From the
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
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.
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) ).
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
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
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
Illumination conditions have strong effects on the ratio of
phosphorylation at Ser
Phosphorylation of Ser
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
-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) .
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.
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) .
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) .
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) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.