©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural and Enzymatic Aspects of Rhodopsin Phosphorylation (*)

(Received for publication, July 14, 1995; and in revised form, October 4, 1995)

Hiroshi Ohguro (1)(§) Maria Rudnicka-Nawrot (1) Janina Buczyko (1) Xinyu Zhao (1) (3)(¶) J. Alex Taylor (2) Kenneth A. Walsh (2) Krzysztof Palczewski (1) (3)(**)

From the  (1)Departments of Ophthalmology, Biochemistry (2), and (3)Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Photoactivated rhodopsin (Rho*) is phosphorylated near the C terminus at multiple sites, predominantly at Ser, Ser, and Ser. We systematically examined the sites of phosphorylation upon flash activation of Rho in rod outer segment (ROS) homogenates. Addition of an inhibitory antibody against rhodopsin kinase (RK) lowered phosphorylation at Ser, Ser, and Ser, without changing the ratio between phosphorylation sites. In contrast, no effect of protein kinase C was detected after stimulation (by a phorbol ester), inhibition (with H7), or reconstitution of protein kinase C with purified ROS membranes. The stoichiometry and the ratio between different phosphorylation sites in purified Rho were also reproduced using RK, purified to apparent homogeneity from ROS or from an insect cell expression system. Thus, we conclude that light-dependent phosphorylation of Rho is mediated primarily by RK. Depalmitoylation of Rho at Cys and Cys altered the conformation of the C terminus of Rho, as observed by phosphorylation by casein kinase I, but did not affect phosphorylation by RK. The sites of phosphorylation were influenced, however, by the presence of four conserved amino acids at the C terminus of Rho. The accumulation of phosphorylated Ser observed in vivo could result from slower dephosphorylation of this site as compared with dephosphorylation of Ser and Ser. These data provide a molecular mechanism for the site-specific phosphorylation of Rho observed in vivo.


INTRODUCTION

G protein-coupled receptors, a large family of topologically homologous proteins, share several characteristics that include seven transmembrane alpha-helical segments and two domains, extra- and intracellular. Intracellular loops are involved in the interaction with G proteins, G protein-coupled receptor kinases, and arrestins, while extracellular fragments are important for protein folding and, in some cases, for the binding of large polypeptide hormones or calcium (reviewed by Baldwin(1994)). In addition, most of G protein-coupled receptors are co- and/or post-translationally modified by glycosylation at the N terminus and by palmitoylation and phosphorylation on the cytoplasmic surface. Among this group of proteins, Rho possesses a special status because of its high expression in retinal rod photoreceptors and strict cellular compartmentalization to ROS, (^1)the precision in its activation by light, and its rapid physiological response (Hargrave and McDowell, 1992).

In rod photoreceptor cells, during transition from active Rho* to inactive opsin, the receptor assumes three relatively stable conformations: Meta I, II, and III (Baumann and Reinheimer, 1973). Meta II binds to and activates hundreds of photoreceptor-specific G proteins (transducins), thus initiating the signal-amplifying cascade of reactions (Hargrave et al., 1993). This activated state of Rho, along with Meta I, serve as substrates for RK (Paulsen and Bentrop, 1983; Pulvermüller et al., 1993), a major protein kinase in ROS. The dissociation of RK from Rho* is followed by the binding of p44 and/or arrestin, thereby preventing continuous G protein activation (Wilden et al., 1986; Palczewski et al., 1994b).

Studies in vitro have shown that Rho* is phosphorylated at multiple Ser and Thr residues. Depending on the experimental conditions, the initial sites of phosphorylation were identified at Ser and Ser (McDowell et al., 1993; Ohguro et al., 1993; Papac et al., 1993), prior to multiple phosphorylation. The binding of arrestin to Rho* and the reduction of the photolyzed chromophore, all-trans-retinal, by membrane-bound retinol dehydrogenase limit the high stoichiometry of phosphorylation (Ohguro et al., 1994b). In vivo analysis however, stands in sharp contrast with in vitro studies, because only monophosphorylated species were found at Ser, Ser, and Ser (Ohguro et al., 1995). Kawamura(1994) and, subsequently, other investigators (Gorodovikova et al., 1994a, 1994b) proposed that RK is under a calcium mediated inhibition through recoverin, a retina-specific calcium-binding protein (Dizhoor et al., 1991; Lambrecht and Koch, 1991; Polans et al., 1991). PKC was also proposed to phosphorylate Rho in a light-independent manner in a reconstituted system (Greene et al., 1995) or in the whole retina when PKC was hyperactivated by a phorbol ester (Newton and Williams, 1993).

To obtain additional insight into the structural and functional aspects of Rho phosphorylation, we systematically tested whether RK, PKC, or another kinase is responsible for phosphorylation of flash-activated Rho in ROS homogenates and for effects of Rho palmitoylation on the stoichiometry and selectivity of Rho phosphorylation. We also examined the role of the C-terminal four amino acids in the specificity of phosphorylation, the effect of phosphatase on accumulation of phosphorylated species of Rho, and the effect of calcium on Rho phosphorylation/dephosphorylation in vitro and in vivo.


EXPERIMENTAL PROCEDURES

Materials

Bovine ROS were prepared from fresh bovine retinas (Papermaster, 1982). To eliminate endogenous kinases and other soluble proteins, ROS were treated with 5 M urea (Shichi and Somers, 1978) and extensively washed with 10 mM BTP buffer, pH 7.5, containing 100 mM NaCl. Calmodulin and recoverin were purified from bovine retinas (Polans et al., 1993), while RK was isolated from ROS (Palczewski, 1993). PrP 2A holoenzyme and catalytic subunit were partially purified from fresh bovine ROS (Palczewski et al., 1989a, 1989b). The catalytic subunit of PrP 2A was also purified to homogeneity from rabbit skeletal muscle (Tung et al., 1984). Recombinant PrP 2A was purchased from Upstate Biotechnology Inc. (UBI, Lake Placid, NY). Recombinant protein kinase C was purchased from UBI; the constitutively active PKC (alpha-isoenzyme) was a gift from Dr. Michael Walsh (University of Calgary). An anti-RK antibody (GS19; IgG class) was purified from antisera of rabbits immunized with a peptide encompassing the N-terminal region of RK (Palczewski et al., 1993) by protein G-Sepharose column chromatography according to the manufacturer's protocol (Pharmacia Biotech Inc.). Purified casein kinase I was a gift from Dr. Paul Graves (Indiana University), and PrP inhibitor-1 (Inh-1) was a gift from Dr. T. Ingebritsen (Iowa State University). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7), was purchased from ICN Pharmaceutics. Phorbol myristoyl acetate (PMA; 4-beta- phorbol 12-myristate 13-acetate) and 4-alpha-PMA (4-alpha-phorbol 12-myristate 13-acetate) were purchased from Research Biochemicals International. Poly-Lys were obtained from Sigma. All other reagents used were analytical grades and purchased from Sigma or Fisher Scientific.

Methods

Phosphorylation and Dephosphorylation of Rho* in Vitro

Phosphorylation and dephosphorylation of Rho* were studied using fresh ROS homogenates. Under dim red illumination, fresh ROS were suspended in 300 µl of a standard phosphorylation buffer composed of 20 mM BTP, pH 7.5, containing 60 mM KCl, 20 mM NaCl, 0.5 mM [-P]ATP (300 cpm/pmol), 5 mM MgCl(2), 0.1 mM cGMP, 0.1 mM GTP, 0.4 mM EDTA, and 0.16 mM CaCl(2) (calculated free Ca, 50 nM; Schoenmakers et al.(1992)) at Rho concentration of 5 mg/ml. Phosphorylation was initiated by a flash (500 fc) generated by an electronic flash unit (Sunpak 433D, 1-ms duration) from a distance of 2 cm that bleached 15% of Rho as determined by UV/vis spectroscopy (McDowell, 1993). The samples were kept at 30 °C in the dark, and at 5, 20, 40, and 60 min, an aliquot (70 µl) was mixed with a quenching buffer (300 µl) composed of 250 mM potassium phosphate buffer, pH 7.2, containing 200 mM EDTA, 100 mM KF, 5 mM adenosine, and 200 mM KCl. The phosphorylated and unphosphorylated Asp-Ala peptides containing all phosphorylation sites from the C terminus of Rho were generated by endoproteinase Asp-N digestion, and purified by a C18 reverse phase column (Palczewski et al., 1991). These peptides were dissolved in 100 µl of 100 mM Tris buffer, pH 8.0, and further digested with TPCK-treated trypsin (1 µg, Worthington) for 1 h at 30 °C. Unphosphorylated Asp-Ala peptide was fragmented to Asp-Lys and Thr-Ala, as was peptide monophosphorylated at residue Ser or Ser; peptide monophosphorylated at residue Ser and multiply phosphorylated peptides remained undigested. These various peptides were completely separated from each other using a C18 reverse phase column (2.1 times 250 mm, Vydac 218TP52) employing a linear gradient of acetonitrile (0-30%) in 0.05% trifluoroacetic acid during 60 min at a flow rate of 0.25 ml/min (Ohguro et al., 1993). The ratio of multiple and monophosphorylated forms and the distribution of phosphate groups of monophosphorylated species among the different sites were calculated from the P-radioactive profiles from the C18 column, calibrated with authentic standards. The sequences of the phosphorylated peptide standards were verified by mass spectrometric analysis (Ohguro et al., 1993, 1994b, 1995).

Assays of PrP and RK Activities

PrP activities were determined using P-labeled opsin or phosphorylase a as substrates (Palczewski et al., 1989). Briefly, the assay was performed in 50 mM Tris buffer, pH 7.0, containing 50 mM 2-mercaptoethanol, 0.1 mM EDTA, 1 mg/ml bovine serum albumin, and 0.8 mg/ml P-labeled opsin (or 0.2 mg/ml of P-labeled phosphorylase a) at 30 °C for 5-15 min. The reaction was terminated and proteins were precipitated by adding 100 µl of 20% trichloroacetic acid. The released [P]phosphate was determined by radioactivity counting. RK activity was assayed using urea-washed ROS (Palczewski, 1993).

Expression of RK in Insect Cells

The full-length StuI/BamHI fragment of RK (1847 base pairs; Lorenz et al.(1991)) was subcloned into a baculovirus transfer vector, pVL1393 (Pharmingen). pVL-RK (3 µg) and helper virus carrying a lethal deletion (0.5 µg) were co-transfected into insect Sf9 cells (2 times 10^6) (Invitrogen) in a 60-mm tissue culture dish using a BaculoGold(TM) transfection kit (Pharmingen). A single recombinant baculovirus plaque was amplified to produce a virus stock. Three 150-mm plates containing a monolayer of High Five insect cells (Invitrogen) were infected with the recombinant virus. Cells were harvested 96 h after the infection, collected by centrifugation at 5,000 rpm, homogenized with 10 mM BTP, pH 7.5, containing 0.4% Tween 80, and the suspension was loaded on a DEAE-cellulose column (1 times 10 cm) equilibrated with the same buffer. The column was washed with 10 mM BTP buffer, pH 7.5, containing 0.4% Tween 80, and RK was eluted with 100 mM NaCl in the same buffer. The fractions containing RK activity were combined and mixed with 2 µg of PrP 2A, dialyzed overnight against 10 mM BTP buffer, pH 7.5, containing 0.4% Tween 80 and 1 mM benzamidine (1 liter), and loaded onto a heparin-Sepharose column (1 times 5 cm) equilibrated with the same buffer. After the column was washed with 10 mM BTP buffer, pH 7.5, containing 0.4% Tween 80, 1 mM MgCl(2), and 125 mM NaCl, RK (>98% pure, 300 µg) was eluted with ATP in 10 mM BTP buffer, pH 7.5, containing 0.4% Tween 80, 1 mM MgCl(2), and 100 mM NaCl (for details see Palczewski et al.(1992) and Palczewski(1993)).

Preparation of Phosphorylated Depalmitoylated and Acetylated Rho

Palmitoyl groups were partially removed from Rho without the hydrolysis of chromophore, by treating urea-washed Rho with 1 M hydroxylamine/HCl, pH 7.5, at 30 °C for 2 h (Morrison et al., 1991). The membranes were extensively washed with 10 mM Hepes buffer, pH 7.5, containing 100 mM NaCl. Mixtures of depalmitoylated, monopalmitoylated, and dipalmitoylated Rho were phosphorylated by purified RK in 20 mM BTP buffer, pH 7.5, containing 5 mM MgCl(2), 0.5 mM [-P]ATP (300 cpm/pmol), and 100 mM NaCl for 30 min under a 150-watt lamp from a distance of 30 cm. The reaction was terminated by the addition of 250 mM potassium phosphate buffer, pH 7.2, containing 200 mM EDTA, 100 mM KF, 5 mM adenosine, and 200 mM KCl (300 µl). The membranes were extensively washed with 100 mM sodium borate buffer, pH 8.0, before modification of Lys and Cys residues with acetic anhydride (Ohguro et al., 1994a) and iodoacetic acid (Glazer et al., 1975), respectively. The modified Rho was digested with trypsin (trypsin to Rho ratio, 1:50 (w/w)) for 60 min at 30 °C in 50 mM Tris buffer, pH 8.0, containing 0.1% SDS. The acetylated, phosphorylated, carboxymethylated C-terminal 34-residue peptides (Asn-Ala) containing zero, one, or two palmitolyl groups were separated on a C8 HPLC, pH-stable column (4.6 times 250 mm, Vydac) employing a 40-min linear gradient of acetonitrile (0-70%) in 0.1% ammonium acetate, pH 9.0, at a flow rate of 0.4 ml/min. Each phosphorylated Asn-Ala peptide (with two, one, or zero palmitoylated groups) was subdigested with endoproteinase Asp-N, and the mixture of Asp-Ala peptides, phosphorylated at different positions, purified on a C18 column (Palczewski et al., 1991). These mixtures were then separated into di-, mono-, and unphosphorylated forms by a C18 HPLC column (1 times 250 mm, Vydac 218TP51) employing a linear gradient of acetonitrile (0-20%) in 0.04% heptafluorobutyric acid during 80 min (Ohguro et al., 1995). The di- and monophosphorylated peptides were further digested with Staphylococcus aureus V8 protease (1 µg, Boehringer Mannheim) in 100 mM Tris buffer, pH 8.0, at 30 °C for 2 h, and proteolytic peptides were separated with a C18 reverse phase column (2.1 times 250 mM, Vydac 201HS52) employing a linear gradient of acetonitrile (0-30%) in 0.05% trifluoroacetic acid during 60 min at a flow rate of 0.25 ml/min. Fractions (0.25 ml each) were collected, mixed with a scintillation mixture, and counted.

Rho Phosphorylation by Casein Kinase I

Urea-washed Rho or depalmitoylated urea-washed Rho (100 µg) was phosphorylated by casein kinase I (1 µg) in 57.5 µl of 20 mM BTP buffer, pH 7.5, containing 5 mM MgCl(2), 0.5 mM [-P]ATP, for 30 min under dark or under illumination at 30 °C. At 20, 40, and 60 min, the reaction was quenched by the addition of 10 µl of 1% SDS, 50% glycerol, and 1 mM 2-mercaptoethanol and analyzed by SDS-PAGE. The opsin bands were cut out, dissolved in 30% H(2)O(2), mixed with a scintillation mixture, and counted. Alternatively, P-labeled Rho precipitated with 0.5 ml of 10% trichloroacetic acid was washed twice with the same solution; the pellet was dissolved in 0.5 ml of 88% formic acid, and the radioactivity was determined.

Phosphorylation of Truncated Forms of Rho

To make Rho truncated at Gln, urea-washed Rho (9 mg) was digested in the dark with carboxypeptidase Y (0.3 mg, Boehringer Mannheim) in 3 ml of 50 mM potassium phosphate buffer, pH 6.0, for 7 h at room temperature. After the digestion, the truncated Rho in the membranes was extensively washed with 10 mM BTP buffer, pH 7.5, containing 100 mM NaCl and 5 mM MgCl(2) by repetitive pelleting of the membranes and suspending in the same buffer. Rho truncated at Glu or Lys was prepared by the method described by Palczewski et al.(1991). Briefly, urea-washed Rho (19.3 mg) was digested with TPCK-treated trypsin (0.96 mg, Worthington), or S. aureus V8 (0.19 mg, Boehringer Mannheim) for 1 h at room temperature. The digestion was quenched by soybean trypsin inhibitor (9.6 mg) or 5 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride, respectively. After the digestion, the truncated Rho in membranes was extensively washed with 10 mM BTP buffer, pH 7.5, containing 100 mM NaCl and 5 mM MgCl(2). The three truncated forms of Rho were phosphorylated by purified RK (Palczewski, 1993) and 0.5 mM [-P]ATP (300 cpm/pmol) under illumination by a 150-watt lamp from a distance of 30 cm, for 30 min at 30 °C. The reaction was terminated by the addition of 250 mM potassium phosphate buffer, pH 7.2, containing 200 mM EDTA, 100 mM KF, 5 mM adenosine, and 200 mM KCl (300 µl). Stoichiometry and sites of phosphorylation were determined by further fragmentation of the Asp endoproteinase-derived peptides and mass spectrometric analysis (Ohguro et al., 1994b).

Rho Phosphorylation in Vivo

Rho phosphorylation in vivo was examined using mice as described by Ohguro et al.(1995). Briefly, mice (80-120/experiment) were 1) kept under constant illumination (8, 16, or 32 fc) for 3 h, or 2) kept under constant illumination and exposed to a single flash (500 fc). Immediately, mice were sacrificed by cervical dislocation, and eyes were enucleated, cut into four fragments, and placed into a solution composed of 20 mM KF, 2 mM EDTA, and 5 mM adenosine (80 µl/retina). The disk membranes were prepared by discontinuous sucrose density gradient centrifugation. Rho C termini were obtained by endoproteinase Asp-N proteolysis and purified by C18 HPLC column chromatography as described above. The peptides monophosphorylated at Ser and Ser/Ser, and unphosphorylated peptides were further separated by rechromatography using a C18 HPLC column (1 times 250 mm, Vydac 218TP51) employing a linear gradient of acetonitrile (0-20%) in 0.04% heptafluorobutyric acid during 80 min. We calculated the stoichiometry of phosphorylation from the absorption area at 225 nm for unphosphorylated peptide and peptides phosphorylated in different positions (Ohguro et al., 1995).

Electrospray, Tandem (MS/MS), and Matrix-assisted Laser Desorption Mass Spectrometry

Electrospray mass spectra were obtained by a Sciex API III triple quadropole mass spectrometer fitted with a nebulization-assisted electrospray ionization source (PE/Sciex, Thornhill, Ontario, Canada) as described by Ohguro et al. (1993). For tandem mass spectrometry, precursor ions were selected with the first of three quadropoles (Q1) for collision-induced dissociation with argon in the second quadropole (Q2), and product ions were scanned by the third quadropole (Q3). Matrix-assisted laser desorption mass spectrometry was performed using a Finnigan Laser Mat TOF mass spectrometer.


RESULTS

In vitro and in vivo studies reveal that light-dependent Rho phosphorylation occurs at the C-terminal Ser, Ser, and Ser (Ohguro et al., 1993, 1994b, 1995; McDowell et al., 1993; Papac et al., 1993). The specificity and extent of phosphorylation could result from 1) reactions catalyzed by two or more protein kinases, 2) different states of palmitoylation of Rho at the C terminus, 3) dynamically altering conformations of Rho* that RK has phosphorylated at different sites, 4) random catalysis by RK; 5) selective phosphatase activity, or 6) metabolism of all-trans-retinal.

Is Rho* Phosphorylated by Protein Kinases Other Than RK?

All phosphorylation sites on opsin were identified within the C-terminal D-A peptide by digesting with trypsin and analyzing by the mass spectrometric methods described by Ohguro et al.(1994). Under standard conditions, ROS were exposed to a single flash, and multiple phosphorylated (61.3 ± 3.9%) and monophosphorylated (38.7%) forms were observed (Table 1). The dominant, singly phosphorylated species were modified at either Ser or Ser, whereas only 14.3 ± 0.7% of the monophosphorylated forms were phosphorylated on Ser. The reaction was not limited by the amount of RK or affected by dilution, because similar results were obtained after flashes that bleached 5% or 10% of Rho, and for Rho concentrations ranging from 1 to 10 mg/ml (data not shown).



Although the extent of phosphorylation was decreased by the inclusion of a specific anti-RK antibody (Palczewski et al., 1993) that inhibits secondary phosphorylation of Rho*, the ratio among initial phosphorylation sites was not changed (Table 1). Similar results were observed with H7, a potent inhibitor of PKC (Hidaka et al., 1984) and a weak inhibitor of RK (Palczewski et al., 1990). Active and inactive phorbol myristoyl acetate isomers (PMA and 4-alpha-PMA) inhibited phosphorylation of Rho* to a similar degree, most likely by perturbing the membrane structure, rather than affecting PKC.

Purified Rho (urea-washed ROS lacking endogenous kinase activity) was phosphorylated by homogeneous RK, purified from ROS or from an insect cell expression system (Fig. 1) at all three Ser residues (Table 1), as in ROS homogenates. Minor variability in the initial sites of phosphorylation may relate to the presence of Tween 80 in preparations of RK. Rho purified in dodecyl-beta-maltoside on concanavalin A-Sepharose (Litman, 1982), was also phosphorylated by homogeneous RK at these three Ser residues (data not shown). No phosphorylation of Rho was detected by recombinant PKC (alpha-isoenzyme) or a constitutively active form of PKC (Allen et al., 1994), despite high specific radioactivity of [-P]ATP (more than 1000 cpm/pmol) and high enzymatic activities of PKCs toward myelin basic protein (2 nmol of phosphate transferred/reaction/10 min). These data show that Rho* in ROS homogenates is phosphorylated at different C-terminal Ser residues exclusively by RK, but not by PKC or any other protein kinases.


Figure 1: Purification of RK from High Five insect cells. A, SDS-PAGE analysis. Aliquots from each step of purification (DEAE-cellulose, lane a; heparin-Sepharose, lane b) was analyzed for protein content using SDS-PAGE (8 µg of total proteins) stained with Coomassie Blue (see ``Experimental Procedures''). Autophosphorylated and dephosphorylated RK is indicated as P(i)-RK and RK, respectively. B, Western blot analysis. Recombinant RK obtained from heparin-Sepharose was identified on Western blots with a panel of antibodies (Palczewski et al., 1993) as exemplified using GS19 antibody. C, autoradiography. Additionally, P-labeled RK (autophosphorylated) obtained from heparin-Sepharose was identified using SDS-PAGE and autoradiography.



Does Palmitoylation Affect Rho Phosphorylation?

Dipalmitoylation of Cys and Cys residues rigidly attaches the C-terminal region of Rho to disk membranes (Ovchinnikov et al., 1988; Papac et al., 1992; Moench et al., 1994), modifying accessibility of this region for the interaction with soluble proteins. Before we could analytically dissect the phosphorylation sites on depalmitoylated Rho, we had to solve a difficult technical problem. The usual depalmitoylation protocol employs 1 M NH(2)OH (Morrison et al., 1991), producing a mixture of di-, mono-, and depalmitoylated Rho. Consequently, to identify the form of Rho from which a phosphorylated fragment is derived, the peptide should contain a ``palmitoylation tag.'' To separate depalmitoylated peptide from mono- and dipalmitoylated forms, phosphorylated opsin was acetylated to block Lys residues, carboxymethylated to block free Cys residues, and digested with trypsin at Arg. The peptides heterogeneously palmitoylated at Cys and Cys were separated on a C8, pH-stable, HPLC column to depalmitoylated, monopalmitoylated, and dipalmitoylated Asn-Ala peptides (Fig. 2A, panels a and b-d). The identity of these peptides were confirmed by partial or complete depalmitoylation, Edman degradation, and matrix-assisted laser desorption mass spectrometry.


Figure 2: Analysis of the phosphorylation sites on depalmitoylated or dipalmitoylated Rho. A, separation of the depalmitoylated, monopalmitoylated, and dipalmitoylated C-terminal peptide of Rho (Asn-Ala) on a pH-stable, C8 HPLC column (see ``Experimental Procedures''). a represents the absorption profile; b-d represent the P profile for the following samples. b, the doubly palmitoylated tryptic peptide obtained from P-labeled, carboxymethylated, acetylated opsin was eluted at 60% acetonitrile (fractions 27-30), on a shoulder of the major absorption peak in fractions 25-26; c, the tryptic digest was treated with 2-mercaptoethanol (5 mM) for 30 min at 30 °C prior to injection on the column. This procedure led to partial removal of the palmitoyl group and the elution of the radioactive peptides in fractions 8-12 (depalmitoylated peptide; open circle), 20-23 (monopalmitoylated; closed circle), and 25-26 (dipalmitoylated peptide, closed triangle); d, the tryptic digest was depalmitoylated with hydroxylamine (1 M) for 30 min at 30 °C prior to injection on the column (fractions 8-12, depalmitoylated peptide). The identity of the major phosphorylated component (panel A, b) was determined by a combination of Edman degradation and laser desorption mass spectrometry as follows: the radioactive peak was pooled, dried, and subjected to hydroxylamine treatment (see panel A, d) and rechromatographed on the same column. The radioactive peptide was eluted earlier in the gradient, whereas contaminated peptides were eluted without change late in the chromatography. Edman sequence analysis revealed that the radioactive peptide starts with the sequence: NCMVTTL . . . , which indicates the predicted tryptic peptide beginning at residue 315 from the C terminus of acetylated opsin. The average molecular mass determined for this peptide by laser desorption mass spectrometry was 3916 Da (observed), in accordance with the calculated mass(3900) for the modified C-terminal peptide of Rho (Asn-Ala). B, analysis of the phosphorylation sites of depalmitoylated and dipalmitoylated Rho. Depalmitoylated and dipalmitoylated peptides from partly depalmitoylated Rho were separated as described above, individually digested with Asp-N endoproteinase, and purified on C18 HPLC (see ``Experimental Procedures''). The Asp-N proteolytic peptides were separated into di-, mono-, and unphosphorylated peptides by C18 HPLC. Next, the differently phosphorylated peptides were subdigested with S. aureus V8 protease, and proteolytic peptides were separated into TSQVAPA and ASTTVSK*TE (where K* is acetylated Lys) using C18 HPLC. The peptides were monitored by absorbance at 215 nm (panel a) and their identity confirmed by MS/MS. b, P-radioactive profile for monophosphorylated peptide obtained from depalmitoylated Rho (open circle) and dipalmitoylated Rho (closed squares). Peptide TSQVAPA phosphorylated at Ser is marked by an open circle (fractions 25-27), while peptide ASTTVSK*TE phosphorylated at Ser or Ser is marked by a closed circle (fractions 29-32). c, P-radioactive profile for diphosphorylated peptides obtained from depalmitoylated Rho (open circle) and dipalmitoylated Rho (closed squares).



To identify phosphorylation sites, a mixture of partially depalmitoylated Rho (Morrison et al., 1991) was phosphorylated with purified RK. The distribution of phosphate on di-, mono-, and depalmitoylated tryptic Asn-Ala peptides was analyzed by further digestion of individual species with endoproteinase Asp-N. Di-, mono-, and unphosphorylated Asp-Ala mixture of peptides were separated to individual phosphorylated components by HPLC chromatography in the presence of heptafluorobutyric acid (Ohguro and Palczewski, 1995). Di- and monophosphorylated peptides were further digested with S. aureus V8. No differences in phosphorylation for di- and depalmitoylated Rho were observed (Fig. 2B). Similar results were obtained for monopalmitoylated Rho* (data not shown). Phosphorylation was also not altered for depalmitoylated Rho after modification of Cys and Cys by carboxymethylation or S-sulfenylsulfonylation (negative charged groups), or carboxamidomethylation (neutral groups). Importantly, neither the rate of dephosphorylation by catalytic or holo-PrP 2A nor the binding of arrestin was affected by depalmitoylation (data not shown).

In contrast, casein kinase I phosphorylated depalmitoylated Rho/opsin decisively faster than Rho*, while Rho was not phosphorylated (Fig. 3). As determined by mass spectrometric analysis and proteolytic fragmentations, the phosphorylation was restricted to the C-terminal region of Rho, which contains a consensus sequence for casein kinase I, Asp-Asp-Glu-X-Ser (Agostinis et al., 1989). (^2)Apparently, depalmitoylation removed some conformational or accessibility constraints to casein kinase I.


Figure 3: Phosphorylation of depalmitoylated Rho but not palmitoylated Rho by casein kinase I. Urea-washed Rho or depalmitoylated urea-washed Rho (100 µg) was phosphorylated by casein kinase I (1 µg) at 30 °C in the dark or under 150-watt illumination. At each indicated time, an aliquot was precipitated with 10% trichloroacetic acid, and the stoichiometry was determined by P counting (A) or mixed with SDS-PAGE sample buffer and analyzed by SDS-PAGE. After staining and destaining of the gels, Rho phosphorylation was analyzed at 60 min by autoradiography (B).



How Does the C-terminal Fragment of Rho Affect Phosphorylation?

Among the opsin family of proteins, the sequence of the C-terminal four amino acids is highly conserved and mutations within this region have been frequently associated with retinitis pigmentosa (Macke et al., 1995). These observations suggest that this tetrapeptide may contribute to a conformation important for phosphorylation. To test this hypothesis, three Rho* proteins truncated at Gln, Glu, and Lys were phosphorylated by RK, and the C-terminal peptides purified by HPLC after digestion with endoproteinase Asp-N. For each truncated Rho, multi-, mono-, and unphosphorylated peptides was further separated by HPLC using heptafluorobutyric acid (Ohguro et al., 1995), and then monophosphorylated peptides were rechromatographed under standard conditions on a C18 column. The radioactive profiles showed that C-terminal truncation reduced multiple phosphorylation (Table 2). A single major monophosphorylated peptide (peak a, Fig. 4A) was obtained from Gln Rho, whereas two species of monophosphorylated peptides (peaks b and c and peaks d and e) were purified from Glu Rho and Lys Rho, respectively (Fig. 4A). MS/MS spectra of the monophosphorylated peptides a-e (Fig. 4B) identified the major phosphorylation sites of Gln Rho (Ser, minor at Ser), Glu Rho (Ser, Ser), and Lys Rho (Ser, Thr) (Table 2). These data demonstrate that the C-terminal conformation has a strong influence on the interaction with RK and may regulate the sites of phosphorylation within Rho*.




Figure 4: Analysis of the phosphorylation sites on truncated Rho. A, C-terminal peptides were obtained from RK-phosphorylated, Gln-truncated Rho, Glu-truncated Rho, and Lys-truncated Rho by endoproteinase Asp-N cleavage and conventional C18 HPLC. Each product was further separated into multiply phosphorylated, monophosphorylated, and unphosphorylated peptides by a C18 HPLC microbore column using heptafluorobutyric acid (see ``Experimental Procedures''). Panel A illustrates HPLC of the monophosphorylated peptides during rechromatographed. B, MS/MS spectra of monophosphorylated peptides a-e from panel A. a, MS/MS spectrum of DDEASTTVSKTETSQ, the singly phosphorylated (M+2H) ion at m/z 839.8. b and c, MS/MS spectra of singly phosphorylated (M+2H) ions at m/z 681.7 from corresponding to DDEASTTVSKTE. d and e, MS/MS spectra of singly phosphorylated (M+2H) ions at m/z 567.2 corresponding to DDEASTTVSK. The phosphorylation site (closed circle above sequence) was confirmed by fragment b and y ions in each panel. The ions designated with open circles indicate beta-elimination of phosphate group.



Does Rho Phosphatase Preferentially Dephosphorylate Specific Sites on Phosphorylated Rho?

Addition of a generic PrP inhibitor, KF, increased the extent of phosphorylation in ROS suspensions, presumably by preventing dephosphorylation, while an activator of a latent form of PrP 2A (Cai et al., 1995), Co, accelerated dephosphorylation (Fig. 5A). Addition of extra PrP 2A affected only minimally the phosphorylation level at 5 min, but accelerated the dephosphorylation of opsin, particularly at Ser (Fig. 5B). These differences in the preferred sites of opsin dephosphorylation may be a factor in the accumulation in vitro of species phosphorylated at Ser or Ser.


Figure 5: Dephosphorylation of Rho* in ROS homogenates. A, the time course of Rho phosphorylation after flash was performed as described under ``Experimental Procedures'' in the presence of KF (50 mM), CoCl(2) (0.2 mM), or PrP 2A (620 pmol of released phosphate/min/phosphatase activity). At 5-, 20-, 40-, and 60-min time points, the reaction was quenched and the membranes were collected by centrifugation, washed sequentially with 200 mM KCl and 10 mM Hepes buffer, pH 7.5, and digested with endoproteinase Asp-N overnight at room temperature. Rho phosphorylation was determined by P radioactivity associated with the HPLC-purified C-terminal peptide. B, percent of monophosphorylated Rho at different sites, Ser, Ser, or Ser was determined by radioactive profiles of the trypsin digests of the C-terminal peptides as described under ``Experimental Procedures.''



How Do Calcium and Recoverin Affect Rho Phosphorylation in Vitro?

At micromolar concentrations of free calcium, recoverin has been reported to inhibit RK activity in a reconstituted system (Kawamura, 1993; Kawamura et al., 1994). We confirmed these results and found that phosphorylation of Rho* is typically inhibited by 10-20% in physiological range of free calcium (50-600 nM; Gray-Keller et al.(1994)), without changes in the sites of phosphorylation (data not shown). We also found that recoverin/Ca activated PrP 2A holoenzyme, but not the catalytic subunit or calmodulin (Fig. 6). The activation was less than that by poly-Lys. In contrast, dephosphorylation of phosphorylase a with holo-PrP 2A was unaffected by recoverin, while it was stimulated by poly-Lys (data not shown). These data suggest that the calcium effect mediated by recoverin is specific for the holo-enzyme and phosphorylated opsin.


Figure 6: Activation of PrP 2A by recoverin and Ca. The effects of recoverin and/or Ca on PrP 2A catalytic subunit and holoenzyme was studied in 60 µl of 50 mM Tris buffer, pH 7.0, containing 50 mM 2-mercaptoethanol, 0.1 mM EDTA, 1 mg/ml bovine serum albumin, and 0.8 mg/ml P-labeled opsin). The final concentrations were as follows: 100 µM Ca, 16 µM calmodulin, 1 mM (free calcium 50 nM) EGTA, 1 mM MgCl(2); 1.37 µM recoverin; 100 nMPrP inhibitor I (Inh-1); 0.4 mg/ml poly-Lys. The reaction was carried out for 15 and 8 min for holoenzyme and catalytic subunit, respectively, at 30 °C (maximum dephosphorylation was less than 10% of total phospho-opsin). Experiments were performed in triplicate.



Do Different Background Illuminations Affect in Vivo Phosphorylation of Mouse Rho?

To decipher the effect of calcium on Rho phosphorylation in vivo, we tested the level of phosphorylation using light-adapted mice. The idea is that continuous background illumination closes the cation channels (Lagnado and Baylor, 1992) and, in turn, lowers the internal free calcium in photoreceptor outer segments. Background illumination at 8 fc led to phosphorylation of Ser and Ser/Ser at similar levels of 3.3 and 3.1% of total Rho (Table 3). A single intense flash produced a modest increase in the phosphorylation of Ser and a more significant increase at Ser/Ser. The latter increase is tempered as the background illumination is increased, and consequently free calcium decreased. These data suggest that as calcium is lowered, further phosphorylation in response to flash of light was decreased, in contrast to observed in vitro effects of recoverin on Rho phosphorylation.




DISCUSSION

Phosphorylation of Rho*: One or Many Protein Kinases

Results of Rho phosphorylation in vivo (Ohguro et al., 1995) suggested that upon illumination, Ser, Ser, and Ser are phosphorylated. All three sites were phosphorylated using purified native or recombinant RK and native ROS disk membranes stripped of endogenous protein kinases, or highly purified Rho. Furthermore, a specific RK-inhibitory antibody did not change the ratio of phosphorylation sites in ROS homogenates.

No evidence was found for involvement of PKC in Rho phosphorylation, as an activator or an inhibitor; addition of purified constitutive active or recombinant alpha-isoforms of PKCs did not produce any significant effect. In previous studies, the site(s) of PKC phosphorylation was not identified; however, it was confined to the C-terminal region of Rho phosphorylated by RK (Greene et al., 1995). It is worth noting that this region lacks, even loosely defined, a consensus sequence for PKC (Kennelly and Krebs, 1991), and that phosphorylation described in previous reports was performed with Rho at low nanomolar concentrations (Kelleher and Johnson, 1986; Newton and Williams, 1991), which may produce highly fragmented forms of membrane vesicles. If the sites of phosphorylation by PKC are identical to those modified by RK, based on the amounts of enzymatic activities, the contribution of PKC would be 200-fold less than of RK (Palczewski, 1993; Newton, 1993). It is unlikely that our preparations of ROS lost PKC, because they retained soluble proteins such as arrestin, and they were prepared without buffering calcium, conditions that should promote association of PKC to ROS membranes. It should be noted that electrophysiological experiments with intact Gecko ROS to which several different inhibitors of PKC, including H7, H8, staurosporin, or chelerythine, were introduced, did not affect electrical light responses. (^3)We conclude that it is not necessary to implicate any protein kinase other than RK in the phosphorylation of Rho.

Rho Phosphorylation: Mono- or Multiple Phosphorylated Forms

Multiple Ser and Thr residues at the C-terminal region of Rho, cone pigments, or other G protein-coupled receptors are considered hallmarks for multiple phosphorylation (Baldwin, 1994; Hargrave and McDowell, 1992; Premont et al., 1995). Indeed, in vitro multiple phosphorylation of Rho* was readily observed (Wilden, 1995; Wilden and Kühn, 1982; Aton et al., 1984; Aton, 1986; Adamus et al., 1993); however, only monophosphorylated species are detected in vivo. How does one reconcile these findings? The reason could be inherent in the methods we used to identify phosphorylated sites in vivo, and multiple phosphorylation could be an artifact of in vitro biochemical procedures. Our analysis of in vivo phosphorylation relies on an isolation of ROS membranes that requires 10-20 s (after the flash) before further phosphorylation/dephosphorylation is inhibited, potentially sufficient time for rapid dephosphorylation of multiply phosphorylated Rho. Alternatively, intensive bleaches could overproduce Rho* before it decays to opsin, and this may not be phosphorylated by RK to high stoichiometry.

Arguments against these suppositions are based on evidence that dephosphorylation of Rho is a slow process in vitro and in vivo (minutes) (Palczewski et al., 1989a, 1989b; Fowles et al., 1989; King et al., 1994; Kühn, 1974; Ohguro et al., 1995). Prerequisites to dephosphorylation include binding of p44/arrestin (Hofmann et al., 1992) and reduction of photolyzed chromophore by retinol dehydrogenase (Palczewski et al., 1994a) to remove arrestins (Palczewski et al., 1989b). In addition, slow decay of Meta II (t= 16 s in frog at 30 °C, and even slower release of all-trans-retinal) (Baumann and Reinheimer, 1973) and accumulation of all-trans-retinal in the first minutes following illumination (Zimmerman, 1974), suggest that dephosphorylation occurs on a time scale of minutes, rather than seconds. Finally, the minimum estimated enzymatic activity of RK in vivo would predict the introduction of 3 P(i)/Rho* during 3 s after a bleach that generated 0.4% Rho*, well within the detection limit of our methods. A single phosphorylation is sufficient for the quenching of phototransduction (Bennett and Sitaramayya, 1988), and a prolonged, low intensity illumination (3 h) did not produce multiply phosphorylated species at detectable levels in the mouse retina. The multiple phosphorylation of Rho* could be an artifact of biochemical procedures that uses very dilute ROS preparations with compromised membrane structures, intense bleaches without proper recycling of the visual chromophore, and long phosphorylation time courses.

At intense bleaches in vivo, less than half of the Rho* is phosphorylated (Ohguro et al., 1995), suggesting that some steric hindrance, such as the binding of arrestins, prevents phosphorylation of each Rho molecule. This would be consistent with a report that only 25% of Rho* is accessible to transducin (Fung, 1983). This mechanism could limit phosphorylation of Rho* at high levels of illumination but allow each Rho* to be phosphorylated, at low levels of illumination; however, as ROS membranes are fragmented and arrestins are removed or diluted in vitro, this accessibility could be enhanced.

Requirements for Rho Phosphorylation: Kinase and Rho Conformations

Why is Rho phosphorylated in a heterogeneous manner at these three Ser residues? During photobleaching of Rho, three stable intermediates, Meta I, II, and III, are substrates for RK (Paulsen and Bentrop, 1983), and all appear to be phosphorylated at similar sites (Ohguro et al., 1994b). (^4)Upon binding of RK to Rho*, the enzyme becomes more active either by simple proximity to the C-terminal region (Palczewski et al., 1991), or by actual activation of RK by Rho* (Brown et al., 1993; Pullen et al., 1993; Dean and Akhtar, 1993). We believe that the proximity of the C-terminal peptide, despite its poor affinity for RK (Palczewski et al., 1989c), is sufficient to account for the increased catalysis (Fowles et al., 1988; Palczewski et al., 1991). A weak interaction between the C-terminal peptide of Rho and the active site of RK would promote a more random phosphorylation at the three sites.

Analysis of phosphorylation of C-terminally truncated forms of Rho (Ohguro et al., 1993, 1994b, 1995) or corresponding peptides (Fig. 7) (Pullen and Akhtar, 1994) revealed that the four terminal amino acids (VAPA) are critical in the selectivity of phosphorylation. Multiple phosphorylation appears to be sequential (as described previously by Ohguro et al.(1993)) with a neighboring hydroxyl-containing amino acid being next to the primary site of phosphorylation. Interestingly, Sung et al.(1994) discovered that truncation of Rho at Gln, as found in retinitis pigmentosa, prevented phosphorylation at Ser (Fig. 7), and produced 15% longer time-to-peak in the electrophysiological responses. The complete truncation of the C terminus at Gly leads to more prolonged responses, suggesting that Rho phosphorylation is needed for normal inactivation of the phototransduction cascade (Chen et al., 1995b). Phosphorylation at different sites may have important physiological consequences in serving different inactivation pathways of Rho*.


Figure 7: Preferential phosphorylation of the C-terminal region of Rho. Sites of phosphorylation of Rho were identified by Ohguro et al. (1993, 1994b, 1995, and present work); sites of phosphorylation of the peptides were taken from Pullen and Akhtar(1994). The symbols bullet represent observed pattern of phosphorylation of the underlined residues for monophosphorylated and multiply phosphorylated species. ↕ and &cjs3707; represent frequent mutations or truncations at the C terminus that are associated with retinitis pigmentosa, respectively (Macke et al., 1995).



Dephosphorylation of Rho

Dephosphorylation of Rho is a slow process in biochemical assays and in vivo (Palczewski et al., 1989a, 1989b; Fowles et al., 1989; King et al., 1994). In this paper, we investigate dephosphorylation of Rho in ROS homogenates supplemented with purified PrP 2A (Fig. 5). We found that dephosphorylation occurs in the following order: Ser < Ser < Ser. Considering continuous illumination as a collection of many small flashes, one might predict an accumulation of phosphorylation at slowly dephosphorylating Ser, as compared to Ser/Ser. Indeed, in vivo studies show that Ser remains phosphorylated longer after illumination than Ser/Ser (Ohguro et al., 1995), in agreement with the proposed explanation.

Rho Phosphorylation: Calcium and Recoverin

A mammalian rod photoreceptor of the retina contains 10^8 Rho molecules that participate in signal transduction. Under low bleaching conditions Rho activation causes the photoreceptor cell to undergo hyperpolarization of the plasma membrane. However, in most daytime activities, our rod cells are either saturated or operate in a desensitized mode due to Ca-dependent adaptation to background illumination (Lagnado and Baylor, 1993). It has been proposed that the adaptation processes are moderated, in part, by a calcium-binding protein, recoverin, which inhibits RK activity when in a complex with Ca (Kawamura, 1993). The data in Fig. 6indicate that recoverin stimulates Rho phosphatase activity. Others have reported interactions of recoverin with immobilized opsin used for its purification (Dizhoor et al., 1991), interaction with GCAP that led to temporary misidentification of recoverin as a GC activator (Dizhoor et al., 1991; Lambrecht and Koch, 1991), binding to RK (Gorodovikova et al., 1994a, 1994b; Chen et al., 1995a), and interference with transducin activation. (^5)In these studies, an attractive hypothesis, interaction of recoverin with RK (Kawamura et al., 1993), was investigated indirectly in vivo (Table 3). In response to adaptation to background illumination, the photoreceptor cells lower their endogenous free calcium. In light-adapted animals, we found that a flash produced a decrease in Rho phosphorylation, rather than the increase expected if RK inhibition by recoverin was abolished. These data suggest that the recoverin effects may play only a minor role in a complex regulation of Rho* phosphorylation in vivo.

These studies emphasize the importance of correlating in vitro and in vivo studies. From a combination of both approaches, we interpret Rho phosphorylation as a marker of conformational change in Rho* and RK, and we suggest that heterogeneity of phosphorylation may serve many different physiological functions during light responses in rod photoreceptors.


FOOTNOTES

*
This research was supported in part by United States Public Health Service Grants EY08061, EY01730, and EY09339 and by a grant from Research to Prevent Blindness (to the Department of Ophthalmology). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Ophthalmology, Sapporo Medical University, School of Medicine, S-1, W-17, Chuo-ku, Sapporo 060, Japan.

Predoctoral fellow of the Merck Foundation.

**
Recipient of a Jules and Doris Stein Research to Prevent Blindness Professorship. To whom correspondence should be addressed: University of Washington, Ophthalmology, Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-543-4414; :palczews{at}u.washington.edu.

(^1)
The abbreviations used are: ROS, rod outer segment(s); BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; PKC, protein kinase C; PrP, protein phosphatase; Rho, rhodopsin; Rho*, photolyzed rhodopsin; RK, rhodopsin kinase; transducin, G-protein of the rod cell; HPLC, high performance liquid chromatography; MS/MS, tandem mass spectrometry; fc, footcandles; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; PMA, phorbol 12-myristate 13-acetate; H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine.

(^2)
Phosphorylation at the first site led to subsequent phosphorylation that was confined to the C terminus. This sequential phosphorylation stemmed from formation of a new consensus sequence that involved phospho-Ser/Thr (Litchfield et al., 1990). Small amounts of highly heterogeneous phosphorylated Rho did not allow precise identification of the phosphorylation sites.

(^3)
P. B. Detwiler, unpublished results.

(^4)
Note that minor variabilities between the initial phosphorylation sites were observed in previous studies (Ohguro et al., 1993, 1994b; Papac et al., 1993; McDowell et al., 1993; Palczewski et al., 1995). It is probable that they are a consequence of experimental conditions, including different time courses of phosphorylation, illumination regimes, and different preparations and concentrations of Rho and RK.

(^5)
K. P. Hofmann, unpublished results.


ACKNOWLEDGEMENTS

We thank J. Preston Van Hooser for technical assistance during these studies and Dr. A. H. Milam for comments on the manuscript.


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