(Investigative Ophthalmology and Visual Science. 2001;42:1439-1443.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
Activation of Arrestin: Requirement of Phosphorylation as the Negative Charge on Residues in Synthetic Peptides from the Carboxyl-Terminal Region of Rhodopsin
J. Hugh McDowell1,
Phyllis R. Robinson2,
Ron L. Miller1,
Michael T. Brannock2,
Anatol Arendt1,
W. Clay Smith1 and
Paul A. Hargrave1,3
1 From the Departments of Ophthalmology and
3 Biochemistry and Molecular Biology, University of Florida, Gainesville; and
2 Biological Sciences, University of Maryland, Baltimore County.
 |
Abstract
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PURPOSE. To determine whether substitution of the potential phosphorylation
sites of bovine rhodopsin’s carboxyl-terminal region with the acidic
residues aspartic acid, glutamic acid, or cysteic acid promotes the
activation of arrestin.
METHODS. Three peptide analogues of the 19-residue carboxyl-terminal region of
rhodopsin (330-348) were synthesized: the fully phosphorylated peptide
(7P-peptide), the peptide with all potential phosphorylation sites
substituted with glutamic acid (7E-peptide), and the peptide with the
phosphorylation sites substituted with cysteic acid (7Cya-peptide). The
peptides were tested in assays in which the 7P-peptide had previously
been shown to have an effect. Rhodopsin with glutamic acid (Etail) or
aspartic acid (Dtail) substituted for the phosphorylation sites in
rhodopsin were constructed and expressed in COS-7 cells and tested in
an in vitro assay.
RESULTS. Earlier work has demonstrated that the 7P-peptide activates
arrestin, showing induction of arrestin binding to light-activated
unphosphorylated rhodopsin, inhibition of the light-induced
phosphodiesterase (PDE) activity in rod outer segments (ROS) with
excess arrestin, increase in the initial rapid proteolysis of arrestin
by trypsin, and enhanced reactivity of one of arrestin’s sulfhydryl
groups with inhibition of the reactivity of another. None of
these effects was observed in the presence of 7E-peptide or
7Cya-peptide. The 7Cya-peptide inhibited the PDE activity in ROS, but
the same effect was observed both in the presence and the absence of
excess arrestin. Because none of the other effects was observed with
the 7Cya-peptide, the authors conclude that the 7Cya-peptide does not
activate arrestin, but acts, probably nonspecifically, through some
other part of the transduction system. Considerable arrestin-mediated
rhodopsin inactivation was observed with both the Etail and the Dtail
mutant, although these substitutions did not yield rhodopsins that were
equivalent to phosphorylated rhodopsin.
CONCLUSIONS. These results, taken together, suggest that the negative charge due to
phosphates in the carboxyl-terminal region of rhodopsin are required
for the full activation of arrestin and that acidic amino acids
(carboxyl and sulfonic) do not mimic the negative charge of
phosphorylated residues.
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Introduction
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Visual arrestin inactivates the light-activated visual
G-protein receptor rhodopsin by binding to phosphorylated rhodopsin,
preventing further activation of the G-protein, transducin (reviewed in
Ref. 1
). Phosphorylation of rhodopsin is required for
arrestin to bind to light-activated rhodopsin. Earlier work has
suggested that phosphorylation of rhodopsin is necessary, primarily to
change arrestin’s conformation to an active form that can bind to and
quench rhodopsin. A synthetic peptide that is identical with the fully
phosphorylated carboxyl-terminal region of rhodopsin (330-348 of bovine
rhodopsin), 7P-peptide, activates arrestin.2
The
7P-peptide has been shown to affect the conformation of arrestin by
three additional assays. In the presence of 7P-peptide3
(1) the rate of initial tryptic proteolysis of arrestin is enhanced,
and further cleavage is suppressed (heparin has a similar
effect4
); (2) arrestin inhibits light-induced
phosphodiesterase (PDE) activity bypassing the need for phosphorylated
rhodopsin3
; and (3) the reactivity of one of the
sulfhydryls of arrestin is greatly enhanced, whereas the reactivity of
another is reduced.
In the current work, we asked whether other negatively charged residues
(glutamic acid or cysteic acid) in the synthetic peptide would yield
similar results. In addition, to test whether negatively charged amino
acid residues could substitute for the phosphorylated residues in
vitro, we replaced the seven potentially phosphorylated residues with
glutamic acid and aspartic acid residues and measured the ability of
arrestin to inhibit the activation of transducin by these mutant
rhodopsins.
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Materials and Methods
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Peptide 330-348 from the carboxyl-terminal region of bovine
rhodopsin containing four phosphothreonine (PThr) and three
phosphoserine (PSer) residues was previously synthesized.5
Two new analogues of this peptide were synthesized for the present
study. In one peptide, cysteic acid was substituted for each of the
phosphorylated residues6
7
: DDEAXXXVXKXEXXQVAPA, where
X is cysteic acid (7Cya-peptide). In the other peptide, glutamic acid
was substituted for each of the phosphorylated residues:
DDEAEEEVEKEEEEQVAPA. This peptide (7E-peptide) was made on an automated
peptide synthesizer (model 431A; Perkin Elmer-Applied Biosystems,
Foster City, CA) on resin (FmocAla-Pam; Bachem, Torrence, CA). The
peptide was purified by reversed-phase HPLC (2.5 x 25-cm
Partisil-10 ODS-3 column; Whatman, Clifton, NJ) with a linear gradient
from 100% A (0.1% acetic acid in water) to 30% B (0.1% acetic acid
in acetonitrile) in 40 minutes at 10 ml/min, with the eluent monitored
at 230 nm. The peptide displayed the correct mass spectrum and amino
acid composition and was essentially homogeneous by HPLC. The effect of
the peptides on the light-activated PDE activity in the presence of
excess arrestin was determined as described earlier.3
The ability of the peptides to induce arrestin binding to
light-activated, unphosphorylated rhodopsin was tested using a
centrifuge assay. Disks from rod outer segments (ROS) were prepared
according to the method of Smith et al.8
Arrestin was
prepared by the method of Buczylko and Palczewski9
with
modifications as described earlier.2
The reaction mixture
contained 22 µM rhodopsin in disks, 18 µM arrestin, and
approximately 0.5 mM of the peptide in 0.1 M
NaPO4 buffer prepared by mixing 0.1 M
NaH2PO4 and 0.1 M
Na2HPO4 to pH 7.0 and then
adjusting to a concentration of 0.1 M NaCl. Aliquots (200 µl)
were prepared and either bleached for 2 minutes on a light box or kept
in the dark. The samples were centrifuged at 39,000g for 20
minutes at 4°C. The pellets were washed once with 0.1 M
NaPO4-0.1 M NaCl buffer. The pellets were then
dissolved in a 2x SDS-PAGE sample buffer. SDS-PAGE was performed under
reducing conditions, according to the method of Laemmli.10
The effect of the peptides on the reactivity of the sulfhydryl groups
of arrestin was determined as described earlier.3
Limited
proteolysis using trypsin in the presence of the peptides was performed
as described earlier.2
Rhodopsins with the seven serine and threonine residues in the
carboxyl-terminal region replaced with glutamic acid (Etail) or
aspartic acid (Dtail) were prepared by a method described
earlier.11
The arrestin-mediated quenching of these mutant
rhodopsins was measured as previously described.11
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Results
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Effect of Peptide Analogues on PDE Activity
Light-induced PDE activity in ROS preparations in the presence of
cGMP was measured as a decrease in pH (Figs. 1A
1B
1C
; trace a). In the presence of arrestin and adenosine triphosphate (ATP),
this activity was markedly reduced (Figs. 1A
1B
1C
; trace b). The
reduction in PDE activity was due to the phosphorylation of the
bleached rhodopsin by endogenous rhodopsin kinase followed by arrestin
binding. These reactions blocked the binding and activation of
transducin. Addition of 7P-peptide without added arrestin had little
effect on PDE activity (Fig. 1A
, trace c). However, in the presence of
both 7P-peptide and arrestin, PDE activity was reduced (Fig. 1A
, trace
d) almost to the same extent as with ATP (trace b). Note that there
should be no phosphorylation of rhodopsin under conditions depicted in
traces c and d, because there was no ATP present in these samples. The
7Cya-peptide reduced the PDE activity, both in the presence (Fig. 1B
,
trace f) and the absence (Fig. 1B
, trace e) of added arrestin. This
inhibition in the absence of added arrestin suggests that the
7Cya-peptide did not act in the same manner as the 7P-peptide. In
contrast, the 7E-peptide had only a slight effect on PDE activity in
the presence of added arrestin (Fig. 1C
, trace h) and almost no effect
in the absence of added arrestin (Fig. 1C
, trace g).
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Figure 1. PDE activity in an ROS preparation in the presence of negatively
charged peptides. PDE activity was determined in the presence of excess
arrestin, as described earlier.3
PDE activity cleaved
cGMP, resulting in a release of H+, shown as an upward
deflection in the curves. Traces a and b,
displayed in all three panels, show the PDE activity of the preparation
in the absence (trace a) and presence (trace
b) of ATP. (A) Trace c: PDE activity in
the presence of 7P-peptide (190 µM), but with no added arrestin or
ATP. Trace d: PDE activity in the presence of added arrestin
and 7P-peptide, but no ATP. (B) Trace e: PDE
activity in the presence of 7Cya-peptide (318 µM), but with no added
arrestin or ATP. Trace f: PDE activity in the presence of
added arrestin and 7Cya-peptide, but no ATP. (C) Trace
g: PDE activity in the presence of 7E-peptide (200 µM), but with
no added arrestin or ATP. Trace h: PDE activity in the
presence of added arrestin and 7E-peptide, but no ATP.
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Effect of Peptide Analogues on Binding to Bleached,
Unphosphorylated Rhodopsin
The peptide analogues were tested for their ability to induce
arrestin binding to bleached, unphosphorylated rhodopsin in a
centrifuge binding assay (Fig. 2)
. All samples have disc membranes and added arrestin with no added ATP.
The odd-numbered samples were kept in the dark, and the even-numbered
samples were bleached. The ratio of the arrestin band to the rhodopsin
monomer band was determined by gel scans (NIH Image 1.62; National
Institutes of Health, Bethesda, MD; available in the public domain at
http://www.nih.gov/od/oba) and is shown in the bar graph under the
photograph of the gel. With no peptide present (Fig. 2
, lanes 1, 2),
there was a small increase in arrestin binding to the disc membranes
after bleaching. There was considerably more light-induced arrestin
binding in the presence of the 7P-peptide (lane 4). However, neither
the 7Cya-peptide (lane 6) nor the 7E-peptide (lane 8) showed any
additional light-induced binding when compared with conditions with no
peptide present. These results indicate that only phosphorylated
residues in the rhodopsin carboxyl-terminal peptide serve to activate
arrestin, allowing it to bind to bleached, but unphosphorylated,
rhodopsin.
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Figure 2. Binding of arrestin to ROS in the presence of negatively charged
peptides with (+) and without (-) light. Lanes 1,
2: arrestin binding to unphosphorylated ROS in the
absence of negatively charged peptides; lanes 3,
4: arrestin binding in the presence of 7P-peptide (415
µM); lanes 5, 6: binding in the
presence of 7Cya-peptide (251 µM); and lanes 7,
8: binding in the presence of 7E-peptide (377 µM).
Low-molecular-weight standards are shown at the left of
the gel, and an arrestin standard at the right. The
bar graph gives the quantitative results from scanning
the gel.
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Effect of Peptide Analogues on the Sulfhydryl Reactivity of
Arrestin
The negatively charged peptide analogues were tested for their
ability to enhance the rate of reactivity of one sulfhydryl group of
arrestin and reduce the reactivity of another. In Figure 3
, trace a shows the reaction of the sulfhydryl groups of arrestin with
5,5'-dithiobis (2-nitrobenzoic acid; DTNB) with no peptide additions
and shows the relatively slow reaction of arrestin’s sulfhydryl groups
with DTNB. Neither 7Cya-peptide (trace b) nor 7E-peptide (trace c) had
much effect on the sulfhydryl reactivity. Only 7P-peptide (trace d)
enhanced the reactivity of one sulfhydryl group and inhibited another.
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Figure 3. Reactivity of the sulfhydryl groups of arrestin in the presence of
negatively charged peptides. The reactivity of the sulfhydryl groups of
rhodopsin was assessed using DTNB, as described earlier.3
Trace a contains no peptides. The reactivity was
measured in the presence of 86 µM 7Cya-peptide (trace
b), 140 µM 7E-peptide (trace c), and 66 µM
7P-peptide (trace d).
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Effect of Peptides on Limited Proteolysis of Arrestin by Trypsin
Arrestin was submitted to limited proteolysis by trypsin in the
presence of the synthetic peptides (Fig. 4)
. Only the 7P-peptide (lane 3) and bleached, phosphorylated rhodopsin
(lane 6) affected the limited proteolysis, by enhancing an initial
cleavage followed by inhibition of further digestion. Digestion in the
presence of 7Cya-peptide (lane 4) and 7E-peptide (lane 5) was
essentially identical with the digestion with no additions (lane 2).
This shows that both 7P-peptide and bleached, phosphorylated rhodopsin
affect the conformation of arrestin. However, no conformational changes
were observed in the presence of the other peptide analogues.
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Figure 4. Limited proteolysis of arrestin in the presence of negatively charged
peptides. Arrestin was submitted to limited proteolysis using trypsin,
as described earlier.2
Low-molecular-weight standards are
on the left. Lane 1: undigested arrestin.
The remaining lanes contain arrestin samples submitted to limited
proteolysis with no added peptide (lane 2), 170 µM
7P-peptide (lane 3), 284 µM 7Cya-peptide (lane
4), 351 µM 7E-peptide (lane 5), and freshly
bleached, phosphorylated ROS (lane 6).
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Effect of Aspartic Acid and Glutamic Acid Mutants of the Rhodopsin
Carboxyl-Terminal Region
Rhodopsin mutants were prepared by substituting acidic residues
for the phosphorylation sites in the carboxyl-terminal region of
rhodopsin. Two mutants were prepared: one with glutamic acid
substituted for each of the three serine and four threonine residues
and the other with aspartic acid substitutions. The ability of arrestin
to quench the activation of transducin by these mutant rhodopsins is
shown in Figure 5
. For both mutants, considerable quenching of the activation was
observed, but only approximately 50% to 60% of the quenching in the
wild type.
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Discussion
|
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A 19-amino-acid peptide that is identical with the completely
phosphorylated carboxyl terminus of bovine rhodopsin was shown to
activate arrestin, allowing it to bind to bleached, unphosphorylated
rhodopsin and to quench transducin activation. Several effects of this
peptide on arrestin conformation were observed. The same peptide in
which glutamic acid residues were substituted for the phosphorylated
residues failed to show any of these effects. Substituting cysteic acid
for the phosphorylated residues produced a peptide that inhibited
light-induced PDE activity, but this occurred both in the presence and
absence of added arrestin. Because none of the other effects on the
conformation of arrestin was observed in the presence of this peptide,
we conclude that the cysteic acid peptide inhibits the PDE through some
other mechanism, not by activating arrestin. Similarly, the 7 glutamic
acid peptide also failed to promote any apparent conformational change
in arrestin. This suggests that the function of phosphorylation is more
than to add negative charges to the carboxyl region of rhodopsin.
The number of phosphorylated residues required to allow arrestin to
fully quench the rhodopsin activation has been the target of several
studies. In one study, only one or two sites were observed to be
phosphorylated (serine 338 and serine 334).12
However, if
only serine 33811
13
or only serine 338 and
33413
were available, little quenching was observed,
suggesting that multiple phosphorylation is required for full quenching
of activated rhodopsin. Replacing one site, serine 343, with glutamic
acid yields a rhodopsin equivalent to wild type in its ability to bind
arrestin, whereas replacing threonine 340 with glutamic acid yields
reduced binding of arrestin.14
Replacing all four
threonines with alanines in the carboxyl-terminal tail also results in
reduced suppression of activated rhodopsin by arrestin.11
In the present study, both of the mutants that contain all the
potential phosphorylation sites converted to negatively charged
residues still did not quench as effectively as when native rhodopsin,
kinase, and ATP were present. This indicates that these negatively
charged residues are not equivalent to phosphoserine or
phosphothreonine in their ability to promote arrestin quenching of
transducin activation.
Although the charged residues were clearly not equivalent to
phosphorylated residues, this finding suggests that other negatively
charged residues can at least partially substitute for phosphorylated
residues in rhodopsin.
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Conclusions
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These results, taken together, suggest that negative charge alone
in the carboxyl-terminal region of rhodopsin is insufficient for the
maximum activation of arrestin, giving rise to its quenching of
transducin activation. Full effectiveness is achieved only with
phosphate. Carboxylic and sulfonic acids are less effective.
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Footnotes
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Supported by National Institutes of Health Grants EY06225, EY06226, and EY08571 (PAH); a departmental award from Research to Prevent Blindness (PAH); a Senior Scientific Investigator Award (PAH); a Career Development Award (WCS); and National Institutes of Health Grant EY 10205-02 (PRR).
Submitted for publication December 18, 2000; accepted February 7, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: J. Hugh McDowell, Department of Ophthalmology, Box 100284 JHMHC, Gainesville, FL 32610. jmcdowel{at}ufl.edu
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References
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Palczewski, K. (1994) Structure and functions of arrestins Protein Sci 3,1355-1361[Abstract]
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Puig, J, Arendt, A, Tomson, FL, et al (1995) Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin FEBS Lett 362,185-188[Medline][Order article via Infotrieve]
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McDowell, JH, Smith, WC, Miller, RL, et al (1999) Sulfhydryl reactivity demonstrates different conformational states for arrestin, arrestin activated by a synthetic phosphopeptide, and constitutively active arrestin Biochem 38,6119-6125[Medline][Order article via Infotrieve]
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Palczewski, K, Pulvermüller, A, Buczylko, J, Hofmann, KP (1991) Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin J Biol Chem 266,18649-18654[Abstract/Free Full Text]
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Arendt, A, McDowell, JH, Abdulaeva, G, Hargrave, PA (1996) Synthesis of multiphosphorylated peptide from a C-terminal bovine rhodopsin sequence Protein Peptide Lett 3,361-368
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Arendt, A, McDowell, JH, Miller, RL, Smith, WC, Hargrave, PA. (2000) Substitution of cysteic acid for the seven phosphorylated residues in bovine rhodopsin’s C-terminal peptide Fields, GB Tam, JP Barany, G eds. Peptides for the New Millennium: Proceedings of the 16th American
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Top
Abstract
Introduction
) demonstrated the
maximum suppression, whereas wild-type rhodopsin in the presence of ATP
but with no kinase (
) gave the minimal suppression. The arrestin
suppression of mutant rhodopsins are shown for Dtail (
) with all
phosphorylation sites converted to aspartic acid residues and for Etail
(
), with all phosphorylation sites converted to glutamic acid.