©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rhodopsin Phosphorylation and Dephosphorylation in Vivo(*)

Hiroshi Ohguro (1), J. Preston Van Hooser (1), Ann H. Milam (1), Krzysztof Palczewski (1) (2)(§)

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rhodopsin is an important member of the superfamily of G protein-coupled receptors. In vitro studies have suggested that multiphosphorylation of rhodopsin is a pivotal step in phototransduction. Because the in vitro biochemical experiments were conducted under non-physiological conditions, we investigated the phosphorylation of mouse rhodopsin in vivo and determined the sites of phosphorylation and the time course of dephosphorylation. We found that a single phosphate group is incorporated into the rhodopsin molecule in a light-dependent manner, primarily at Ser after flashes and at Ser after continuous illumination. Dephosphorylation of these sites had different kinetics and spatial distribution in rod outer segments. Dephosphorylation of Ser was complete within 30 min, while Ser was dephosphorylated much slower (requiring up to 60 min), correlating with the regeneration of rhodopsin. These results suggest that phosphorylation of Ser and Ser plays different roles in phototransduction.


INTRODUCTION

Phosphorylation and dephosphorylation of G protein-coupled receptors are widespread regulatory mechanisms occurring in many tissues(1) . In the vertebrate retina, light triggers rhodopsin photolysis, leading to its activation. The photoactivated rhodopsin catalyzes a nucleotide exchange on the G protein, transducin, that initiates the amplification pathway of the light signal(2, 3, 4) . Rhodopsin is regenerated from the active state by a cascade of reactions that involves phosphorylation by a specific kinase, rhodopsin kinase(5, 6, 7, 8) , and possibly by protein kinase C(9) ; binding of a regulatory protein, arrestin(10) ; reduction of the photolyzed chromophore all-trans-retinal; regeneration with 11-cis-retinal; and dephosphorylation by protein phosphatase 2A(11) . Thus, phosphorylation and dephosphorylation are crucial mechanisms of inactivation of the active form of rhodopsin and its restoration to the dark state(8, 9, 10, 11, 12, 13) . It was shown in vitro that phosphorylation occurred at multiple sites within the C terminus of rhodopsin(14, 15) . However, the functional significance of the multiple phosphorylations of photolyzed rhodopsin is questionable, because at least two mechanisms, binding of arrestin and reduction of photolyzed chromophore by retinol dehydrogenase, prevent high stoichiometric phosphorylation(16) . Recently, we developed methods for directly detecting phosphate incorporation into rhodopsin using selective cleavage of the C terminus of rhodopsin that contains all potential phosphorylation sites(17) , chromatographic separation, and mass spectrometric analysis(16, 18) . These methods enabled us to investigate the phosphorylation and dephosphorylation of rhodopsin in living animals. Results of our in vivo study strongly suggest that rhodopsin phosphorylation is involved in both quenching of phototransduction and dark adaptation.


MATERIALS AND METHODS

All procedures involving animals were performed according to the approved procedures by the University of Washington Animal Care Committee (according to The American Veterinary Medical Association Panel on Euthanasia(38) ).

Chromatographic Separation of Phosphorylated/Unphosphorylated Peptides from Mouse Rhodopsin

In vitro, mouse rod outer segments (ROS)()(19) from 200 retinas were illuminated with continuous light for 45 min in the presence of 1 mM [-P]ATP and analyzed as described previously(18) . In vivo, mice were kept under different illuminations and sacrificed by cervical dislocation. The eyes were dissected, cut in two, and placed into 80 µl/retina of a solution that contained a high concentration of protein kinases and phosphatase inhibitors (20 mM potassium fluoride (KF), 2 mM EDTA, 5 mM adenosine). The procedure at this point takes less than 5 s. The disc membranes were isolated by sucrose centrifugation(20) . Rhodopsin was cleaved with endoproteinase Asp-N (17), and the C-terminal peptide was purified(16, 18) . Rechromatography of the mixture of phosphorylated/unphosphorylated peptides was performed using acetonitrile gradient (0-20%, during 80 min) in the presence of 0.04% heptafluorobutyric acid, pH 2.3, on a C HPLC column (1.0 250 mm, Vydac 218TP51). For the flash experiments, dark adapted mice were subjected to a flash from an electronic flash unit (Sunpak, 433D, 1-ms duration) from a distance of 2 cm.

To determine how much rhodopsin was bleached under different illuminations, the eyes (20/each data point) were cut in two and homogenized with 10 mM Hepes buffer, pH 7.5, containing 10 mM dodecyl -maltoside and 20 mM hydroxylamine. The samples were vortexed for 2 min and centrifuged at 100,000 g for 3 min (Optima-TLX ultracentrifuge, Beckman), and the spectra were recorded before and after complete bleaching.

Immunolabeling of Phosphorylated Rhodopsin

The retinas were fixed immediately post mortem in 4% paraformaldehyde in 0.13 M phosphate buffer, pH 7.4, for 4 h at 4 °C, dissected, and stored overnight in 30% sucrose in the same buffer at 4 °C. Cryostat sections (12 µm) were processed for indirect immunofluorescence using mouse monoclonal antibody RT 97 at a 1:5 dilution (21) and secondary fluorescein isothiocyanate-labeled goat anti-mouse IgG at a 1:50 dilution. Comparable results were obtained with an antibody against phosphorylated rhodopsin (1:100)(22) . As a positive control, adjacent sections from each experiment were processed with antibody 4D2 against the N terminus of rhodopsin (1:20)(23) , which labeled ROS throughout in all lighting conditions. Control sections treated with non-immune mouse IgG or secondary antibody alone showed only labeling of serum in blood vessels.

Mass Spectrometry

Electrospray mass spectra (ES/MS) and tandem mass spectra (MS/MS) were obtained using a Sciex API III triple quadrupole mass spectrometer fitted with a nebulization-assisted electrospray ionization source (PE/Sciex, Thornhill, Ontario) as described by Ohguro et al.(16, 18) .


RESULTS AND DISCUSSION

Phosphorylated and unphosphorylated C-terminal regions of mouse rhodopsin obtained by endoproteinase Asp-N (17) were separated by using an HPLC C18 column and heptafluorobutyric acid as a counterion (Fig. 1). In vitro, all P-labeled mono- and multiphosphorylated peptides were eluted between a peptide derived from transducin (at 27 min) and the unphosphorylated C-terminal peptide of rhodopsin (at 52 min). In vivo, no phosphorylation was detected on rhodopsin from dark adapted mice, while a single or three consecutive flashes produced greater amounts of a phosphorylated peptide eluting at 40 min (peak A) and lesser amounts of a phosphorylated peptide eluting at 44 min (peak B). In contrast, strong or dim continuous illumination for 30 min resulted in production of greater amounts of peak B relative to peak A. Mice exposed to strong light for 30 min and transferred to the dark for 10 min had decreased amounts of peak A and similar amounts of peak B, as compared with the sample without dark incubation. Subsequent exposure of the latter group of mice to three consecutive flashes led to substantial phosphorylation at both positions (Fig. 1), suggesting that each site is independently phosphorylated in a light-dependent manner. Less than 30% of rhodopsin was phosphorylated in vivo in all conditions of illumination.


Figure 1: Elution profiles of phosphorylated and unphosphorylated peptides (DDDASATASKTETSQVAPA) derived from the C terminus of mouse rhodopsin. Left panel, in vitro multiphosphopeptides, monophosphopeptides, and unphosphopeptide of rhodopsin were eluted from the HPLC column at 30, 40, and 52 min, respectively. In vivo, only peak C was observed in the sample from mice kept in the dark for 2 h. Different amounts of peptide derived from transducin -subunit were eluted at 27 min (indicated by an arrow) and reflected variable contamination of ROS membranes by transducin. The single flash (500fc) produced mostly monophosphorylated species (A = 7.3 ± 0.2% and B = 4.5 ± 0.7% of the total (number of experiments, n = 3; number of retinas, n` = 500; percent of photolyzed rhodopsin, R* 15%)). Triple consecutive flashes at 20-s intervals yielded 10.6 ± 1.3% A and 6.2 ± 1.5% B (n = 3, n` = 480, R* = 43%). Right panel, dim (three 50-watt lamps from 100 cm; 32 fc) or strong continuous illumination (two 150-watt lamps from 30 cm; 2000 fc) for 30 min resulted in A = 6.1 ± 0.4%, B = 8.0 ± 1.1% (n = 3, n` = 413, R* <5%) or A = 8.4 ± 1.3%, B = 13.0 ± 2.4% (n = 4, n` = 240, R = 52%), respectively. For 10 min dark adapted animals that had been exposed to strong continuous illumination (A = 2.0%, B = 11.0%), flashes generated A = 9.4 ± 1.1% and B = 16.3 ± 0.6%. The intensity of illumination was expressed in fc. Solid and dottedlines represent UV absorption at 215 nm and P radioactivity, respectively.



The primary site of phosphorylation in vitro (Fig. 1) (16, 18) was at Ser for monophosphorylated species (minor at Ser) (data not shown but see also Refs. 24-26). ES/MS showed that peaks A and B (Fig. 1) are monophosphorylated rhodopsin C termini (predicted mass, 1945.9; observed mass, peak A, 1945.0 and peak B, 1945.2). Staphylococcus aureus V8 protease cleaved peak A and peak B into two peptides: monophosphorylated DDDASATASKTE (predicted mass, 1290.5; observed mass, 1290.6) and unphosphorylated TSQVAPA (predicted mass, 673.4; observed mass, 673.1). MS/MS spectra of the doubly charged ions corresponding to monophosphorylated DDDASATASKTE from peaks A and B revealed peak A as a peptide monophosphorylated at Ser and B as a peptide monophosphorylated at Ser (Fig. 2). These observations were also confirmed by 1) trypsin digestion (while peak A was resistant to proteolysis, peak B was fragmented to two peptides: monophosphorylated DDDASATASK and TETSQVAPA) and 2) coelution with the authentic standard phosphopeptides (data not shown). As a minor component (less than 15%), the monophosphorylated species of rhodopsin at Ser was co-eluted with peak A (phosphorylated at Ser). ES/MS revealed that peak C (Fig. 1) is unphosphorylated at the rhodopsin C terminus (DDDASATASKTETSQVAPA, predicted mass, 1865.9; observed mass, 1865.0).


Figure 2: Tandem mass spectra of rhodopsin phosphopeptides derived from peak A (upper panel) and peak B (lower panel). MS/MS spectra of (M+2H) precursor ion (m/z 645.8) of the monophosphorylated DDDASATASKTE from peak A (upper panel) and B (lowerpanel) yielded ions y-y, b-b and ions produced by phosphate elimination (opencircles), indicating the phosphorylation sites of peak A and peak B at Ser and Ser, respectively (closedcircles).



Dephosphorylation of rhodopsin was studied in mice exposed to three flashes and after strong continuous illumination (Fig. 3). After three flashes, there was further phosphorylation in the dark, which peaked at 5 min for Ser and 20 min for Ser. Continued phosphorylation in the dark may be explained by the formation of metarhodopsin III in vivo after an intense pulse of light, a long lived (10-20 min) substrate for rhodopsin kinase(27) . Dephosphorylation of Ser was nearly completed after 30 min, while dephosphorylation of Ser was much slower, and only 50% of photolyzed rhodopsin was regenerated during 60 min (Fig. 3, leftpanel). After continuous illumination, phosphorylated Ser was dominant over Ser. In the dark, Ser was dephosphorylated more rapidly, while the phosphate group at Ser was removed more slowly. During the first 15 min, 67% of opsin was regenerated (Fig. 3, rightpanel). Dephosphorylation seems to be spatially controlled because the tips of the ROS were dephosphorylated later than the bases (Fig. 4), as visualized using antibodies to phosphorylated neurofilament antibody (21) and phosphorylated rhodopsin (22) on sections of mouse and rat retinas (latter not shown).


Figure 3: Dephosphorylation of rhodopsin at different sites in vivo after three consecutive flashes (left panel) and after continuous illumination (right panel). Each data point represents 100 retinas obtained from pigmented mice of mixed genetic types and gender, which were fully dark adapted and exposed individually to either three consecutive flashes at 20-s intervals or continuous illumination for 30 min and then transferred to dark. At the indicated time, the mice were sacrificed by cervical dislocation, the rod disc membranes were isolated, and the analysis of the sites of phosphorylation was performed. The dottedline with closedcircles represents the observed time course of regeneration that was measured from direct rhodopsin content of mouse eyes.




Figure 4: Immunolabeling of mouse retinas with an antibody against phosphorylated neurofilament protein that also recognizes phosphorylated rhodopsin. Mouse retinas were fixed in constant darkness (leftpanel), constant light (middle panel), or in darkness at 15 min following a single flash of light (right panel). ROS (*) in constant darkness showed no labeling (leftpanel), while ROS (*) fixed in constant light were strongly labeled throughout (middle panel). ROS fixed in darkness at 15 min following a flash of light showed immunolabeling of the distal tips (arrowheads) (right panel). Results similar to middle or rightpanels, respectively, were obtained in mouse retinas fixed immediately or after 30 min in the dark after one flash (data not shown). s, serum in choroidal blood vessels recognized by secondary antibody; arrows, neurofilaments in inner retina, which were labeled in all lighting conditions; scalebar, 100 µm.



Results obtained in these in vivo studies demonstrate that rhodopsin phosphorylation is a light-dependent phenomenon, as shown previously in frog retina in vivo(28) . We found only monophosphorylated species of rhodopsin, although as many as seven to nine phosphoryl groups per photolyzed rhodopsin were found during in vitro studies(14, 15) . The presence of multiple Ser and Thr at the C terminus of many G protein-coupled receptors, considered as putative phosphorylation sites, could result from the high frequency of Ser and Thr in proteins (approximately 15%). Both Ser and Ser, the residues phosphorylated in vivo, are well conserved in vertebrate rhodopsins.

The lower limit of the estimated rate of phosphorylation after a flash is 12% of total rhodopsin per 30 s or 12 µM/s, making this rate compatible with fast reactions of phototransduction. As found in vivo in frogs, the rate of dephosphorylation is slow and is related to bleaching conditions (Fig. 3). Dephosphorylation of rhodopsin at Ser correlates with the time course of regeneration and dark adaptation, suggesting that release of arrestin from phosphorylated photolyzed rhodopsin precedes dephosphorylation (11) and increases the sensitivity of the rods(29) .

Illumination conditions have strong effects on the ratio of phosphorylation at Ser to Ser, suggesting that the specificity of rhodopsin kinase, probably altered by autophosphorylation(30) , may govern two independent processes related to these sites, recovery and adaptation. Alternatively, phosphorylation of metarhodopsin I, II, and III (16, 31, 32) or phosphorylation of non-photolyzed rhodopsin (33) may produce rhodopsin phosphorylated at different sites. The molecular basis for phosphorylation at these sites could also be explained by the action of two different kinases, rhodopsin kinase and protein kinase C, differential binding of arrestin and/or its splice variant p, or selectivity of phosphatase(s) for specific sites, as Ser is closer to the membranes and presumably less accessible. The role of protein kinase C in rhodopsin phosphorylation in vivo remains controversial(9) . Several arguments suggest that in vitro phosphorylation of rhodopsin by protein kinase C is not involved in physiological processes of the photoreceptors. For example, both Ser and Ser that are phosphorylated in vivo are also phosphorylated by rhodopsin kinase in vitro(16) , while in vitro protein kinase C phosphorylates rhodopsin at Thr residue(s)(34) .

Phosphorylation of Ser persists at the tips of ROS, presumably as a result of slow diffusion of protein phosphatase from the inner segments or a decrease in the rate of rhodopsin recycling at another step. Interestingly, the ROS tips have weaker electrophysiological responses (35) and remain saturated for a longer period of time than the ROS bases(36) . The ROS tips are also shed soon after the onset of light in rats maintained in cyclic light(37) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants EY09339, EY08061, EY01311, and EY01730, by an award from Research to Prevent Blindness, Inc. to the Department of Ophthalmology at the University of Washington, and by The Foundation Fighting Blindness, Inc. 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.

§
Recipient of a Jules and Doris Stein Research to Prevent Blindness award. To whom correspondence should be addressed: Dept. of Ophthalmology RJ-10, School of Medicine, University of Washington, Seattle, WA 98195. Tel.: 206-543-9074; Fax: 206-543-4414. E-mail: palczews@u.washington.edu.

The abbreviations used are: ROS, rod outer segment(s); HPLC, high pressure liquid chromatography; ES/MS, electrospray mass spectra; MS/MS, tandem mass spectra; fc, footcandle(s).


ACKNOWLEDGEMENTS

We thank Dr. Ken Walsh for mass spectroscopic analysis, Dr. Jack Saari for help during the manuscript preparation, Dr. Grant Balkema for monoclonal antibody RT97, Dr. Paul Hargrave for anti-phosphorylated rhodopsin monoclonal antibody, Dr. Robert Molday for monoclonal antibody 4D2, and Jean Chang and Dan Possin for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.