ability of the modified proteins to associate with the membranes is much more prominent for farnesylated as compared to geranylgeranylated proteins [16,27]. Despite the importance of α-carboxymethylation of isoprenylated proteins, little is known about the molecular mechanisms underlying its regulation. The loss of isoprenylcysteine carboxyl methyltransferase (ICMT), the only enzyme thought to be capable of α-carboxymethylation of isoprenylated proteins, is embryonically lethal in mice [28]. On the other hand, pharmacological ICMT inhibition suppresses oncogenic potential of Ras and Raf mutants, and ICMT inhibitors are considered as promising anti-cancer agents [23]. Whether α-carboxymethylation of isoprenylated proteins is reversible and may potentially serve as a dynamic regulatory mechanism has remained controversial, and the isoprenylcysteine carboxyl methyltesterase (ICME) has not been unambiguously identified in animals. ROS small G proteins and the γ subunit of transducin have been reported to be reversibly methylated [17]. An increase in methylation of unidentified membrane proteins has been documented upon treatment of pre-B lymphocytes with lipopolysaccharide [29] and upon treatment of PC-12 cells with nerve growth factor [30]. An increase in methylation of small G proteins has been observed upon activation of human neutrophils [31] and upon stimulation of endothelial cells with TNFα [32]. The presence of a methylesterase in bovine rods that specifically recognises isoprenylated substrates has been reported [33]. Both soluble and membrane-associated ICME activities have been detected in the brain [34]. At least two ICME enzymes have been described in bovine adrenal medulla [35]. These methylesterases have not been identified at molecular level; mammals do not have close homologues of recently identified plant ICME [36]. A liver ICME activity has been recently ascribed to the endoplasmic reticulumassociated non-specific carboxylesterase [37]. Thus, it seems plausible that different esterases may demethylate isoprenylated proteins in different tissues and cell types. On the other hand, no demethylation of KRas could be detected [38], and it has been proposed that carboxymethylation of isoprenylated proteins may be a constitutive modification [23]. In this work, we assessed how GRK1 methylation can be regulated. We found that the methylation status of GRK1 is affected by nucleotide binding and by the levels of free Ca 2+ via recoverin. These findings provide the first evidence for the mechanisms of regulation of the methylation status of this G protein-coupled receptor kinase.

2.2. Buffers Isotonic buffer: 10 mM Tris‐HCl, pH 7.5; 120 mM KCl; 5 mM MgCl2. Hypotonic buffer: 5 mM HEPES, pH 7.5. Buffers for chromatography: A, 10 mM HEPES, pH 7.5; 0.05% Tween 80; B, buffer A supplemented with 1 M NaCl; C, 10 mM HEPES, pH 7.5; 0.1 M KCl. Prior to use, all buffers were supplemented with 1 mM dithiothreitol and 1 mM benzamidine. Free Ca2+ concentrations were set using CaCl2/EGTA buffers as described in detail previously [39]. 2.3. Preparation of rod outer segments (ROS) Rod outer segments (ROS) were prepared from dark-adapted fresh bovine eyes using modified procedure of Sitaramayya [40] and extracted with the isotonic buffer as described previously [39]. ROS were stored at − 25 °C in the dark in isotonic buffer containing 50% glycerol at 200–250 μM rhodopsin concentration. For some experiments, ROS were washed with hypotonic buffer containing 6 M urea, followed by extensive washes with water. 2.4. Purification of GRK1 ROS pellets were resuspended in the hypotonic buffer at 25–30 μM rhodopsin using a tightly fitting hand-driven glass/teflon homogeniser. Membranes were pelleted (60 Ti Beckman rotor, 55000 rpm for 25 min), and supernatants recentrifuged to remove possible membrane contamination. Hypotonic extracts (50–70 ml) were applied directly at a flow rate of 1 ml/min onto a Fractogel TSK DEAE column (0.5 × 3 cm) equilibrated in buffer A. The column was washed with buffer A until the absorbance at 280 nm returned to baseline. Proteins were eluted with a linear gradient of buffer B (0–300 mM NaCl in 60 min at a flow rate 0.2 ml/min). Fractions containing GRK1 were pooled, diluted 5 fold with the buffer A and further purified on a Heparin Sepharose column (0.5 × 1.5 cm) using the same buffer system. 2.5. Methylation and demethylation of ROS proteins Crude ROS (25–45 μM rhodopsin) in a buffer containing 80 mM HEPES (pH7.5), 85 mM KCl, 3.5 mM MgCl2, 5–10% glycerol were incubated with 0.3 μM [ 3H]SAM at room temperature in the dark. The reaction was stopped after 1–2 h (see figure legends) by addition of 1/3 (v/v) of the electrophoresis sample buffer (20 mM Tris‐HCl, pH 6.8; 25% glycerol; 10% SDS; 0.25 M dithiothreitol). Alternatively, labelling was stopped by addition of 1 mM (final concentration) nonlabelled SAM and the samples were allowed to demethylate for up to 2 h. The reactions were stopped by addition of the electrophoresis sample buffer. 2.6. Analysis of methylated proteins

2. Experimental 2.1. Materials ATP and GTP were obtained from Boehringer Mannheim; AMP-PNP, ADP, AMP, ITP, ebelactone B, dithiothreitol, benzamidine and S-adenosyl-L-methionine (SAM) were from Sigma. Formic acid and Fractogel TSK DEAE-650(S) were from Merck. Trichloroacetic acid and glycerol were from Prolabo. S-Adenosyl-L-[methyl- 3H] methionine ([3H]SAM, 75 Ci/mmol), was from DuPont-NEN. [γ33P] ATP and [ 14C]-labelled molecular weight markers were from Amersham. Phenyl Sepharose CL 4B, Heparin Sepharose and a Superose HR12 prepacked column were from Pharmacia Biotech.

Proteins were separated by SDS-PAGE [41]. Gels were stained with Coomassie R 250 and destained for 4–12 h in 7.5% acetic acid, 25% ethanol. For fluorography, gels were incubated with Amplify (Amersham) for 30 min, vacuum-dried and exposed with XAR-5 film (Kodak) at − 70 °C for 3–7 days. Autoradiograms were scanned and densitometry performed using Image J software (http://rsb.info. nih.gov/ij/). For quantitation of radioactive methyl incorporation, the gels were soaked in water for 30 min to remove acetic acid, the bands of interest were excised and placed into open 1.5 ml microcentrifuge tubes containing 250 μl of 2 M NaOH. The vapourphase equilibrium procedure was carried out essentially as described [42]. Hydrolysis was performed in 20 ml scintillation vials containing 5 ml of scintillation fluid (ACS II, Amersham).

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2.7. Purification of the Ca 2+-binding proteins from retina The supernatants from ROS isolation were collected, recentrifuged and used as a source of Ca 2+-binding proteins. Chromatographic procedure involved 3 steps: (1) Phenyl Sepharose chromatography. The column (5 × 3.5 cm) was equilibrated in isotonic buffer diluted 1:1 with water and supplemented with 50 μM CaCl2. After sample application (2.5 ml/h) the column was washed with 10 mM HEPES (pH 7.5), 80 mM NaCl, and bound proteins were eluted with the same buffer containing 0.1 mM EGTA (1 ml/min). (2) Fractogel TSK DEAE chromatography. The pooled fractions from Phenyl Sepharose containing the proteins of interest were diluted 3 fold with buffer A. Chromatography was performed as described above for hypotonic ROS extracts. Recoverin, calretinin and calmodulin were eluted at 80–100, 120–140 and 240–280 mM NaCl, respectively. (3) Final purification of the three Ca 2+-binding proteins and buffer change were achieved by gel-filtration on a Superose 12 column (flow rate 0.4 ml/min) equilibrated in isotonic buffer. Recoverin and calmodulin were homogeneous as judged by SDS-PAGE (Coomassie R-250 staining, 20–25 μg of protein per 0.75 × 4 mm well). Calretinin contained ~5% impurities of recoverin and of an unidentified ~32 kDa protein. Identities of the purified proteins were confirmed by partial sequencing of the products of tryptic digestion. 2.8. Assay for GRK1 activity Urea-washed ROS membranes (20 μM rhodopsin) were bleached under room light at 0 °C for 10 min and incubated with 200 μM [γ- 33P] ATP for 4–10 min in a total volume of 25–50 μl. Reactions

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were stopped by addition of 150 μl ice-cold 10% trichloroacetic acid and 10 μl 2% bovine serum albumin. Precipitated proteins were pelleted by centrifugation for 5 min in an Eppendorf centrifuge, washed once with 150 μl 10% trichloroacetic acid, dissolved in 100 μl 80% formic acid, 20% ethanol and counted for [γ-33P]. 2.9. Data representation Except where stated otherwise, all experiments shown in the figures were repeated at least three times using 3 independent ROS preparations. For time course plots, representative experiments are shown rather than averaged data from different experiments, since absolute rates of GRK1 methylation and demethylation varied considerably between different ROS preparations (possibly due to variability in the loss of soluble methylesterase(s) upon breaking the ROS from the neuron body), however the phenomena reported here were observed in all 3 ROS isolations. Statistical analysis was performed using Student's t-test where appropriate. A level of p b 0.05 was considered significant. 3. Results 3.1. Reversible methylation of ROS proteins When preparations of crude ROS were incubated with [ 3H]SAM, several major methylated polypeptides (~ 90 kDa, ~ 60 kDa and two or three bands in the 20–27 kDa region) were observed (Fig. 1A), in line with published observations [17,18,21] (the methylated γ subunit of transducin was not resolved from the non-incorporated radiolabel

Fig. 1. Methylation of rod outer segment (ROS) proteins. (A) Time course of ROS proteins methylation. Methylation was performed with crude dark-adapted ROS (30 μM rhodopsin; see 2.5) in two samples from independent ROS isolations. Aliquots were withdrawn at indicated time intervals, quenched and analysed by SDS-PAGE and autoradiography. Lane M, [14C]-labelled molecular weight markers. (B) Quantification of the autograph shown in (A). Data for GRK1 and PDE6 are the means of the two samples. Data are normalised to the maximum methylation attained for GRK1. GRK1 (C) and PDE6 (D) demethylation is inhibited by ebelactone B. Crude ROS (45 μM rhodopsin) were methylated for 1.5 h followed by addition of vehicle (2% isopropanol, empty circles) or ebelactone B (0.5 mM; filled circles). After additional 30 min, methylation was stopped by addition of excess non-labelled SAM (1 mM). Aliquots were withdrawn from the reaction mixtures, proteins separated by SDS-PAGE and methylation of the GRK1 band analysed using vapour-phase equilibrium procedure (see 2.6). Data are means ± S.D. (n = 4).

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under the SDS-PAGE conditions used). At moderate ionic strength and at 20–40 μM rhodopsin concentration, 85–90% of all major methylated proteins remained bound to the membranes (data not shown), in agreement with an early report [21]. The ~90 kDa methylated band has been previously identified as the α and β subunits of the cGMPspecific phosphodiesterase (PDE6) [19–21]. The 20–27 kDa methylated bands represent small G proteins [43]. In addition, methylation of a weaker 35 kDa band was observed in some preparations (see Fig. 4A). This band was similarly methylated in crude ROS and in ROS exctracts devoid of membranes (data not shown) and probably represented the catalytic subunit of protein phosphatase 2A [44]. The 62 kDa protein methylated in crude ROS has been previously identified as rhodopsin kinase (GRK1) [22]. Using a similar [ 3H] methylation procedure, Ong and co-workers [20] found that stoichiometry of PDE6 [ 3H] methylation plateaus at 1–2 methyl groups per 100 molecules, likely because most PDE6 molecules are already endogenously methylated. GRK1: PDE6 stoichiometry can be estimated as approximately 1:15 from published GRK1: rhodopsin and PDE6: rhodopsin ratios of ~1: 1000 [13], [22] and ~1.5: 100 [40], respectively. Densitometry of the [3H] autoradiograms (Fig. 1B) showed the ratio of [ 3H] GRK1 to [3H] PDE6 signals of approximately 2:1 in the linear part of the time course (within 60–80 min), which corresponds to up to 30–60% total GRK1 [ 3H] methylated in these experiments. It should be noted that in some experiments that used independent ROS preparations (see Fig. 4A) the level of GRK1 [ 3H] methylation was severalfold lower. Since reversibility of methylation of isoprenylated proteins has been a matter of controversy (see Introduction), we examined whether GRK1and PDE6 methylation is reversible. When [3H] methylation was stopped by addition of excess non-labelled SAM, gradual loss of labelling was observed for both GRK1 (Fig. 1C) and PDE6 (Fig. 1D). Typically, 20–40% GRK1 demethylation was observed in 1 h. Demethylation of PDE6 was slower (5–10% in 1 h; Fig. 1D and data not shown), which suggested that GRK1 may be subject to dynamic methylation to a

greater extent than PDE6. GRK1 and PDE6 demethylation was strongly inhibited by ebelactone B (Fig. 1C and D, respectively, filled circles), an efficient irreversible inhibitor of the ROS α-carboxymethyl esterase [33]. These observations are in line with a report of demethylation of ROS small G proteins by an endogenous methylesterase [33]. GRK1 is well known to undergo a rapid autophosphorylation, which results in a characteristic shift in its electrophoretic mobility [45]. We examined whether GRK1 autophosphorylation and methylation might affect each other. Addition of ATP to partially purified GRK1 resulted in a gradual accumulation of a 67 kDa phospho-GRK1 band with a concomitant decrease in the 60 kDa band of non-phosphorylated GRK1 (Fig. 2A, left panel). Upon addition of ATP to the [3H]SAM-labelled crude ROS, a similar shift in the electrophoretic mobility of methylated GRK1 was observed, which occurred with a similar time course (Fig. 2A, right panel). Since ROS methyltransferase is a membrane protein [16,46], while ROS methylesterase is partially soluble [34], purified GRK1 is expected to be at least partially demethylated. Thus, these data suggest that methylated and non-methylated GRK1 undergo autophosphorylation qualitatively similarly, although they do not rule out moderate quantitative effects of GRK1 methylation on its autophosphorylation. In particular, higher proportion of GRK1 was phosphorylated when the assay was performed in crude ROS as compared to purified GRK1 (Fig. 2A). However, this difference is not necessarily due to the extent of GRK1 methylation and may reflect some loss of kinase activity during purification. To test whether GRK1 autophosphorylation might affect its methylation, we preincubated crude ROS with 2 mM ATP to ensure a complete conversion of GRK1 into autophosphorylated form, and then followed its methylation with [ 3H]SAM. Autophosphorylated GRK1 did undergo methylation, though at a lower rate than in the absence of ATP (Fig. 2B). The presence of ATP did not affect methylation of PDE6 (Fig. 2B) and small G proteins (not shown), suggesting that ICMT activity is not affected by ATP. These data suggested that GRK1 autophosphorylation does not preclude its methylation, but may reduce its efficiency.

Fig. 2. Reciprocal effects of GRK1 methylation and autophosphorylation. (A) Effect of methylation on GRK1 autophosphorylation. Non-labelled ATP (1 mM) was added to the partially purified GRK1 (left part, Coomassie staining) or to crude ROS labelled with [3H]SAM (right part, autoradiography). Aliquots were withdrawn and quenched at indicated time intervals. Lanes 0, respective samples prior to ATP addition. (B) Effect of ATP on GRK1 methylation. Methylation was performed in crude ROS in the absence (solid lines, empty symbols) or in the presence (dashed lines, filled symbols) of ATP (2 mM). Circles, PDE6; diamonds, GRK1. pGRK1, autophosphorylated GRK1. Plots show quantification of the autoradiographs from 3 (A) and 4 (B) replicates, respectively (means ± S.D.).

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3.2. Effect of nucleotides on GRK1 methylation We examined the possible role of autophosphorylation in inhibiting GRK1 methylation in more detail. Addition of ATP in the low millimolar range inhibited GRK1 methylation by 40–60% (Fig. 3A). Preincubation of ROS with ebelactone B decreased the effect of ATP approximately twice, but did not abolish it (Fig. 3A), indicating that both inhibition of GRK1 methylation and stimulation of its demethylation may contribute to the effect of ATP. ATP addition accelerated GRK1 demethylation severalfold (Fig. 3B). In the presence of ebelactone B, stimulation of GRK1 demethylation by ATP was abolished (Fig. 3B). Therefore, ATP is likely to stimulate GRK1 demethylation by the same ebelactonesensitive enzyme(s) that is(are) active without ATP, rather than to activate additional esterase(s) or protease(s). These findings suggested that GRK1 (auto)phosphorylation may regulate its methylation status. However, when ATP was replaced by its non-hydrolysable analogue, AMP-PNP, a qualitatively similar effect was observed (Fig. 3C). The characteristic shift in the electrophoretic mobility of [ 3H]-methylated GRK1, indicative of phosphorylation, was observed in the samples incubated with ATP, but not with AMP-PNP (data not shown), which confirms the absence of ATP contamination and GRK1 phosphorylation in the presence of AMP-PNP. The effect of ATP was similar in the presence of EGTA (data not shown), ruling out a possibility that it was due to the ATP-dependent inhibition of GRK1-recoverin interaction [47]. The approximately twofold higher apparent affinity for ATP as compared to AMP-PNP (Fig. 3C) is in line with the 35% inhibition of GRK1 activity in the presence of equal concentrations of ATP and AMP-PNP reported by Dean and Akhtar [48]. It should be noted however that accurate estimates of IC50 values are not possible from these data, since the extent of nucleotide metabolisation during sample incubation was not examined. Millimolar concentrations of ADP, AMP, GTP or ITP also inhibited GRK1 methylation with apparent affinities for these nucleotides decreasing as follows: ATP≈ GTP>AMP-PNP> ADP> AMP > ITP; adenosine was without effect (data not shown). These observations indicated that the inhibition of GRK1 methylation observed in the presence of nucleotides was due primarily to nucleotide binding, although they do not exclude a possibility of GRK1 phosphorylation contributing to the effect. 3.3. Demethylation of GRK1 is inhibited by recoverin/Ca 2+ We also examined whether methylation of ROS proteins would be affected by Ca 2+, which is known to play an important and

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multifaceted role in light adaptation [49]. Methylation of GRK1, but not of other methylated ROS proteins, was slightly decreased (by 10–20%) at nanomolar as compared to micromolar calcium levels (data not shown). Since concentrations of all proteins in in vitro experiments are much lower than their physiological levels, we checked whether addition of the known ROS Ca2+-binding proteins could enhance the Ca2+-dependence. Indeed, addition of micromolar concentrations of recoverin purified from bovine retinas led to a considerable (2–3 fold) increase in GRK1 methylation at high as compared to low [Ca 2+], while no effect was observed on methylation of other proteins (Fig. 4A,B). Addition of purified calmodulin or calretinin at similar concentrations could not mimic the effect of recoverin (not shown). To determine whether recoverin/Ca2+ affects GRK1 methylation or demethylation, we examined the effect of ebelactone B in the presence of recoverin at low or at high [Ca 2+]. The Ca 2+-dependent difference in [ 3H] labelling was completely abolished by preincubation with ebelactone B (Fig. 4B, left panel). This indicated that recoverin/Ca2+ regulates GRK1 demethylation, rather than methylation. To confirm this conclusion, we directly followed the time course of GRK1 demethylation at low and at high [Ca 2+] in the absence or in the presence of added purified recoverin (Fig. 4C). In the presence of EGTA, recoverin had no effect on the rate of GRK1 demethylation. However, at high [Ca2+] recoverin decreased the rate of GRK1 demethylation severalfold. In the absence of added recoverin, a modest inhibition of GRK1 demethylation was observed at high Ca2+, probably due to the presence of endogeneous recoverin. Two other EF-hand Ca2+-binding proteins, calmodulin and calretinin, failed to produce this effect. Neither Ca2+ concentration nor addition of recoverin affected methylation (Fig. 4B) and demethylation (data not shown) of PDE6. These results demonstrate that Ca2+-bound recoverin specifically inhibits demethylation of GRK1. Since crude ROS preparations might contain some residual levels of endogenous ATP, we checked the possibility that recoverin might affect GRK1 methylation status by regulating, in a Ca 2+-dependent manner, its autophosphorylation. Recoverin has been suggested to inhibit directly the catalytic activity of GRK1 [13]. If this is the case, the effect of recoverin should be independent of the substrate, i.e. rhodopsin or GRK1 itself. However, we found no significant difference in GRK1 autophosphorylation (either in crude ROS or partially purified GRK1) in the presence or in the absence of recoverin at high or at low Ca 2 + concentrations, while GRK1 activity towards rhodopsin was inhibited by ~ 40% in the presence of recoverin/Ca 2+ (data not shown). These results indicate that the mechanism of regulation of GRK1 demethylation by recoverin does not involve control of GRK1

Fig. 3. Effects of ATP and AMP-PNP on GRK1 methylation status. (A, B) ATP suppresses GRK1 methylation. Methylation (A) and demethylation (B) were performed in crude ROS for 1.5 h and 1 h, respectively, in the absence (NA) or in the presence of ATP (1 mM) and/or ebelactone B (EbB; 0.5 mM). [3H] label incorporation was analysed using vapour-phase equilibrium procedure. **, p b 0.01; *, p b 0.05 in a two-tailed t-test. (C) Comparison of the effects of ATP and AMP-PNP on GRK1 methylation. Crude ROS were methylated for 1.5 h in the absence or in the presence of various concentrations of ATP (circles) or AMP-PNP (triangles). Data shown are means of two different experiments (values obtained in each experiment are indicated by horizontal lines).

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Fig. 4. Regulation of GRK1 methylation status by recoverin/CA2+. (A) Recoverin specifically enhances methylation of GRK1. Crude ROS were methylated for 1.5 h at high (1.6 μM, empty circles, dashed line) or low (50 nM, filled circles, solid line) free Ca2+, in the presence of purified recoverin (10 μM). Ca2+ concentrations were set using CaCl2-EGTA buffers as described previously [39]. (B) Effect of ebelactone B on GRK1 and PDE6 methylation at high and low Ca2+. ROS were supplemented with purified native recoverin (10 μM) and methylation was performed for 1.5 h in the presence of 1 mM EGTA or 100 μM CaCl2, and ebelactone B (100 μM) or vehicle (2% isopropanol). Protein methylation was quantified by the vapour-phase equilibrium procedure. **, pb 0.01; *, p b 0.05 in a two-tailed t-test. (C) Recoverin/Ca2+ inhibits GRK1 demethylation. Methylation was performed for 1.5 h without any addition of CaCl2 or EGTA (i.e. at micromolar level of free Ca2+), and stopped by addition of a mixture containing (final concentrations) non-labelled SAM (1 mM), EGTA (1 mM; filled symbols) or CaCl2 (50 μM; empty symbols), purified recoverin (10 μM; triangles, solid lines), and the time course of GRK1 demethylation was followed. Circles, dashed lines: buffer was added instead of recoverin. The experiment shown is representative of three experiments that used two independent ROS preparations.

autophosphorylation. These data also confirm, using native proteins, previous observations with purified recombinant recoverin and GRK1 [47,50], and show that recoverin does not affect GRK1 catalytic activity per se. As expected for a methylesterase inhibitor, preincubation with ebelactone B increased incorporation of [ 3H] label into GRK1 at low [Ca 2+], however it had an opposite effect at high [Ca 2+] (Fig. 4B, left panel). Similarly, ebelactone B also reduced [ 3H] methylation of PDE6 (Fig. 4B, right panel). The most plausible explanation of this “inverse” effect of ebelactone B is that considerable fractions of both GRK1 and PDE6 are endogenously methylated in ROS, and that prior removal of unlabelled methyl groups is required before [ 3H] methyl groups can be incorporated. 3.4. Role of membrane targeting in the regulation of GRK1 methylation Since recoverin associates with the membranes in the presence of Ca 2+ [51] and targets GRK1 to the membranes [52], and since ATP or AMP-PNP binding releases GRK1 at least from the membranes containing photoactivated rhodopsin [48], we hypothesised that the regulation of GRK1 methylation status by recoverin and nucleotides described above might be due to their effects on its association with

the membranes. Since the methylesterase is partially soluble [34], and ICMT is an integral membrane protein [16,46], GRK1 partitioning into the cytoplasm would favour its demethylation and would prevent its methylation. The N-terminal acylation of recoverin is known to be essential for its Ca 2+-dependent membrane association [53], but not for its ability to inhibit GRK1 [13,51]. To determine whether recoverin acylation is necessary for the regulation of GRK1 demethylation, we examined the effects of myristoylated [54] and non-myristoylated recombinant recoverin, as well as recombinant recoverin purified as a fusion protein with the N-terminally attached fragment of protein A [55]. The fusion protein and non-myristoylated recoverin used in these experiments were active as Ca2+-dependent inhibitors of GRK1 and were about 70% as potent as the natural or recombinant myristoylated recoverin (data not shown). Recoverin purified from retinal extracts and recombinant myristoylated recoverin strongly inhibited GRK1 demethylation in the presence of Ca2+, while addition of non-myristoylated recoverin or fusion protein slightly accelerated GRK1 demethylation (Fig. 5A). The latter effect is probably due to the competition between added non-myristoylated recoverins and endogenous recoverin. Indeed, as expected if non-myristoylated recoverins bind to GRK1 but are inactive with respect to the inhibition of its demethylation, both

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Fig. 5. Role of ROS membranes in the regulation of GRK1 methylation. (A) Recoverin myristoylation is required for the regulation of GRK1 methylation status. Methylation and demethylation were performed as in Fig. 4C. Demethylation reactions contained 50 μM CaCl2 and native (5 μM) or recombinant (10 μM) recoverin as indicated: Myr, myristoylated; NMyr, non-myristoylated; PrA, N-terminally fused with protein A fragment. For each replicate, the rate of GRK1 demethylation is expressed relative to a sample containing 1 mM EGTA, considered as 100%. (B) The presence of membranes is required for the regulation of GRK1 methylation status by ATP and by recoverin/Ca2+. Crude ROS (40 μM rhodopsin) were methylated for 1.5 h. Methylation was stopped by addition of excess of non-labelled SAM (1 mM), membranes pelleted by centrifugation and GRK1 demethylation in the supernatants was allowed to proceed for 40 min with or without addition of urea-washed ROS membranes (20 μM rhodopsin), ATP (2 mM) or purified native recoverin (10 μM) as indicated. (C, D) Both methylation and demethylation of GRK1 are inhibited by illumination. Reactions were performed as in Fig. 1A,B in the dark (filled circles) or under room illumination (empty circles). To follow demethylation time course (C), methylation was performed in the dark for 1.5 h. GRK1 methylation was quantified by the vapour-phase equilibrium procedure. In (A) and (B), data shown are means of 3 experiments that used 3 independent ROS isolations; error bars, S.D.; ***, p b 0.001 in a two-tailed t-test.

non-myristoylated recoverins could reverse to some extent the effect of the myristoylated form when added together (data not shown). These data show that recoverin myristoylation is required for its effect on GRK1 demethylation. To directly examine whether the presence of membranes would affect GRK1 demethylation and its regulation by recoverin/Ca 2+ and ATP, we performed protein methylation in preparations of crude ROS using [ 3H] SAM, removed the membranes by centrifugation and assessed the effects of exogenously added purified native recoverin or ATP on GRK1 demethylation in the absence or in the presence of urea-washed ROS membranes. Reconstitution of soluble ROS exctract with urea-washed ROS membranes reduced the rate of GRK1 demethylation twofold, and additional presence of recoverin inhibited demethylation severalfold (Fig. 5B). Yet, addition of purified recoverin in the absence of membranes had no effect on GRK1 demethylation (Fig. 5B). ATP also failed to affect GRK1 demethylation in ROS extracts containing no membranes (Fig. 5B). When added together with urea-washed ROS membranes, ATP restored the rate of GRK1 demethylation to the levels observed in solution (Fig. 5B). Taken together, these data indicate that regulation of GRK1 demethylation by recoverin/Ca 2+ and nucleotides requires the presence of membranes and, at least in the case of recoverin, likely relies on regulation of GRK1 shuttling between membranes and the cytoplasm.

Although ATP and AMP-PNP are similarly efficient in releasing GRK1 from the membranes containing photoactivated rhodopsin [48], we observed the effects of nucleotides on GRK1 methylation in dark-adapted ROS. We checked whether the presence of ATP would affect GRK1 redistribution in our ROS preparations, but did not find any significant changes in GRK1 extractability in the absence or in the presence of ATP (data not shown). Furthermore, the absence of detectable rhodopsin phosphorylation with [ 33P] ATP in the darkadapted ROS confirmed the absence of photoactivated rhodopsin in our samples (data not shown). Thus, even though membranes are required for the effect of nucleotides on GRK1 methylation status, this effect does not appear to require membrane-cytoplasmic redistribution of GRK1. Upon rhodopsin activation, GRK1 translocates to the ROS membranes [56]. Therefore, ROS illumination would be predicted to favour GRK1 methylation and reduce its demethylation. Indeed, GRK1 demethylation was strongly reduced when ROS where illuminated after [3H] methylation was performed in the dark (Fig. 5C). When methylation was performed in ROS illuminated just before the start of the reaction, it was also inhibited as compared to the dark-adapted samples (Fig. 5D), which would be consistent with the requirement for removal of endogenous methyl groups for efficient incorporation of the [3H] label as observed with ebelactone B. Alternatively, the C terminus of GRK1

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may be less accessible for both processing enzymes when the kinase is in the complex with activated rhodopsin. PDE6 methylation and demethylation was unaffected by illumination (data not shown). 4. Discussion We report here the first evidence that the methylation status of GRK1, a protein that plays an important and well established role in a desensitisation of photoreception, may be controlled by such physiologically relevant parameter as free Ca 2+ concentration, via a Ca 2+‐ binding protein recoverin. Our findings indicate that the methylated form of GRK1 is stabilised by recoverin in the presence but not in the absence of Ca 2+. We also report that GRK1 methylation is antagonised by nucleotide binding, rather than by GRK1 autophosphorylation. The effects of recoverin/Ca 2+ and nucleotides are specific for GRK1 and are not observed for other methylated ROS proteins, indicating that the activities of the processing enzymes (methyltransferase and methylesterase) are unlikely to be modified by recoverin and nucleotides, and that the regulation is at the substrate level. An important question is whether a considerable fraction of GRK1 is endogenously methylated in the ROS, i.e. whether regulation of its methylation status might have any appreciable physiological consequences. Two lines of evidence indicate that a substantial proportion of GRK1 is endogenously methylated and can be dynamically methylated at least in vitro. First, two conditions that inhibit GRK1 demethylation by different mechanisms (preincubation with ebelactone B and ROS illumination) both result in concomitant inhibition of its [ 3H] methylation. The most plausible explanation of these observations is that a considerable fraction of GRK1 molecules is endogenously methylated, and that removal of unlabelled methyl groups is necessary for efficient incorporation of the [ 3H] label. Indeed, if only a small fraction of GRK1 were methylated in vivo, then inhibition of its demethylation would have no noticeable effect on GRK1 availability for [ 3H] methylation. Only 1–2% of endogenous PDE6 has been found to exist in a non-methylated state and be able to be [ 3H] methylated using a procedure similar to ours [20]. Based on (i) this estimate, (ii) the relative intensity of [ 3H] signals from PDE6 and GRK1 in our experiments, and (iii) the known stoichiometry between the two enzymes, we could estimate that up to 30–60% of GRK1 could be dynamically methylated in our preparations. The mechanism of the regulation of GRK1 methylation status reported here appears to rely largely upon its redistribution between the membranes and the soluble phase. Indeed, both recoverin/Ca 2+ and nucleotides failed to affect demethylation of [ 3H]-methylated GRK1 in solution. We found that reconstitution with urea-washed ROS membranes is sufficient to restore the effect of recoverin, and that even addition of urea-washed membranes alone moderately inhibits GRK1 demethylation. The simplest mechanistical explanation of these data is that membrane targeting makes GRK1 less available for methylesterase(s), which is (are) partially soluble [34], and more available for ICMT, which is an integral membrane protein [16,46]. Such a mechanism is also consistent with the observations that (i) ROS illumination, which causes GRK1 to translocate to the membranes, impedes its demethylation, and (ii) recoverin myristoylation is required for its effect on GRK1 demethylation. Indeed, recoverin myristoylation is not necessary for its interaction with GRK1 [12,47] but is required for its membrane targeting [53]). The following considerations suggest that nucleotide binding to GRK1, rather than its autophosphorylation, affects its methylation status: (i) not only ATP, but also AMP-PNP, ADP and several other tested nucleotides are active in this respect; (ii) ATP and GTP show similar efficiency in respect to GRK1 methylation, while GRK1 active site is highly selective for ATP over GTP [57]; (iii) apparent IC50 for adenine nucleotides are at least 2 orders of magnitude higher than their concentrations effective for catalysis (ATP) or competitive inhibition (ADP, AMP) [57]. The latter two observations would also suggest

a possible involvement of a nucleotide binding site distinct from the catalytic centre. ATP and AMP-PNP concentrations necessary to reduce GRK1 methylation are similar to the concentrations reported to release GRK1 from bleached ROS membranes, presumably by dissociating its complex with photoactivated rhodopsin [48]. Nucleotide-induced GRK1 release from the membrane would be in line with the observed reduced methylation, and the requirement for the membranes for nucleotide effect seems to support such possibility. However, we observed the effects of nucleotides on GRK1 methylation in the darkadapted ROS, and control experiments did not reveal any traces of activated rhodopsin, as evidenced by the absence of detectable rhodopsin phosphorylation with [ 33P] ATP. Moreover, the amounts of GRK1 recovered in the soluble fraction of dark-adapted ROS were similar irrespective of nucleotide presence. Thus, the precise mechanism how nucleotides affect GRK1 methylation status in a membrane-dependent manner remains to be determined. What could be the functional significance of the regulation of GRK1 methylation status by nucleotide binding and recoverin? In vivo concentrations of ATP, ADP and AMP in bovine ROS have been estimated as 0.43–0.46, 1–1.3 and 1.6 mM, respectively, and are not significantly affected by illumination [58]. In addition, ROS contain concentrations of GTP comparable to those of ATP [59]. Therefore, cumulative nucleotide concentrations in vivo would likely be saturating for their effect on GRK1 (see Fig. 3C), and thus nucleotide binding might rather play a “structural” than a regulatory role. In contrast, the control of GRK1 methylation status by recoverin may contribute quantitatively to its Ca 2+-dependent inhibition. In darkadapted rods, where cytoplasmic Ca2+ concentration is high [60], GRK1 would be associated with recoverin. This association would target it to the membranes, where it would be preferentially methylated. This, in turn, would further strengthen GRK1 association with the membranes, since the affinity of farnesylated proteins for the membranes is considerably higher in methylated than in non-methylated form [16,27]. Thus, both GRK1 and its inhibitor would be concentrated on the membranes. This would make GRK1 inhibition more efficient, since the complex of recoverin/Ca2+ and GRK1 is stabilised by membrane association [52]. Recoverin/Ca2+ is necessary to prevent spurious phosphorylation of hundreds of inactive rhodopsin molecules by GRK1 activated by a single photoexcited receptor, a phenomenon known as “high gain phosphorylation” [61,62]. Therefore, stabilisation of the methylated form of GRK1 by recoverin/Ca2+ would increase the efficiency of its inhibition under conditions of dark adaptation. This positive feedback loop would further enhance signal amplification upon light perception by dark-adapted rods. Unlike elusive mammalian ICME(s) (see Introduction), ICMT is a well known integral membrane protein, considered to be the only mammalian enzyme capable of carboxyl methylation of isoprenylated proteins, since Icmt−/− cells lack the ability to methylate small G proteins and N-acetyl-S-geranylgeranylcysteine [28]. ICMT is thought to be restricted to the endoplasmic reticulum [46,63], which is present in the rod inner but not outer segments (e.g. [64]). Since we and others observe methylation of GRK1 and other isoprenylated proteins in isolated ROS, this would imply that either it is performed by a distinct enzyme, or that ICMT is located in the rods beyond the ER, in membranes such as the disks and/or plasma membrane. Since the conclusion about only one ICMT form in mammals has been made on the basis of the absence α-carboxymethylation of small G proteins in the absence of ICMT, the existence of other enzyme(s) with ICMT-like activity acting on different substrates and in different locations cannot be excluded. Indeed, protein α-carboxymethylation at the plasma membrane in neutrophils [65] and renal epithelial cells [66] has been reported. Recent development of specific ICMT inhibitors [67] could help future identification of the enzyme(s) responsible for ROS ICMT activity. In conclusion, this study provides the first evidence for dynamic regulation of α-carboxymethylation of GRK1 in the rod photoreceptor

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