Rhodopsin phosphorylation and its role in photoreceptor function

Vision Research 38 (1998) 1341 – 1352

Rhodopsin phosphorylation and its role in photoreceptor function James B. Hurley *, Maribeth Spencer, Gregory A. Niemi Department of Biochemistry and Howard Hughes Medical Institute, Uni6ersity of Washington, Seattle, WA 98195, USA Received 18 August 1997; received in revised form 17 October 1997

Abstract Light-stimulated phosphorylation of rhodopsin was first described 25 years ago. This paper reviews the progress that has been made towards (i) understanding the nature of the enzymes that phosphorylate and dephosphorylate rhodopsin (ii) identifying the sites of phosphorylation on rhodopsin and (iii) understanding the physiological importance of rhodopsin phosphorylation. Many important questions related to rhodopsin phosphorylation remain unanswered and new strategies and methods are needed to address issues such as the roles of Ca2 + and recoverin. We present one such method that uses mass spectrometry to quantitate rhodopsin phosphorylation in intact mouse retinas. © 1998 Published by Elsevier Science Ltd. All rights reserved. Keywords: Rhodopsin; Phosphorylation; Photoreceptor

1. Introduction

2. Review of rhodopsin phosphorylation

In the first reports describing light-stimulated phosphorylation of vertebrate rhodopsin three fundamental questions were recognized [1 – 3]: 1. What enzymes catalyze phosphorylation and dephosphorylation of rhodopsin and how are they regulated? 2. How many phosphorylation sites are there on rhodopsin and where are they located? 3. What is the physiological role of rhodopsin phosphorylation? Part A of this paper reviews answers to these questions that have developed during 25 years following the discovery of rhodopsin phosphorylation. Part B describes a mass spectrometry method that we recently developed to address unresolved questions about the regulation of rhodopsin phosphorylation and its physiological role in photoreceptor function.

2.1. Enzymes that phosphorylate and dephosphorylate rhodopsin

* Corresponding author. Fax: +1 206 6852320; e-mail: [email protected].

2.1.1. Rhodopsin kinase The stimulatory effect of light on rhodopsin phosphorylation is transient; it decays slowly in darkness with a t1/2 : 40 min following illumination [4–6]. Both metarhodopsin I and II are phosphorylated [7]. Opsin, the end product of the bleaching pathway, is not efficiently phosphorylated although the presence of all-trans retinal enhances its ability to be phosphorylated in vitro [8]. The kinase responsible for this activity is rhodopsin kinase (RK), a 63 kDa single subunit enzyme. In darkness, RK can be extracted with aqueous buffers from homogenates of photoreceptor outer segments [9–11] but it is not as effectively extracted when the membranes have been exposed to light [12]. Centrifugation and light-scattering studies have suggested that RK and photo-activated rhodopsin (R*) form a complex [13]. The Km’s of RK are 4 mM for ATP and 4 mM for R* [14]. RK, also referred to as GRK1, was the first member of a family of G-protein coupled-receptor kinases (GRK’s) to be recognized [15,16]. GRK’s have a conserved central kinase domain, an N-terminal domain involved in receptor recognition and a C-terminal domain required for membrane localization. Membrane

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attachment requires either direct prenylation of the C-terminus of the GRK as in the case of GRK1 (RK), interaction of the GRK with a prenylated protein, Gbg, as in the case of GRK2 (bARK1), or an electrostatic interaction as in the case of GRK5. RK and GRK6 undergo autophosphorylation and GRK5 is a substrate for protein kinase C [17]. RK is the most specifically expressed of the GRK’s; it has been detected immunologically only in rods and cones [18] and in the pineal gland [19,20]. RK and the gene encoding it have been characterized and effective methods to purify it have been developed [21 – 23]. The purified protein was sequenced and cDNA encoding RK was isolated and characterized [24]. The human RK gene is on chromosome 13 band q34 [25] and mutations in it have been identified that are linked to Oguchi disease, a form of stationary night blindness [26]. The carboxy-terminus of RK isolated from bovine retinas is farnesylated [27,28], a modification that appears to enhance the interactions between RK and R* [29]. Hydrophobic modifications also occur on other peripheral membrane proteins involved in phototransduction [30].

2.1.2. Phosphorylation of cone opsins Cone opsins are recognized and phosphorylated by RK in vitro [31] and RK has been localized by immunocytochemical analysis to both rods and cones [18]. But a recent finding suggested that the roles of GRKs in rods and cones may be different. Mutations in RK that cause Oguchi disease affect only scotopic vision; cone-mediated photopic vision is not greatly affected by lesions in the RK gene [26]. This suggests either that cone function relies on a different kinase than RK or that opsin phosphorylation is less critical to cone function than it is to rod function. 2.1.3. Regulation of RK acti6ity Mechanism of RK activation. Ku¨hn suggested that the effect of light on rhodopsin phosphorylation may reflect increased accessibility of phosphorylation sites rather than stimulation of RK catalytic activity [9]. But it was later found that the ability of RK to recognize and phosphorylate short synthetic peptides is enhanced by R* [32–34]. It was also shown that RK interacts with the second and third cytoplasmic loops of rhodopsin, regions that are different from the domain phosphorylated by RK [34,35]. These findings suggest that R* stimulates catalytic activity of RK by an allosteric mechansim analogous to the mechanism by which it activates transducin. The allosteric model for RK activation may be correct to a first approximation, but differences have been detected in structural requirements for activation of transducin versus activation of RK [36]. Furthermore, a

peptide resembling one of the cytoplasmic loops of rhodopsin stimulates transducin 20X but it only stimulates RK 1.5X [34]. One report has suggested that constitutively active rhodopsins differ in their ability to stimulate transducin and RK [37], but this result was not confirmed by subsequent studies in which the same mutants were shown to be effective activators of both transducin and RK [38–40]. Alternatively, R* may enhance phosphorylation by bringing together RK and the rhodopsin C-terminus [40]. In support of this idea, removing the C-terminus of R* significantly enhances the ability of RK to phosphorylate a synthetic peptide [34]. Truncation of R* could expose a docking site for the peptide thereby enhancing the interactions between RK and the peptide substrate. ‘High-gain phosphorylation’ of rhodopsin. Bownds observed that photo-activation of a rhodopsin molecule can stimulate incorporation of 20 phosphates [1]. Since so many sites on a single rhodopsin seemed unlikely, it was suggested that a single R* promotes phosphorylation of sites on neighboring, non-photolyzed rhodopsins. This observation was later confirmed and extended and the reaction has been referred to either as ‘high-gain phosphorylation’ or ‘trans-phosphorylation’ [41–43,23,44,45]. High-gain phosphorylation has also been detected using purified RK preparations so it does not reflect the activity of kinases other than RK [23]. A mechanism for high-gain phosphorylation has been proposed based on the finding that an ATP/RK complex dissociates from R* in a state capable of phosphorylating unbleached rhodopsins [44]. It would be useful to test this and other models using reconstitution assays but a recent study with recombinant rhodopsin and RK in reconstituted phospholipid vesicles found no evidence for trans-phosphorylation [40]. Autophosphorylation. In addition to phosphorylating rhodopsin, RK also undergoes autophosphorylation [46], a reaction that is not affected by R* [47]. 21 Ser, 488Ser and 489Thr have been identified as autophosphorylation sites on RK [22]. The effects of RK autophosphorylation have been studied but its role is still unclear. Centrifugation analysis suggested that autophosphorylation of RK does not affect its interaction with R* [47,48]. But a light-scattering study also showed that autophosphorylation weakens the RK/R* interaction [13] possibly by increasing kd. It has been reported that ATP tightens the complex of phosphorylated RK with phosphorylated R* in membranes without increasing ka [13]. But a separate study found that RK becomes soluble when activated by ATP and R* [44]. Alanine and aspartate substitutions designed to mimic constitutively unphosphorylated and constitutively phosphorylated forms of RK have been analyzed. Both types of substitutions reduced the specificity of RK [49]. More extensive studies will be

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needed to resolve the significance of RK autophosphorylation. Regulation of RK by Ca2 + and recoverin. Kawamura and Murakami [50] discovered that a 26 kDa Ca2 + -binding protein, S-modulin, imparts Ca2 + -sensitivity to light-stimulated phosphodiesterase activity. Kawamura subsequently showed that S-modulin functions in the presence of Ca2 + by inhibiting light-stimulated phosphorylation of rhodopsin [51]. Avian [52] and mammalian [53 – 55] homologues of S-modulin were discovered independently and are usually referred to as visinin and recoverin. Initial reports characterized recoverin as a guanylyl cyclase activator [53,55] but this activity most likely derived from related proteins, GCAP-1 and GCAP-2, which may have contaminated preparations used in those studies [56–58]. The inhibitory effect of recoverin on RK in vitro has been confirmed by several independent laboratories [59 – 63,23,45]. Recoverin also prolongs the photoresponse [50,64], consistent with its ability to inhibit rhodopsin phosphorylation in vitro. Three-dimensional structures of Ca2 + -bound and Ca2 + -free forms of recoverin have been described [65– 67]. Recoverin is fatty acylated at its N-terminus [68]. In Ca2 + -free recoverin the fatty acyl residue is buried within a hydrophobic pocket whereas in Ca2 + -bound recoverin, it is solvated and available to anchor recoverin to membranes [69 – 71,66,72]. This mechanism for regulating exposure of a fatty acyl residue has been referred to as a ‘calcium-myristoyl switch’ [70]. The K0.5 for the recoverin-mediated effect of Ca2 + on RK activity is about 2 mM [23,73,63]. N-terminal myristoylation is not essential for the activity of recoverin but it enhances the ability of recoverin to inhibit RK [23,73,74]. Minor differences in Ca2 + and myristoylation dependence described in those studies may reflect differences in the extent of bleaching and in methods used to purify RK and recoverin. The ratio of recoverin to rhodopsin in frog photoreceptors has been estimated at 1:174 [63]. The interaction of recoverin with RK is direct and inhibitory and it is specific [23,75]. Recoverin does not interact with a different GRK, b-ARK-1 [23] and RK is not effectively inhibited by a different Ca2 + -binding protein, calmodulin [76,77]. Calmodulin has an inhibitory effect on other members of the GRK family [77,78]. Other factors that influence the activity of RK. Rhodopsin is doubly palmitoylated at 322Cys and 323Cys near its carboxy-terminus [79]. The relationship between palmitoylation and phosphorylation of rhodopsin has been investigated but the results of those studies are inconclusive. One study found that a substitution which prevents palmitoylation reduces rhodopsin phosphorylation [80] but a different study found that depalmitoylation of rhodopsin does not affect phosphorylation [81].

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2.1.4. Protein kinase C An activity that has the Ca2 + - and phospholipid sensitivity of protein kinase C (PKC) has been detected in vertebrate photoreceptors [82–84]. Phorbol myristoyl acetate (PMA), an activator of PKC, has been shown to both enhance [83] and inhibit [85] light-stimulated phosphorylation of rhodopsin in intact retinas. These actions of PMA depend upon many conditions and the mechanisms by which it exerts these effects on rhodopsin phosphorylation are unknown. The stimulatory effect of PMA appears to be transient [86] and it does not stimulate rhodopsin phosphorylation in darkness in intact retinas [85–87]. However, under in vitro conditions, PKC phosphorylates both bleached and unbleached rhodopsin [82,87,88]. These findings suggest that PKC functions in a complex, perhaps indirect manner in photoreceptors. A separate study, however, has failed altogether to support a role of PKC in phototransduction [81]. Two inhibitors, sangivamycin and calphostin C, have been used to assess RK and PKC function in photoreceptors. One study [89] used sangivamycin to investigate RK function in vivo. But that study should be interpreted with caution since it has been reported that PKC is more sensitive than RK to this inhibitor [88]. PKC is more sensitive than RK to another inhibitor, calphostin C. The idea that PKC regulates phototransduction is supported by the finding that 1 mM calphostin C reduces by 50% the extent of light-stimulated rhodopsin phosphorylation in intact retinas [86]. 2.1.5. Dephosphorylation of rhodopsin The activity that dephosphorylates rhodopsin has characteristics of protein phosphatase 2A (PP2A). Both PP1 and PP2A have been detected in outer segment preparations but only PP2A recognizes and dephosphorylates rhodopsin [90,91]. PP2A holoenzyme has been purified from photoreceptors as a 2A catalytic subunit and two larger polypeptides [92]. A separate study has raised the possibility that rhodopsin phosphatase activity in photoreceptors derives from a novel enzyme, CAOP, which requires Ca2 + and a 25 kDa protein for full activity [93]. CAOP activity appears to be different from PP2A; it is much less sensitive than PP2A to the phosphatase inhibitor, okadaic acid. Furthermore, recoverin reportedly stimulates PP2A [81], but it does not substitute for the 25 kDa protein associated with CAOP [93]. The suggestion has also been made [93] that the vertebrate rhodopsin phosphatase may be related to RDGC, a Ca2 + -dependent enzyme that dephosphorylates Drosophila rhodopsin [94,95]. A mammalian homologue of RDGC was recently identified but it appears to be localized primarily to photoreceptor inner segments [96].

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2.2. Number and location of phosphorylation sites on rhodopsin As much as 40% of rhodopsin typically remains unphosphorylated in most experiments even under conditions of intense illumination [97 – 100]. The mechanistic and structural basis for this is unknown [81]. It might represent a pool of rhodopsin that is inaccessible to RK or it may represent the maximum level of phosphorylation that can be achieved under steady-state phosphorylation and dephosphorylation conditions. Within the pool of rhodopsin available for phosphorylation, as many as 8 – 9 phosphates have been detected on a rhodopsin molecule [101,102]. Phosphorylation sites have been localized to the cytoplasmic C-terminal tail of rhodopsin where there are seven hydroxy-amino acids [103]. The most favored sites were originally reported to be 338Ser and 343Ser followed by 334Ser, 335Thr and 336Thr [103]. More recently, phosphorylation sites on rhodopsin were confirmed using more advanced analytical sequencing and mass spectrometry methods. The consensus of three separate studies is that 338Ser and 343 Ser are major sites of rhodopsin phosphorylation in vitro and that additional sites on the rhodopsin C-terminus may also be phosphorylated. McDowell and colleagues reported that phosphorylation occurs primarily at 338Ser and 343Ser [104]. Threonine phosphorylation was also detected at positions 335, 336, 340 and 342. In other studies, Papac and colleagues also identified 338Ser and 343Ser as major sites of phosphorylation in outer segment homogenates [105] and Ohguro and colleagues reported that phosphorylation occurs initially at 338Ser followed by 343Ser or 336Thr [106]. Mutagenesis studies suggest that phosphorylation may be cooperative. Glutamic acid substitutions within the C-terminal region of rhodopsin enhance the rate of phosphorylation [107]. 343 Ser phosphorylation may be detected at low levels because it is preferentially dephosphorylated [105,81]. In support of this idea, the predominant site of phosphorylation was 338Ser when ATP was used as the kinase substrate but when ATPgS was used thiophosphorylation of rhodopsin was detected at both 338 Ser and 343Ser [108]. Since thiophosphorylation is resistant to dephosphorylation these findings suggest that 338Ser and 343Ser are normally phosphorylated with similar efficiency but that 343Ser may be more susceptible to dephosphorylation. The substrate specificity of RK has also been examined using synthetic peptides. For example, acidic peptides were found to be preferred substrates for RK [109,110]. However, the Km’s for these peptides are three orders of magnitude greater than for the same sequences within intact rhodopsin. So there is some

uncertainty about whether recognition of these short peptides by RK accurately represents recognition of the same sequence in the context of rhodopsin. Nevertheless, the results of an extensive analysis of 30 different peptide substrates for RK are generally consistent with studies using intact rhodopsin [111]. Phosphorylation occurred first at 343Ser followed by 338 Ser, 336Thr, 342Thr and finally 334Ser, 335Thr and 340 Thr. RK requires at least one residue on the amino terminal side and at least five on the carboxyl side of the peptide phosphorylation site and a neutral residue is preferred 4 amino acids to the carboxyl side of the site. Another approach to understanding the substrate specificity of RK has been to use mutant forms of rhodopsin in which C-terminal phosphorylation sites have been altered [107]. Surprisingly, it was found that most serines and threonines in the carboxyl teriminus can be substituted without significantly reducing the extent of phosphorylation. For example, a Ser338Ala/Ser343Ala mutant was phosphorylated at 72% of the rate at which normal rhodopsin is phosphorylated even though the two most favored phosphorylation sites were removed. The sites of PKC catalyzed phosphorylation have also been investigated [112]. Both 334Ser and 338Ser were found to be major phosphorylation sites for PKC in vitro. Preferred sites of rhodopsin phosphorylation in vitro are reportedly different from the ones preferred in vivo. Ohguro and colleagues exposed mice to light, isolated photoreceptor disk membranes and cleaved C-termini from rhodopsin for quantitation by chromatographic and optical methods [100]. Only monophosphorylated species of rhodopsin modified at either 334Ser or 338Ser were detected in the study. The result is surprising because in vitro studies have consistently detected multiple phosphorylation of rhodopsin. The detection of only monophosphorylated species in vivo is also inconsistent with the observation that light-adapted retina sections are immunoreactive with an antibody that recognizes only multiply phosphorylated rhodopsin [39,100,113]. Our own studies, described below, have detected significant amounts of multiple phosphorylation of rhodopsin in vivo. Given this concern as a caveat, 338Ser was found to be phosphorylated relatively quickly in vivo [100]. The site was maximally phosphorylated within 5 minutes following light exposure, then it was dephosphorylated within 15 minutes. 334Ser was phosphorylated to a similar extent in vivo but four times more slowly. 334 Ser is not a good substrate for RK in vitro but it is recognized by PKC, so these findings suggest that the slower reaction may represent endogenous PKC activity [112].

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2.3. Physiological role of rhodopsin phosphorylation The physiological relevance of rhodopsin phosphorylation was established by Ku¨hn who used metabolic labelling methods to show that light stimulates rhodopsin phosphorylation in vivo [114,115]. The idea that there is a relationship between phosphorylation and photosensitivity was suggested by Bownds on the basis of biochemical studies of phototransduction [116,117]. Further support for this came from observations that dephosphorylation of rhodopsin correlates with dark adaptation [114] and with rhodopsin regeneration [100]. The physiological role of rhodopsin phosphorylation has been studied in transgenic mouse rods by expressing a mutant rhodopsin in which all known phosphorylation sites were removed by truncation at 334Ser [118]. Individual rhodopsin molecules were excited by single photons and photoresponses were recorded. A fraction of the responses, corresponding to the fraction of rhodopsins in the cell that were truncated, showed prolonged recovery. These findings are consistent with the idea that phosphorylation followed by arrestin binding, is necessary to quench the ability of R* to activate transducin.

2.3.1. Effects of phosphorylation on R* acti6ity Rhodopsin phosphorylation was linked to phototransduction by observations that ATP quenches lightstimulated cGMP hydrolysis and that this effect requires RK [119,120]. Phosphorylation reduces the ability of R* to stimulate transducin and phosphodiesterase activity [121 – 126]. It does this by shortening the lifetime of active R*, i.e. the state of rhodopsin which stimulates phosphodiesterase activity [126]. It has been suggested that RK may also compete with transducin for binding to R* [13,127]. However, evidence against this idea has come from a different study in which addition of purified RK to a phosphodiesterase assay did not slow the rate at which phosphodiesterase is activated by R* [128]. 2.3.2. Arrestin binding Phosphorylation increases the affinity of R* for arrestin [129] which accelerates even further the rate of R* inactivation [126]. Only the lifetime of R* is affected by arrestin; later steps in phototransduction do not seem to be affected. Arrestin has no effect on phosphodiesterase activity stimulated by the constitutively activated GTPgS form of transducin [124] and it has no effect on phosphodiesterase activity stimulated by a single cycle of transducin activation [130]. The affinity of arrestin for R* increases with the extent of phosphorylation at the rhodopsin C-terminus [126]. Structures necessary for phosphorylation-dependent arrestin binding have not yet been defined pre-

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cisely but 340Thr and 343Ser on the rhodopsin C-terminus appear to be important [107]. Phosphorylated peptides promote arrestin binding to R* [131] perhaps by disrupting an intramolecular interaction within arrestin that constrains its activity [132,130]. The relationships between phosphorylation and arrestin binding may be different in invertebrates. Vino´s and colleagues [95] reported that removal of the C-terminal tail of Drosophila rhodopsin eliminates phosphorylation in vivo but causes no signficant abnormalities in the photoresponse. These findings suggest that Drosophila arrestin does not require R* phosphorylation in order for it to quench activation. It has been suggested that the C-terminus of Drosophila rhodopsin normally competes with arrestin for binding to intracellular loops of rhodopsin [95]. Phosphorylation of the C-terminus may, by reducing the affinity of this interaction, allow arrestin to compete more effectively.

2.3.3. High-gain phosphorylation and Ca 2 + dependence The Ca2 + -dependence of high-gain phosphorylation [42] and its sensitivity to recoverin [23,45,133] suggest that high-gain phosphorylation may regulate photoreceptor sensitivity by pre-phosphorylating unbleached rhodopsin in light-adapted photoreceptors. However, a recent study found that high-gain phosphorylation does not phosphorylate a significant fraction of the unbleached rhodopsin pool in vivo [134]. Ohguro and colleagues attempted to demonstrate Ca2 + -dependent rhodopsin phosphorylation in intact mouse eyes by using background illumination to manipulate free Ca2 + levels within photoreceptors. No difference in light-stimulated phosphorylation was found between dark-adapted and light-adapted eyes [81]. However, the strategy used in those experiments cannot be considered a valid test of the hypothesis that Ca2 + regulates rhodopsin phosphorylation in vivo because intracellular Ca2 + levels fall within seconds upon illumination of rods [135–137]. Phosphorylation detected more than 10–20 s following intense illumination of dark-adapted retinas [81] therefore occurs mostly while the intracellular free Ca2 + concentration is low. Recoverin would not be expected to inhibit rhodopsin phosphorylation under those conditions. A more critical evaluation is needed of the physiological importance of Ca2 + , recoverin and high-gain phosphorylation. Concluding remarks: Enzymes that phosphorylate and dephosphorylate rhodopsin have been identified and characterized, sites of phosphorylation on rhodopsin are now known and the biochemical and physiological consequences of rhodopsin phosphorylation have been characterized. However, as noted in this review there are many issues that are still unresolved, e.g. the roles of kinase autophosphorylation and highgain phosphorylation, the identity of the enzyme that dephosphorylates rhodopsin, the role of phosphoryla-

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tion in cone function and the physiological roles of Ca2 + , recoverin and high-gain phosphorylation. New strategies and methods such as the one described in the following section will help to resolve these issues.

3. A new method for analyzing phosphorylation of rhodopsin in intact retinas

3.1. Introduction We recently developed a method to analyze rhodopsin phosphorylation in mouse retinas ex vivo. The method is based on mass spectrometry methodology and does not require metabolic labelling of retinas. Phosphorylation and dephosphorylation reactions are blocked during the analysis by extracting retinas in 6 M urea. The following sections provide experimental details about the method. In a separate report we will describe the use of this method to investigate regulation of rhodopsin phosphorylation by Ca2 + and recoverin in intact retinas.

3.2. Materials and methods 3.2.1. Phosphorylation of rhodopsin ex 6i6o C57/B6 × 127SvEv mice were dark-adapted overnight and two retinas were dissected into 100 ml Ringer solution (120 mM NaCl, 4.8 mM KCl, 25.2 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4.H2O, 1.3 mM CaCl2, 11 mM glucose, 1 mM glutamine, pH 7.35). The retinas were bleached using a 100 W Halogen light source, neutral density filters, a Schott BG-28 filter, :325 – 600 nm bandwidth and fiber optics. After 20 minutes incubation in darkness, the retinas were triturated with a micropipette tip in 1 ml 6 M deionized urea, 20 mM Tris, 5 mM EDTA, pH 7.35, frozen on dry ice and stored at − 70oC. These procedures were carried out only under infrared illumination with the aid of night-vision goggles. The urea-extracted membranes were thawed under room light and spun at 125000×g for 30 min. They were resuspended in H2O, centrifuged at 55000×g, washed in 200 mM KCl and then in 200 mM KCl, 10 mM Hepes, pH 7.35. The pellets were resuspended, triturated with a micropipette tip and digested with 10 ng Endoproteinase Asp-N (Boerhinger-Mannheim) in 20 ml 200 mM KCl, 10 mM Hepes, pH 7.35 for 18 – 24 h at room temperature. The digested membranes were spun at 125000×g for 30 min. and the peptides were recovered in the supernatant. 3.2.2. Liquid chromatography-electrospray mass spectrometry (LC-MS) of Asp-N peptides Then 5–10 ml (equivalent to 0.5 – 1 retina) of peptides were separated in a capillary column packed with Vydac 218TPC18 silica (20– 30 mM) using a 0 – 80% acetonitrile gradient in the presence of 0.05% trifluoroacetic acid. Chromatographed peptides were infused into a Sciex API

III triple-quadrapole mass spectrometer fitted with a nebulization-assisted electrospray ionization source (PE/ Sciex, Thornhill, Ontario).

3.2.3. Phosphorylation and purification of rhodopsin Asp-N peptides from bo6ine rod outer segments (ROS) Bovine ROS were prepared from 100 dark-adapted frozen retinas [4]. After thawing the retinas in the dark, ROS were purified in room light in buffer containing 2.3 mM ATP and 500 nM Okadaic acid (Sigma). The ROS membranes were resuspended in 6 M buffered urea, washed and digested with 4 mg endoproteinase Asp-N by scaling up the procedure described above for mouse retinas. Nonphosphorylated, mono-, di- and tri-phosphorylated rhodopsin C terminal Asp-N peptides were purified on a Vydac C18 column using a 0–30%/80min acetonitrile gradient (in the presence of 0.05% TFA) and identified by mass using a Sciex API III triple-quadrapole mass spectrometer. The peptides were quantitated by amino acid analysis. Synthetic non- and monophosphorylated peptides corresponding to the mouse rhodopsin C-terminus were purchased from Anaspec, (San Jose, CA) 3.3. Results 3.3.1. Use of LC-MS to determine phosphorylation states of rhodopsin To analyze rhodopsin phosphorylation ex vivo we dissected retinas from the eyes of dark-adapted mice, exposed them to light and quenched phosphorylation reactions by homogenizing the retinas in 6 M urea. C terminal peptides were cleaved from rhodopsin (DDDASATASKTETSQVAPA) in the urea-stripped mouse retinal membranes using endoproteinase Asp-N ([34]). We found that we can detect (M+2H)2 + ions of nonphosphorylated (‘0P’)(m/z = 933.6), mono(‘1P’)(973.6), di-(‘2P’)(1013.6) and tri-phosphorylated (‘3P’)(1053.6) peptides by LC-MS using as little as one-half of a mouse retina (Fig. 1A,B and Table 1). The hydrophilic C-terminal peptides from rhodopsin elute Table 1 Phosphorylation after flash % Bleach

0 PO4

1 PO4

2 PO4

3 PO4

0 0.17 1.7 17 100

97.3 96.7 97.2 84.0 61.5

2.7 3.3 2.8 12.7 25.5

3.3 (1.4) 11.9 (2.7)

1.0 (1.5)

(0.5) (0.8) (3.6) (3.6)

(0.5) (0.8) (2.2) (2.7)

Retinas were bleached with full intensity light followed by 20 min incubation in the dark. The percentage unphosphorylated rhodopsin C-termini were determined as described in the text. Each data point represents 1 – 6 samples. S.D. are presented in parentheses.

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from the column before the bulk of other components in the mixture. The identities of the peptides were confirmed by comparing retention times of native peptides with synthetic 0P and 1P mouse rhodopsin C-terminal peptides (data not shown).

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3.3.2. Effects of phosphorylation on peptide detection To quantitate rhodopsin phosphorylation by LC-MS we first needed to determine relative efficiencies with which MS detects non-phosphorylated and phosphorylated peptides. We found that equimolar concentrations (10–50 pmol each) of the 0P and 1P synthetic peptides derived from the mouse rhodopsin C-terminal sequence were detected at a ratio of 1:0.5 (data not shown). 2P and 3P synthetic peptides from mouse were unavailable, so we purified 0P, 1P, 2P and 3P peptides from an Asp-N digest of bovine photoreceptor membranes. The peptides were quantitated by amino acid analysis and equimolar amounts (10–50 pmol) were analyzed by LC-MS (Fig. 1C). The peptides were detected at average ratios of 1.0 (0P): 0.5 (1P): 0.25 (2P): 0.05 (3P). We subsequently used these values as correction factors when quantitating peptides from mouse retinas. The ratios were found to be constant within the range of signals we use to quantify peptides isolated from mouse retinas (4–50× 106 counts). 3.3.3. Rhodopsin phosphorylation following illumination Retinas were flashed with unattenuated light (730 mW/cm2) for 0, 0.025, 0.25, 2.5, or 25 s to bleach 0, 0.17, 1.7, 17 and 100% of the rhodopsin in the retina sample. The retinas were incubated in darkness following the flash, then they were homogenized with 6 M urea. The results of an analysis that used a 20 min. postflash dark incubation are shown in Table 1. 2–3% of C-terminal peptides were consistently found to be monophosphorylated when retinas were isolated from fully dark adapted mice then incubated in darkness for 20 min. No phosphorylation above the dark level was detected with this protocol when the retinas were exposed to light bleaching 0.17% or 1.7% of the

Fig. 1.

Fig. 1. LC-MS of C terminal peptides from mouse rhodopsin. A. Total ion current of an endoproteinase Asp-N digest of 100% bleached mouse retinal membranes. The hydophilic rhodopsin C terminal peptides elute in the first peak before other retinal contaminants. B. The shaded area in A is expanded in in this panel. Rhodopsin peptides were identified by mass/2 and extracted from the total ion current. Total counts under each peak (shaded area) were calculated by MacSpec 3.3. 100% relative intensity =1572000 for no PO4, 306000 for 1 PO4, 104000 for 2 PO4 and 30000 for 3 PO4. C. To determine the relative ionization efficiency of phosphopeptides by LC-MS, nonphosphorylated and phosphorylated rhodopsin C terminal peptides were purified by HPLC from bovine ROS. The peptides were quantitated by amino acid analysis. Equimolar concentrations of tri-, di-, mono- and non-phosphorylated peptides were analyzed by LC-MS and the area under the peaks integrated. The areas are listed above the peaks in the figure. 100% relative intensity =2192000 for no PO4, 1504000 for 1 PO4, 960000 for 2 PO4 and 192000 for 3 PO4.

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rhodopsin in the retinas. However, when retinas were flashed with light bleaching 17% of the rhodopsin 13% of C-termini were monophosphorylated and 3% were doubly phosphorylated. Exposure of retinas to illumination that bleached 100% of the rhodopsin (25 sec at maximum intensity), yielded 25% mono-phosphorylation, 12% double phosphorylation and 1% triple phosphorylation of C-termini.

3.4. Discussion Conventional methods for measuring rhodopsin phosphorylation using bleached and unbleached ROS homogenates with radio-labeled ATP are useful for identifying factors that influence rhodopsin phosphorylation in vitro but they do not necessarily demonstrate how rhodopsin phosphorylation is regulated within a functional photoreceptor cell. In this report we have demonstrated that mass spectrometry can be used to directly quantitate rhodopsin phosphorylation that occurs in photoreceptor cells within intact mouse retinas. The urea extraction step included in our method irreversibly quenches biochemical reactions so that the extent of phosphorylation at the time of quenching can be accurately determined. The sensitivity of LC-MS allows rhodopsin phosphorylation to be measured from as little as one half of a mouse retina. There are notable differences between the results of our study versus studies of in vivo phosphorylation reported previously by another group. The previous studies analyzed phosphorylation in intact mouse eyes by purifying disk membranes and quantitating rhodopsin C-terminal peptides using HPLC and optical detection methods [100,81]. No phosphorylation was detected in dark adapted mouse retinas in those studies. In contrast, we consistently detect 2 – 3% of C-termini from dark-adapted retinas to be monophosphorylated. Furthermore, only monophosphorylated C termini were detected in the previous studies following illumination that bleached 15–43% of rhodopsin. Our method reliably detected significant levels of doubly phosphorylated C-termini at 17% bleach levels. At higher bleach levels we consistently detected triply phosphorylated C-termini as well. The detection of multiply phoshorylated rhodopsin in intact retinas in our study is more consistent with observations that RK catalyzes multiple phosphorylation of rhodopsin in vitro and with the observation that light-adapted retinas are immunoreactive with an antibody that recognizes only multiply phosphorylated species of rhodopsin [113]. The differences between our findings and those of a previous study by Ohguro could reflect differences in the ways C-terminal peptides were isolated for analysis. Both methods require dissection of retinas from mouse eyes but the previous study also requires the dissection following illumination and then purification of disk

membranes before C-terminal peptides are isolated. Purification of disk membranes was done in the presence of kinase and phosphatase inhibitors but it is possible that the inhibitors were not 100% effective during the isolation of disk membranes. Other possible explanations include lower concentrations of Asp-N used in the previous study and the possibility that minor peptides may have been lost during preparative reverse phase purification done prior to analytical HPLC analysis. These differences might account for the absence of multiply phosphorylated species in the previous studies. In the method we describe here, retinas are homogenized directly in 6 M urea which is expected to irreversibly inactivate all biochemical reactions within seconds. The samples were then analyzed directly by mass spectrometry. Our method consistently detects phosphorylation of rhodopsin in fully dark-adapted retinas. 2.49 1.3% of rhodopsin C-termini were found to be mono-phosphorylated in the seven normal dark-adapted mouse retinas we have examined. This finding was unexpected but our conclusion is based on the identification of mono-phosphorylated peptides using two independent criteria, reverse phase LC elution time and mass. It is therefore unlikely that the signals we detect derive from peptides other than monophosphorylated rhodopsin C-termini. However, we are developing more sensitive methods to sequence the phosphorylated peptide to confirm its identity. We expect that not all the rhodopsin C-termini detected in our experiments are derived only from rhodopsin in outer segments. Since whole retinas were used, we also may be detecting C-termini from newly synthesized rhodopsin in the inner segment of the photoreceptor or perhaps from rhodopsin associated with pieces of pigment epithelium unavoidably left attached to the retina. We developed this method to investigate how rhodopsin phosphorylation is regulated in vivo. We recently used it to study the kinetics of rhodopsin phosphorylation after bleaching and to monitor the effects of Ca2 + and recoverin on rhodopsin phosphorylation under physiological conditions. Intracellular Ca2 + levels were controlled by manipulating the composition of extracellular solutions and recoverin and rhodopsin kinase expression were controlled by inactivation of their respective genes. A detailed description of this investigation will be reported elsewhere. Acknowledgements We wish to thank Lowell Erickson for expert advice on mass spectrometry analyses and Vlad Slepak, Tom Ebrey, Kris Palczewski, Ellen Weiss and Kim Campbell for their comments on this manuscript. This study was supported in part by NIH grant EY06641 to JBH.

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