(Received for publication, April 7, 1997)
From the Departments of Biochemistry and Molecular
Pharmacology and Microbiology and Immunology, Kimmel Cancer
Institute, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107
Quenching of phototransduction in retinal rod
cells involves phosphorylation of photoactivated rhodopsin by the
enzyme rhodopsin kinase followed by binding of the protein arrestin.
Although it has been proposed that the mechanism of arrestin quenching
of visual transduction is via steric exclusion of transducin binding to
phosphorylated light-activated rhodopsin (P-Rh*),
direct evidence for this mechanism is lacking. In this study, we
investigated both the role of rhodopsin phosphorylation in modulating
its interaction with arrestin and transducin and the proposed binding
competition between arrestin and transducin for P-Rh*.
While the -adrenergic receptor kinase promotes significant arrestin
binding to rhodopsin at a phosphorylation stoichiometry of
2 mol/mol,
rhodopsin kinase promotes arrestin binding at a stoichiometry of ~0.9
mol/mol. Moreover, while
-adrenergic receptor kinase phosphorylation
of rhodopsin only modestly decreases transducin binding and activation,
rhodopsin kinase phosphorylation of rhodopsin significantly decreases
transducin binding and activation. Finally, arrestin competes
effectively with transducin for binding to P-Rh* (50%
inhibition at ~1:1 molar ratio of arrestin:transducin) but has no
effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh*), paralleling the functional inhibition by
arrestin on P-Rh*-stimulated transducin activation (50%
inhibition at ~1.7:1 molar ratio of arrestin:transducin). These
results demonstrate that a major role of rhodopsin phosphorylation is
to promote high-affinity arrestin binding and decrease transducin
binding thus allowing arrestin to effectively compete with transducin
for binding to photoactivated rhodopsin.
Phototransduction mediated by the photoreceptor rhodopsin and
hormonal transduction mediated by the -adrenergic receptor (
AR)1 serve as excellent model systems
for investigation of the molecular events underlying agonist-induced
receptor desensitization (reviewed in Refs. 1 and 2). Phosphorylation
of light-activated rhodopsin and hormone-activated
AR by the G
protein-coupled receptor kinases rhodopsin kinase and
-adrenergic
receptor kinase (
ARK), respectively, initiates desensitization and
leads to partial uncoupling of receptor-G protein interaction. Rapid
and complete uncoupling, however, is accomplished by the subsequent
binding of the proteins arrestin and
-arrestin, respectively, to the
phosphorylated form of the agonist-activated receptor.
Many studies have investigated the effect of receptor phosphorylation on modulating interaction with arrestins. Light-induced binding of visual arrestin to rhodopsin was highly enhanced by rhodopsin phosphorylation (3), and this binding suppressed light-induced cGMP phosphodiesterase activation by rhodopsin (4). Although it has been shown that highly phosphorylated rhodopsin is significantly impaired in its ability to activate transducin, addition of arrestin accelerated the recovery process and further suppressed phosphodiesterase activation, most likely by quenching partially phosphorylated rhodopsin species (4-7). Recently, Puig et al. (8) demonstrated that a heptaphosphopeptide from the C terminus of rhodopsin stimulates arrestin binding to photoactivated forms of rhodopsin, suggesting that at least one function of rhodopsin phosphorylation is to promote high-affinity arrestin interaction with other cytoplasmic regions of rhodopsin. Indeed, the third cytoplasmic domain of rhodopsin appears to be involved in arrestin binding (9). The study by Puig et al. (8) is consistent with the recently proposed model of strict selectivity of arrestin binding to phosphorylated light-activated rhodopsin (10). This model proposes that arrestin interaction with both the phosphorylated rhodopsin C terminus and rhodopsin domains that manifest its activation state results in a conformational change from a low-affinity to a high-affinity binding state in which a secondary binding site becomes accessible for interaction with rhodopsin. The corresponding domain in arrestin for interaction with the phosphorylated C terminus of rhodopsin, the "phosphorylation-recognition" domain, has been localized to a discrete region near the middle of the molecule (11).
In hormonal transduction mediated by the AR, arrestins have been
shown to specifically inhibit the signaling of
ARK-phosphorylated
2AR (12-15). Moreover, in vitro synthesized
radiolabeled
-arrestin and arrestin 3 were demonstrated to have
high-affinity and selectivity for binding to the phosphorylated form of
the
2AR and m2 muscarinic acetylcholine receptor (16,
17). Similar to the visual arrestin studies, mutagenesis of
-arrestin and arrestin 3 has also identified regions critical for
recognition of the phosphorylated state of the receptor (17).
The effect of receptor phosphorylation on G protein interaction has not
been well characterized. Although several studies have demonstrated a
modest to potent functional effect of receptor phosphorylation on G
protein activation, it is not known whether this effect is due to
attenuated G protein binding to the receptor or subsequent G protein
activation. Phosphorylated rhodopsin was demonstrated to have a
significantly lower light-induced phosphodiesterase activating capacity
than nonphosphorylated rhodopsin (4, 18), and it was subsequently
proposed that rhodopsin phosphorylation reduces the binding affinity
for transducin (18). ARK phosphorylation of the
2AR
resulted in a modest functional inhibition of
2AR-stimulated Go GTPase activity (12, 15),
and phosphorylation of the m2 muscarinic acetylcholine receptor by
ARK modestly reduced its capacity to stimulate GTP
S binding to
Go (19).
The mechanism of arrestin quenching of rhodopsin signaling is believed to involve steric exclusion of transducin interaction with photoactivated rhodopsin (3, 5, 20). In 1984, Kuhn and co-workers (3) demonstrated that excess transducin displaces arrestin from phosphorylated rod outer segment (ROS) membranes suggesting a binding competition between arrestin and transducin for site(s) on the cytoplasmic surface of photoactivated rhodopsin. A quantitative assessment of this competition, however, could not be made due to the crude nature of the protein preparations. Here, we quantitatively characterized the ability of arrestin to compete with transducin for binding to nonphosphorylated and phosphorylated light-activated rhodopsin (Rh* and P-Rh*, respectively) and correlated these results with the functional effects of arrestin on Rh*- and P-Rh*-stimulated transducin activation.
[-32P]ATP was from NEN Life
Sciences Products. 11-cis-Retinal was generously provided by
Dr. R. K. Crouch (Medical University of South Carolina). Frozen bovine
retinas were purchased from George A. Hormel and Co. The bovine
arrestin cDNA was generously provided by Dr. T. Shinohara (National
Institutes of Health) (21). A monoclonal antibody (F4C1) against a
conserved N-terminal domain of all arrestins (22) was kindly provided
by Dr. L. Donoso (Wills Eye Hospital). A polyclonal antibody (116)
against Gi
subunits (23) was kindly provided by Dr. D. Manning (University of Pennsylvania). All restriction enzymes and most
other molecular biology reagents were from Boehringer Mannheim. All
other chemicals were from Sigma. Q-Sepharose and heparin-Sepharose
resins were from Pharmacia Biotech Inc. Wild-type Spodoptera
frugiperda (Sf9) cells were obtained from American Type Culture
Collection and tissue culture reagents were purchased from Life
Technologies Inc. and Sigma.
Urea-treated ROS membranes were
prepared as described (9) with all manipulations carried out on ice
under dim red light. Briefly, 50 frozen bovine retinas were resuspended
in 50 ml of 10 mM Tris acetate, pH 7.4, 65 mM
NaCl, 2 mM MgCl2, 34% (w/v) sucrose, shaken
vigorously and centrifuged at 2000 × g for 5 min. The
supernatant was diluted with 2 volumes of 10 mM Tris
acetate, pH 7.4, and centrifuged as above. The crude ROS pellet was
resuspended in 30 ml of 10 mM Tris acetate, pH 7.4, 1 mM MgCl2, 0.77 M sucrose and
further purified on a discontinuous sucrose gradient. The interface
between 0.84 and 1.0 M sucrose was collected, diluted 2-fold with 10 mM Tris acetate, pH 7.4, and centrifuged at
48,000 × g for 20 min. The resulting pellet was
resuspended in 50 mM Tris-HCl, pH 8.0, 5 mM
EDTA, 5.0 M urea, sonicated on ice for 4 min, diluted with
2 volumes of 50 mM Tris-HCl, pH 7.4, and centrifuged at
100,000 × g for 45 min. The pellet was washed three
times with 50 mM Tris-HCl, pH 7.4, resuspended in the same
buffer, sonicated on ice, snap frozen in liquid nitrogen, and stored in
the dark at 80 °C. The concentration of rhodopsin was assessed by
absorbance at 498 nm using an extinction coefficient of 40,600. Purity
of the ROS membrane preparation was assessed by SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining.
ROS membranes were
phosphorylated by ARK or rhodopsin kinase.
ARK was purified from
Sf9 insect cells infected with a recombinant baculovirus as described
previously (24). Rhodopsin kinase was purified to ~50% homogeneity
by sequential Q-Sepharose and heparin-Sepharose chromatography from Sf9
cells infected with a recombinant baculovirus generously provided by
Dr. R. Lefkowitz (Duke University Medical Center). ROS membranes
(~250-300 µg of rhodopsin) were incubated with
ARK (~20-40
µg) or rhodopsin kinase (~20-40 µg) in 1 ml of 20 mM
Tris-HCl, pH 7.5, 2 mM EGTA, 5 mM
MgCl2, 1 mM dithiothreitol (DTT), 2 mM ATP at 30 °C for various times under constant
illumination. Phosphorylation reactions were stopped by centrifugation
at 50,000 rpm (TLS55 rotor) for 35 min, 4 °C. The resulting pellets
were washed with 1.5 ml of 20 mM potassium-Hepes, pH 7.2, 2 mM DTT, thoroughly resuspended in 0.5 ml of the same buffer
and sonicated on ice two times for 10 s. Opsin was regenerated by
addition of a 3-fold molar excess of 11-cis-retinal and
incubation in the dark for 40 min at 37 °C, followed by addition of
another 3-fold molar excess of 11-cis-retinal and incubation
in the dark for 2 h at 37 °C. Phosphorylated regenerated
rhodopsin was aliquoted under dim red light and stored in the dark at
80 °C.
Stoichiometry of phosphorylation was measured by incubating 1-2 µCi
of [-32P]ATP with an aliquot of the initial
phosphorylation reaction under the exact conditions as described above
and quenching with SDS sample buffer followed by electrophoresis on a
10% polyacrylamide gel. The gel was dried and autoradiographed and the
radiolabeled rhodopsin bands excised and counted. Phosphorylation
stoichiometries were determined assuming that all of the receptors were
accessible to kinase.
The bovine arrestin cDNA was excised by
HpaI/NheI digestion, blunted with Klenow, and
ligated into the vector pBluebac which was digested with
NheI, blunted with Klenow and treated with phosphatase. To
generate a recombinant baculovirus for expression of arrestin, Sf9
insect cells were cotransfected with 1 µg of the recombinant pBluebac
arrestin DNA and 1 µg of viral DNA using calcium phosphate precipitation (25). Transfected cells were allowed to recover and
produce recombinant phage particles in culture media for ~5 days. The
viral supernatant was then used to infect a fresh monolayer of Sf9
cells which was overlaid with 1% low melting agarose in complete
media. After 4-5 days, isolated viral plaques were selected for
-galactosidase activity (with 5-bromo-4-chloro-3-indoyl
-D-galactoside), eluted into complete media and
amplified by reinfection of another fresh monolayer of Sf9 cells. Sf9
insect cells were cultured on a monolayer or in suspension (spinner
flask, 70 rpm) using TNM-FH medium containing 10% fetal bovine serum
and antibiotics (2.5 µg/ml fungizone, 50 µg/ml streptomycin, 50 µg/ml penicillin).
The recombinant arrestin baculovirus was used to infect a monolayer of
Sf9 cells (2-2.5 × 107 cells) on four 150-mm plates
at a multiplicity of infection of ~10. Cells were harvested ~48 h
after infection by rinsing several times with ice-cold
phosphate-buffered saline and then scraping the cells into 5 ml of
ice-cold 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 200 µg/ml benzamidine (Buffer A). Cells were lysed with a Brinkmann tissue disruptor (3 × 30 s at 20,000-25,000 rpm) and
centrifuged at 20,000 rpm (SS34 rotor) for 30 min, 4 °C. Recombinant
arrestin in the resulting supernatant was then purified by sequential
Q-Sepharose and heparin-Sepharose chromatography. Briefly, the Sf9
supernatant (20 ml, 30 mg) was diluted 2-fold with 2 mM
Tris-HCl, pH 7.4, 0.5 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 200 µg/ml benzamidine and then loaded at 0.5 ml/min on a 5-ml Q-Sepharose column equilibrated with Buffer A. The
column was washed with Buffer A and arrestin eluted at 1 ml/min with a
120-ml linear gradient of NaCl (0-400 mM) in Buffer A. Peak fractions (between 65 and 150 mM NaCl) were pooled (30 ml, 2-3 mg), the salt concentration adjusted to 200 mM
NaCl, and the pooled fractions loaded at 0.25 ml/min on a 1-2-ml
heparin-Sepharose column equilibrated with Buffer A in the presence of
200 mM NaCl. The column was washed with 200 mM
NaCl in Buffer A and arrestin eluted at 0.5 ml/min with a 40-ml linear
gradient of NaCl (200-600 mM) in Buffer A. Peak fractions
(between 370 and 470 mM NaCl) were pooled (10 ml, 100 µg), concentrated to 0.25-0.5 ml with an Amicon concentrator, and
aliquots were snap frozen in liquid nitrogen and stored at 80 °C.
Arrestin levels and purity were assessed by immunoblotting, using the
monoclonal antibody F4C1, and by Coomassie Blue staining of 10%
SDS-polyacrylamide gels.
Isolation of
holotransducin from dark-adapted bovine retinas was performed as
described (26). Briefly, ROS membranes were bleached in white light for
30 min, pelleted, and resuspended in hypotonic buffer (10 mM Hepes, pH 7.5, 1 mM DTT, 0.1 mM
EDTA, 0.3 mM phenylmethylsulfonyl fluoride), repeating the
latter manipulation several times. Transducin was then recovered by
resuspending the membranes in hypotonic buffer containing 100 µM GTP, incubation on ice for 30 min under constant
illumination, and centrifugation. This step was repeated three times
and the supernatants pooled, concentrated, aliquoted, and stored at
80 °C. Purity was assessed by Coomassie Blue staining of a 10%
SDS-polyacrylamide gel.
Arrestin
and transducin alone or together were incubated with rhodopsin or
phosphorhodopsin for 5 min at 30 °C under constant illumination in
10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.75 mM MgCl2, 0.2 mM DTT, 1 mg/ml
bovine serum albumin in a total volume of 50 µl. Samples were then
placed on ice and loaded, under dim red light, onto 0.2 ml of the above
buffer containing 0.2 M sucrose. Following centrifugation
at 100,000 rpm for 30 min at 4 °C, supernatants were removed and the
resulting pellets solubilized in 20 µl of SDS sample buffer,
electrophoresed on a 10% SDS-polyacrylamide gel, and electroblotted
onto nitrocellulose for 1 h at 100 V. Transferred proteins were
then analyzed using the F4C1 monoclonal arrestin antibody and/or the
116 polyclonal Gi antiserum. The 116 polyclonal
Gi
antiserum is directed against a C-terminal region of
Gi
and selectively detects Gi
1 ~ Gi
2 and to a lesser extent Gi
3 and
Gt
(23). Blots were blocked for 30 min with 5% (w/v)
nonfat dry milk in Buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), incubated with a 1:5000 (F4C1)
and/or 1:500 (116) dilution of primary antibody in Buffer B with milk
for 1 h, washed with Buffer B, incubated for 1 h with a
1:2000 dilution of anti-mouse and/or anti-rabbit secondary antibody
(for F4C1 and 116, respectively) in Buffer B with milk, washed with
Buffer B, and then visualized using ECL reagent (Amersham) followed by
autoradiography. Films were scanned by a densitometer (Molecular
Dynamics) and the intensity of immunoreactive bands in experimental
samples compared with the intensity of immunoreactive bands in standard
samples loaded on the same gel. Nonspecific binding was determined in
the absence of rhodopsin and endogenous immunoreactivity determined in
the presence of rhodopsin alone.
Transducin alone or in the presence of
increasing concentrations of arrestin was incubated with rhodopsin or
phosphorhodopsin for 5 or 15 min at 30 °C under constant
illumination in 10 mM Tris-HCl, pH 7.5, 1 mM
EGTA, 0.75 mM MgCl2, 0.2 mM DTT,
0.2 mM AMP-PNP, 1 mg/ml bovine serum albumin, 0.2 µM [-32P]GTP (150-250 cpm/fmol) in a
total volume of 50 µl. Reactions were quenched by addition of 25 µl
of 100 mM sodium phosphate, pH 2.0, and 500 µl of 5%
(w/v) activated charcoal and centrifugation for 15 min at 10,000 rpm in
a microcentrifuge. Aliquots of the resulting supernatants were counted
in a liquid scintillation counter and rhodopsin-stimulated hydrolysis
of GTP by transducin determined. Nonspecific hydrolysis of GTP was
assessed in the presence of rhodopsin alone (endogenous) and transducin
alone (rhodopsin-independent activity). The specific activity
(rhodopsin-stimulated activity) of different transducin preparations
varied in the range of ~30-80 fmol/min/µg protein using 50 nM rhodopsin.
Since direct evidence for competition between arrestins and G
proteins for receptor binding is lacking, we assessed the ability of
purified preparations of arrestin and transducin to bind to rhodopsin
using a centrifugation binding assay (to isolate rhodopsin-bound proteins). Transducin was purified from bovine retinas using a standard
procedure in which transducin is allowed to bind to photoactivated rhodopsin, peripheral proteins are then removed by hypotonic buffer washes and transducin subsequently released by GTP. This protein preparation was estimated to be 95% pure (Fig. 1,
lane 1). Arrestin was overexpressed in Sf9 insect cells
using the baculovirus expression system and purified by sequential
Q-Sepharose and heparin-Sepharose chromatography. This protein
preparation was judged to be
75% pure (Fig. 1, lane 2).
The ROS membrane preparation contained
90% rhodopsin (Fig. 1,
lane 3), and Western analysis indicated negligible
endogenous arrestin and transducin (data not shown). Since the
expression and purification of arrestin in Sf9 insect cells has not
previously been described, we initially investigated the function of
this protein. Purified Sf9-expressed arrestin was found to inhibit
in vitro synthesized [3H]arrestin binding to
P-Rh* with 50% inhibition occurring at an ~1:1 molar
ratio of arrestin:P-Rh* (data not shown), similar to that
previously reported for purified bovine retinal arrestin inhibition of
[3H]arrestin binding to P-Rh* (27).
We next characterized the binding of purified arrestin to
Rh* and P-Rh* preparations containing
increasing phosphorylation stoichiometries (Fig. 2). We
assessed arrestin binding to rhodopsin phosphorylated by both ARK
and rhodopsin kinase since
ARK has been reported to phosphorylate
rhodopsin on similar sites as rhodopsin kinase (28), and several
studies have utilized
ARK-phosphorylated rhodopsin to assess
in vitro synthesized arrestin binding to P-Rh*
(10, 11, 16, 17, 27). As shown in Fig. 2A, arrestin displayed minimal binding to Rh* and increasing binding to
P-Rh* as the extent of phosphorylation by
ARK increased
from 1.6 to 4.6 mol/mol. These results are consistent with the general
effect of rhodopsin phosphorylation on in vitro synthesized
[3H]arrestin binding to
ARK-phosphorylated rhodopsin
(10, 27).
It was recently demonstrated that ARK phosphorylation of rhodopsin
to a stoichiometry of ~2 mol/mol is necessary and sufficient for
high-affinity [3H]arrestin binding to rhodopsin (10). The
arrestin binding differences apparent in Fig. 2A may be
explained in light of this study and the observation that a
phosphorhodopsin preparation, containing a specific average
phosphorylation stoichiometry, is actually a heterogeneous population
of rhodopsin molecules where
67% of the total molecules contain the
average phosphorylation stoichiometry or greater (29). Thus, the
modestly enhanced binding of arrestin to P-Rh* (~4.6
mol/mol) when compared with P-Rh* (~2.7 mol/mol) is
likely due to a greater percentage of rhodopsin molecules in the
P-Rh* (~2.7 mol/mol) preparation containing less than 2 mol/mol stoichiometry. Similarly, the significantly decreased binding
of arrestin to P-Rh* (~1.6 mol/mol) when compared with
P-Rh* (~2.7 mol/mol) is likely due to a significantly
greater fraction of rhodopsin molecules in the P-Rh*
(~1.6 mol/mol) preparation containing a phosphorylation stoichiometry of less than 2 mol/mol.
Interestingly, arrestin binding to rhodopsin phosphorylated by
rhodopsin kinase (RK) to ~0.9 mol/mol was almost as good as its
binding to P-Rh* phosphorylated by RK to 2-3 mol/mol
stoichiometries (Fig. 2B). Moreover, arrestin binding to
P-Rh* phosphorylated by RK to ~0.9 mol/mol was in fact
better than its binding to P-Rh* phosphorylated by ARK
to ~1.6 mol/mol. Thus, high-affinity binding of arrestin to
RK-phosphorylated rhodopsin appears to require incorporation of only 1 mol of phosphate/mol of rhodopsin. These observations clearly indicate
that RK and
ARK differ in their initial site of phosphorylation of
rhodopsin. Indeed, a recent study (30) demonstrating that the initial
C-terminal site phosphorylated by RK and
ARK is different, with RK
phosphorylating Ser338 first and Ser343 second
while
ARK has the reverse preference, sheds light on this observed
difference in arrestin binding. A plausible explanation is that
phosphorylation of Ser338 is necessary and sufficient for
high-affinity arrestin binding. Consistent with the apparent
requirement of ~1 mol/mol phosphorylation stoichiometry for
high-affinity arrestin binding to RK-phosphorylated rhodopsin, several
studies have indicated that one phosphate incorporated into rhodopsin
is sufficient for arrestin function. In a functional assay, total
rhodopsin inactivation is achieved with a phosphorylation extent of
0.5-1.4 mol/mol (5). Moreover, although arrestin most effectively
quenches rhodopsin with a high phosphorylation stoichiometry (~8
mol/mol), it can still partially quench rhodopsin with a
phosphorylation stoichiometry of ~1 mol/mol (7).
The finding that phosphorylation of Ser338 is likely a key modulator of arrestin binding, along with our recent identification of Arg175 as a key phosphorylation-sensitive trigger in arrestin (11, 31), implies that phosphoserine 338 in rhodopsin may directly interact with Arg175 in arrestin. Moreover, several phosphorylation-independent arrestin mutants bound equally well to P-Rh* and 329G-Rh* (truncated rhodopsin lacking the C-terminal phosphorylation sites), but weakly to Rh* (31). This suggests that rhodopsin phosphorylation induces a conformational rearrangement involving the C terminus that promotes high-affinity arrestin binding.
Since the direct binding of transducin to rhodopsin and
phosphorhodopsin has not been well characterized, we next investigated the effect of rhodopsin phosphorylation on this interaction. Similar to
the arrestin binding studies, we compared transducin interaction with
rhodopsin phosphorylated by either ARK or RK. Phosphorylation of
rhodopsin by
ARK to ~4.6 mol/mol modestly reduced transducin binding (Fig. 3A), correlating with the
modest decrease in the ability of
ARK-phosphorylated rhodopsin
(~4.6 mol/mol) to stimulate the GTPase activity of transducin (Fig.
3B). Interestingly, similar to the more potent effects of
rhodopsin phosphorylation by RK on arrestin binding, phosphorylation by
RK also more effectively decreased transducin interaction. Rhodopsin
kinase phosphorylation of rhodopsin to ~0.9 mol/mol significantly
decreased transducin binding (Fig. 3C), correlating with a
significant reduction in RK-phosphorylated P-Rh*-stimulated
transducin GTPase activity (Fig. 3D). The more potent effect
on transducin interaction of rhodopsin phosphorylation by RK compared
with
ARK is highlighted by the observation that the decrease in
transducin binding and activation is more pronounced with the
RK-phosphorylated P-Rh* than the
ARK- phosphorylated
P-Rh*. The significant decrease in transducin activation by
RK phosphorylation of rhodopsin is consistent with the desensitization
observed in arrestin-knockout mice (32).
This is the first study to date to correlate the reduced ability of the phosphorylated receptor to both bind and activate the G protein. Although it has been proposed that phosphorylation of rhodopsin reduces its binding affinity for transducin (18), evidence to support this is lacking. Multiple studies have demonstrated that the rhodopsin domains most critical for transducin binding are the second and third cytoplasmic loops (33-36). Moreover, one study has suggested involvement of the C terminus of rhodopsin in transducin interaction (37), although truncation mutagenesis suggested that the C terminus is not absolutely required for transducin interaction (38). Possible explanations for reduced transducin binding to phosphorhodopsin are that phosphorylation of the C terminus may directly decrease transducin interaction with the C terminus or it may result in a conformational change that alters the accessibility of other cytoplasmic regions of rhodopsin critical for transducin interaction.
The more potent effects on transducin interaction by RK compared with
ARK phosphorylation of rhodopsin again suggests mechanistic differences in the manner in which these G protein-coupled receptor kinases phosphorylate rhodopsin. In light of the study indicating that
RK initially phosphorylates Ser338 (30), these results
strongly suggest that phosphorylation of Ser338 is a key
modulator of both transducin and arrestin interaction. Since
ARK
phosphorylation of rhodopsin to a high stoichiometry only modestly
decreased transducin interaction, the sequential order of
phosphorylation of rhodopsin may be more critical for modulating
transducin interaction than phosphorylation of Ser338 per
se. It is conceivable that initial phosphorylation of
Ser338 is required for a specific conformational change to
occur that significantly decreases transducin interaction. Thus,
monophosphorhodopsin phosphorylated on Ser338 may exist in
a conformation particularly unfavorable for transducin interaction, a
conformation that is subsequently maintained throughout further
phosphorylation of rhodopsin by RK. However, in the case of
ARK
phosphorylation of rhodopsin, this unique change may not occur since
Ser343 is phosphorylated before Ser338.
Using the purified preparations of arrestin and transducin, we next
investigated whether these proteins compete for binding to rhodopsin.
Since ARK-phosphorylated rhodopsin demonstrates both high-affinity
binding to arrestin and a modest reduction in transducin interaction,
we assessed the binding competition with this form of phosphorylated
rhodopsin. Increasing concentrations of arrestin were incubated with
transducin and either Rh* or P-Rh* (~4.6
mol/mol), and rhodopsin-bound arrestin and transducin were then
isolated by centrifugation through a 0.2 M sucrose cushion. The proteins were resolved by electrophoresis and binding quantitated by immunoblotting using arrestin and Gi
antibodies. It
can be clearly observed that as arrestin binding to P-Rh*
increased, transducin binding decreased (Fig.
4A). Arrestin competed with transducin for
binding to P-Rh* with 50% inhibition occurring at a molar
ratio of arrestin:transducin of ~1:1 (~50 nM arrestin)
and maximal inhibition of ~80% at the highest concentration of
arrestin (Fig. 4B). Arrestin, however, did not compete with
transducin for binding to Rh* (Fig. 4B),
consistent with its low affinity for nonphosphorylated rhodopsin (Fig.
2A and Refs. 10 and 27).
To our knowledge, this represents the first time that an arrestin protein has been shown to directly inhibit G protein binding to the phosphorylated form of an agonist-activated receptor, thus providing direct evidence for the binding competition mechanism first proposed by Kuhn et al. (3). Similar to the demonstration that excess transducin displaces arrestin from binding to highly phosphorylated ROS membranes (3), these results suggest that arrestins and G proteins compete for binding to a common site(s) on the phosphorylated receptor. Based on our previous results that the third cytoplasmic domain of rhodopsin is critical for rhodopsin-arrestin interaction (9), at least one of these overlapping binding site(s) is likely to be the third cytoplasmic loop of rhodopsin.
To assess whether the observed binding competition between arrestin and transducin for phosphorhodopsin can fully explain the functional effect of arrestin on phosphorhodopsin-stimulated transducin activation, we correlated the observed binding competition with the functional inhibition of arrestin on phosphorhodopsin-stimulated transducin GTPase activity. Arrestin inhibited P-Rh*-stimulated transducin GTPase activity with 50% inhibition occurring at a molar ratio of arrestin:transducin of ~1.7:1 (~85 nM arrestin) and maximal inhibition of ~70% at the highest concentration of arrestin (Fig. 4C), comparable to the half-maximal value and maximal inhibition observed in the binding competition experiments. Arrestin, however, had no effect on Rh*-stimulated transducin GTPase activity (Fig. 4C), consistent with its inability to compete with transducin for binding to this form of rhodopsin (Fig. 4B). The molar ratio of arrestin:P-Rh* required for 50% inhibition in the functional studies here correlates well with the molar ratio of arrestin:receptor required for 50% inhibition in functional studies reported elsewhere (12-14, 39). These results thus indicate that the observed binding competition between arrestin and transducin for interaction with phosphorylated rhodopsin is able to explain the functional effects of arrestin on inhibiting phosphorhodopsin-stimulated transducin activation.
In summary, we investigated the proposed mechanism of action of
arrestins (involving a binding competition with G proteins for the
phosphorylated agonist-activated receptor) and correlated these results
with the functional effects of arrestins on receptor-stimulated G
protein activation. We also characterized the direct effect of receptor
phosphorylation on interaction with both arrestins and G proteins. Our
results demonstrate: 1) phosphorylation of rhodopsin greatly enhances
arrestin binding and reduces transducin binding and activation; 2) RK
is more effective than ARK at phosphorylating rhodopsin and
modulating its interaction with arrestin and transducin; and 3)
arrestin competes with transducin for binding to P-Rh*
paralleling the functional inhibition of arrestin on
P-Rh*-stimulated transducin activation. Finally, the data
presented here raise the intriguing possibility that phosphorylation of GPRs to different stoichiometries with various G protein-coupled receptor kinases, and then assessment of the effect on GPR interaction with arrestins and G proteins, may be a viable approach for
investigation of the specificity of GPR interaction with G
protein-coupled receptor kinases and arrestins.
We thank Deidre Heyser for purified transducin and Drs. Jon Erickson and Richard Cerione for helpful discussions.