(Received for publication, October 25, 1994; and in revised form, April 20, 1995)
From the
Recoverin (Rv) is a myristoylated Ca Photoactivation of rhodopsin stimulates cGMP hydrolysis within
vertebrate photoreceptors (for review, see Lagnado and Baylor(1992) and
Yarfitz and Hurley(1994)). The resulting loss of intracellular cGMP
slows the influx of Ca Recoverin ( The NH We have characterized
recoverin's effect on rhodopsin phosphorylation in vitro to gain insight as to how Rv functions in vivo. Our aim
was to identify soluble retinal proteins that interact with Rv, and we
found that RK interacts with immobilized Rv in a specific and
Ca
Figure 2:
Purification of RK by Rv affinity
chromatography. A, the immobilized NA-Rv column was used as an
affinity matrix to purify RK from bovine retinal extracts as described
under ``Experimental Procedures.'' NA-Rv was chosen because
it did not bind IRBP and it has an apparent higher capacity for RK.
Before elution with EGTA, two high ionic strength washes were used to
remove tubulin. Inset, purified RK was analyzed by Coomassie
Blue-stained 12% SDS-PAGE. Incubation of eluted RK with 10 µM ATP at 37 °C for 10 min caused the apparent mobility of RK to
shift to 66 kDa, indicating that autophosphorylation had occurred. B, purified recombinant RK was analyzed by Coomassie
Blue-stained 12% SDS-PAGE. Lane1, 2 µg of
recombinant RK purified by NA-Rv column chromatography followed by
DE-52 column chromatography; lane2, 2 µg of
autophosphorylated recombinant RK. Size markers (in kilodaltons) are
indicated to the left.
Figure 3:
Phosphate incorporation into rhodopsin as
a function of RK concentration: reconstitution of purified Rv, RK, and
urea-stripped ROS membranes. Rhodopsin phosphorylation was performed as
described under ``Experimental Procedures.'' The effect of RK
concentration on the amount of phosphate incorporated per bleached
rhodopsin (*R) is shown when no Rv (opentriangles), 35 µM NA-Rv (opencircles), or 8 µM C14:0-Rv (closedcircles) was present. The final concentrations of ATP,
free Ca
Figure 5:
Time course of phosphate incorporation
catalyzed by RK into rhodopsin. Rhodopsin phosphorylation was carried
out as described under ``Experimental Procedures.'' The time
course of phosphate incorporated per bleached rhodopsin (*R)
is shown when no Rv (opentriangles), 15 µM C14:0-Rv (closedcircles), or 30 µM NA-Rv (opencircles) was present in the assay.
The final concentrations of ATP, free Ca
Figure 1:
Identification of
Ca
Figure 4:
Light titration of phosphate incorporation
catalyzed by RK into rhodopsin. Rhodopsin phosphorylation was performed
as described under ``Experimental Procedures.'' The effect of
bleaching level on phosphate incorporation is shown in A as
total phosphate incorporated into rhodopsin and in B as
phosphate incorporated per bleached rhodopsin (*R) when no Rv (opentriangles), 10 µM NA-Rv (opencircles), or 10 µM C14:0-Rv (closedcircles) was present. The final concentrations of ATP,
free Ca
Figure 6:
Effect of free Ca
Figure 7:
Inhibition of rhodopsin phosphorylation by
Rv. Rhodopsin phosphorylation was performed as described under
``Experimental Procedures.'' The effect of increasing NA-Rv (opencircles) and C14:0-Rv (closedcircles) concentrations on relative RK activity (100%
when no Rv was present) is shown. The final concentrations of ATP, free
Ca
Figure 8:
Rv does not inhibit rhodopsin
phosphorylation catalyzed by
Comparison of the extent of RK binding to
immobilized C14:0-Rv versus NA-Rv suggests that the myristoyl
moiety on Rv interferes with RK binding. A typical example of this is
shown in Fig. 1. A possible explanation for this is that there
may be competition between RK and IRBP for binding sites on immobilized
C14:0-Rv. It could also be that immobilized NA-Rv has a higher affinity
for RK than immobilized C14:0-Rv. We found that Rv also binds to
IRBP and tubulin. Ca
Both the Ca
The molecular mechanism by which RK is activated to carry out
high gain phosphorylation is not yet clear. In our reconstituted
system, the extent of high gain phosphorylation is not saturated at 1
µM RK even though the amount of photolyzed rhodopsin is
only 10 nM. This might reflect either a catalytic mechanism by
which photolyzed rhodopsin stimulates RK or, alternatively, a weak
affinity of RK for photolyzed rhodopsin. The reconstitution of high
gain phosphorylation using purified RK reported in this paper lays the
groundwork for a study of the role of high gain phosphorylation in
photoreceptor light adaptation. Experiments that assess the link
between the extent of rhodopsin phosphorylation and the level of
transducin activation are now in progress to examine if high gain
phosphorylation indeed regulates the gain of phototransduction.
Finally, the specific and Ca
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-binding
protein present primarily in bovine photoreceptors. It represents a
newly identified family of neuronal specific
Ca
-binding proteins that includes neurocalcin,
hippocalcin, and guanylyl cyclase-activating protein. To investigate
the function of Rv in photoreceptors, we identified proteins that bind
immobilized Rv in a Ca
-dependent manner. Rhodopsin
kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin
interact with Rv in the presence of Ca
. The
importance of the Rv/RK interaction was further characterized. RK,
purified using immobilized Rv as an affinity matrix, catalyzed the
light-dependent and Ca
-independent incorporation of
phosphates into rhodopsin when reconstituted with urea-stripped rod
outer segment membranes. When only a small fraction (0.04%) of
rhodopsin was photolyzed, as many as 700 phosphates were incorporated
per photolyzed rhodopsin, a phenomenon known as ``high gain''
phosphorylation. When recoverin was added, the activity of RK became
sensitive to free Ca
, with EC
= 3
µM. The N-terminal myristoyl residue of Rv enhances the
inhibitory effect of Rv and introduces cooperativity to the
Ca
-dependent inhibition of rhodopsin phosphorylation.
Rv neither interacts with other members of the G-protein-coupled
receptor kinase family such as
-adrenergic receptor kinase 1 nor
inhibits
-adrenergic receptor kinase 1 activity. The specific and
Ca
-dependent Rv/RK interaction is necessary for the
inhibitory effect of Rv on rhodopsin phosphorylation and may play an
important role in photoreceptor light adaptation.
through cGMP-dependent cation
channels in the photoreceptor plasma membrane. Under these conditions,
unabated Ca
efflux through
Na
/K
,Ca
exchangers
lowers the bulk concentration of intracellular free Ca
from 550 to 50 nM (Gray-Keller and Detwiler, 1994). The
light-induced lowering of intracellular free Ca
is a
signal that promotes recovery from photoexcitation. The calcium signal
is decoded by calcium-binding proteins that participate in several
biochemical reactions. Low Ca
concentrations
stimulate resynthesis of cGMP by the action of
Ca
-binding proteins that stimulate guanylyl cyclase
(Koch and Stryer, 1988; Gorczyca et al., 1994; Dizhoor et
al., 1994). Low Ca
concentrations also stimulate
plasma membrane cation channel activity by relieving the inhibitory
effect of Ca
/calmodulin on cGMP-gated cation channels
(Hsu and Molday et al., 1993). Finally, low Ca
concentrations facilitate phosphorylation and inactivation of
photoactivated rhodopsin (Kawamura and Murakami, 1991; Kawamura, 1993).
The effect of Ca
on rhodopsin phosphorylation is
mediated by a protein referred to as sensitivity-modulating protein
(S-modulin)
or recoverin (Rv) (Kawamura et al.,
1993).
)is a Ca
-binding
protein present only in vertebrate photoreceptors (Dizhoor et
al., 1992), certain retinal cone bipolar cells, and pineal glands
(Milam et al., 1993). It represents a recently identified
family of neuronal specific Ca
-binding proteins that
includes hippocalcin (Kobayashi et al., 1992), neurocalcin
(Okazaki et al., 1992), visinin (Yamagata et al.,
1990), S-modulin (Kawamura and Murakami, 1991), visinin-like protein
(VILIP) (Lenz et al., 1992), frequenin (Pongs et al.,
1993), and guanylyl cyclase activating protein (GCAP) (Gorczyca et
al., 1994). Rv was initially purified from bovine retinas, and it
was thought to be a Ca
-sensitive activator of
guanylyl cyclase, but this function has not been confirmed (Hurley et al., 1993). Rv was also recognized as an antigen associated
with cancer-associated retinopathy (Polans et al., 1991). When
internally dialyzed into rod outer segments (ROS), Rv prolongs the
photoresponse (Gray-Keller et al., 1993). This in vivo effect of Rv is consistent with the in vitro observation
that S-modulin, a frog homologue of Rv, enhances the effect of light on
cGMP phosphodiesterase and inhibits rhodopsin phosphorylation
(Kawamura, 1993). A similar effect of bovine Rv has been reported
(Kawamura et al., 1993), and it has been suggested that Rv
interacts with rhodopsin kinase (RK) since Rv coeluted with a 67-kDa
protein on a sizing column in the presence of Ca
(Gorodovikova and Phillipov, 1993).
terminus of Rv purified from bovine retinas is heterogeneously
acylated by one of four fatty acids, C14:0, C14:1, C14:2, or C12:0
(Dizhoor et al., 1992). N-acylation of Rv plays an
essential role in Ca
-dependent membrane targeting
(Dizhoor et al., 1993) through a novel calcium-myristoyl
protein switch mechanism (Zozulya and Stryer, 1992). However,
N-terminal myristoylation is not required for Rv to inhibit rhodopsin
phosphorylation (Chen and Hurley, 1994; Kawamura et al.,
1994). This suggests that binding of Rv to ROS membranes is not
necessary for its inhibitory effect. A recent report demonstrated that
an N-terminal myristoyl residue lowers the apparent Ca
affinity of Rv, but introduces cooperative binding of
Ca
. Nonacylated Rv (NA-Rv) binds two Ca
with affinities of 0.11 and 6.9 µM, respectively.
Myristoylated Rv (C14:0-Rv) binds two Ca
with an
affinity of 17 µM and a Hill coefficient of 1.75. The
affinity of C14:0-Rv for Ca
in the presence of
membranes was calculated to be 4 µM according to a
concerted allosteric model (Ames et al., 1995). There are
disparate reports about the Ca
dependence of
recoverin's effect on rhodopsin phosphorylation. Original reports
indicated that the IC
for both Rv and S-modulin was
100-200 nM free Ca
, but a recent study
indicates that the IC
may be significantly higher
(Klenchin et al., 1994).
-dependent manner. RK, affinity-purified by
immobilized Rv, phosphorylates rhodopsin in response to light when
reconstituted with urea-stripped ROS membranes. When only a small
fraction of rhodopsin is photolyzed in the reconstituted system, as
many as 700 phosphates are incorporated per photolyzed rhodopsin. These
results suggest that nonphotolyzed rhodopsins are also being
phosphorylated in response to light. Similar ``high gain''
phosphorylation has been reported by Binder et al.(1990) using
electropermeabilized frog ROS. When Rv is added to our reconstituted
system, high gain phosphorylation is reduced in a
Ca
-titratable manner.
Immobilized Recoverin
C14:0-Rv and NA-Rv were
produced as described (Ray et al., 1992). The extent of
myristoylation of the C14:0-Rv preparation was determined to be >99%
using liquid chromatography-coupled electrospray mass spectrometry
(data not shown). CNBr-activated Sepharose CL-4B (Sigma) was washed
with 50 mM HCl for 30 min and incubated with 100 mM sodium borate (pH 8.5) at room temperature for 1 h. The beads were
then washed with 100 mM sodium bicarbonate (pH 8.3) and 200
mM NaCl. CaCl was added to a final concentration
of 1 mM. Rv (1.5 mg/ml of beads) was then added to the slurry.
The slurry was gently shaken at 4 °C for 6 h, the coupling was
blocked by adding Tris-HCl (pH 8.0) to a final concentration of 100
mM, and incubation was continued for another 2 h. The coupling
efficiency was typically >99%. The beads were stored at 4 °C in
ROS-Ca
buffer (20 mM MOPS (pH 7.0), 30 mM NaCl, 60 mM KCl, 2 mM MgCl
, 1 mM dithiothreitol, 1 mM CaCl
, and 200
µM phenylmethanesulfonyl fluoride) with 0.02% sodium
azide.
Retinal Extracts and Recoverin Chromatography
100
frozen bovine retinas (Excel) were suspended and shaken in ROS buffer
(ROS-Ca buffer without 1 mM CaCl
)
containing 47% sucrose and then centrifuged at 30,000
g at 4 °C for 30 min. The supernatant was diluted with an equal
volume of ROS buffer and centrifuged at 30,000
g at 4
°C for 30 min. The supernatant was collected and spun at 100,000
g at 4 °C for another 90 min. All preparative work
for retinal extracts was done in a dark room under infrared
illumination. The supernatant was applied to Rv columns, and the
columns were washed extensively with ROS buffer until no protein washed
off as judged by the Bradford assay. Bound proteins were eluted with
ROS buffer containing 5 mM EGTA.
Preparation of RK and Urea-stripped ROS
Membranes
Retinal extracts prepared from 50 frozen bovine
retinas were applied to a 1 15-cm NA-Rv column. The column was
washed extensively with ROS buffer and two additional high salt washes
(see Fig. 2A) before EGTA elution. The high salt washes
removed most of the weakly bound tubulins. In the experiments described
in Fig. 3-8, Sf9 cell extracts containing recombinant RK
(Premont et al., 1994) were used as starting material.
Affinity-purified RK was further purified by DE-52 column
chromatography. In brief, the EGTA eluate from the NA-Rv column was
diluted 1:1 with Buffer D (20 mM MOPS (pH 7.5), 2 mM MgCl
, 1 mM dithiothreitol, and 50 µM CaCl
) and applied to a 2.5
20-cm DE-52 column
(Whatman). Bound RK was eluted with a 250-ml linear gradient of
0-400 mM NaCl in Buffer D. ROS membranes were washed and
sonicated in 6 M urea in 20 mM Tris-HCl (pH 7.5) and
5 mM EDTA for 10 min in the dark (Shichi and Somers, 1978).
After urea treatment, the ROS membranes were washed extensively with TM
buffer (50 mM Tris-HCl (pH 7.5) and 4 mM MgCl
). Endogenous RK activity was inactivated by urea
treatments.
, and urea-stripped ROS membranes (expressed
as concentration of rhodopsin) in the assay were 500, 100, and 25
µM, respectively.
Rhodopsin Phosphorylation Catalyzed by RK
Purified
recombinant RK, TM buffer, and urea-stripped ROS membranes were mixed
in the dark, with or without Rv, under infrared illumination.
[-
P]ATP (500-2000 cpm/pmol, 500
µM final concentration) was added to make a total volume
of 20 µl. 30 s after ATP was added, a test flash that bleached
0.04% rhodopsin was given, and the reaction was allowed to proceed
after the flash for a time course as depicted in Fig. 5or, in
most cases, for 40 min in the dark at room temperature. Reactions
without flashes were used as blanks. Each reaction was stopped by
adding an equal volume of SDS-PAGE sample buffer. The phosphorylation
of rhodopsin was visualized by autoradiography after 12% SDS-PAGE. The
amount of radioactivity incorporated into rhodopsin was measured by
cutting the rhodopsin bands from the gel, incubating the gel slices
with 30% H
O
at 65 °C for 60 min, and
subsequent liquid scintillation counting. The blank, a sample not
exposed to light (typically 5% of the maximal activity observed and
never >20-25% of the light-exposed samples in experiments
designed to quantitate high gain phosphorylation), was subtracted to
reveal light-dependent phosphorylation.
, RK, and
urea-stripped ROS membrane in the assay were 625 µM, 100
µM, 200 nM, and 10 µM, respectively.
Similar results were obtained from two
experiments.
Rhodopsin Phosphorylation Catalyzed by
ROS buffer, urea-stripped ROS membranes (10
µM rhodopsin), 10 µM Rv, and either 50 nM recombinant RK or 100 nM recombinant -Adrenergic
Receptor Kinase 1
-adrenergic
receptor kinase 1 expressed and purified from Sf9 cells (Kwatra et
al., 1993), with or without 2 µM G
purified from bovine brain extracts (a cofactor required for
maximal activity of
-adrenergic receptor kinase 1) (Pitcher et
al., 1992), were mixed in the dark under infrared illumination.
[
-
P]ATP (2000 cpm/pmol, 50 µM final concentration) was added to make a total volume of 20
µl. 10 s after ATP was added, a test flash that bleached 5%
rhodopsin was given, and the reaction was allowed to proceed after the
flash for 2 min in the dark at room temperature. Reactions not exposed
to light were used as blanks. The reaction was stopped by adding an
equal volume of SDS-PAGE sample buffer. The amount of rhodopsin
phosphorylation was analyzed as described above. Blanks that were
5% of the maximal activity were subtracted to reveal
light-dependent phosphorylation.
Identification of Retinal Proteins That Bind to
Recoverin in a Ca
To
identify proteins that bind Rv, either C14:0-Rv or NA-Rv was linked to
CNBr-activated Sepharose to make an immobilized Rv column. A retinal
extract in 1 mM Ca-dependent Manner
was then passed over the
column. The column was rinsed with 1 mM Ca
,
and then Ca
-dependent Rv-binding proteins were eluted
with 5 mM EGTA. Three proteins (140, 64, and 52 kDa in size)
bound to immobilized C14:0-Rv (Fig. 1A, lane3) in the presence of Ca
and eluted
when Ca
was chelated with EGTA. Only the 64- and
52-kDa proteins bound to immobilized NA-Rv (Fig. 1A, lane5). The 140-kDa protein was further purified by
Mono-Q fast performance liquid chromatography, and its N-terminal
sequence was determined by Edman degradation as
FQPSLVLEMAQVXLDNYXFP. Comparison of this sequence
with PIR Protein Data Base identified the 140-kDa protein as bovine
interphotoreceptor retinoid-binding protein (IRBP). We also purified
the 52-kDa protein(s) by reverse-phase fast performance liquid
chromatography and found the following N-terminal sequence:
MREI(I/V)(H/S). Sequence analysis identified this as the combined
N-terminal sequences of tubulin
- and
-subunits. The
identities of these 52-kDa proteins were further confirmed by
immunoreactivity with monoclonal antibodies against tubulin on
immunoblots (data not shown). The 64-kDa protein was recognized by
antibodies against bovine RK (Fig. 1B) (Palczewski et al., 1993; Inglese et al., 1992). The
identification of the 64-kDa protein as RK was confirmed by purifying
it on an NA-Rv column (Fig. 2A) and demonstrating its
ability to phosphorylate bleached rhodopsin (data not shown). The
purified 64-kDa protein also undergoes autophosphorylation (Fig. 2, A (inset) and B), consistent
with its identification as RK (Kelleher and Johnson, 1990).
-dependent Rv-binding proteins: detection of
proteins that bind to immobilized Rv. A, 12% SDS-PAGE followed
by Coomassie Blue staining; B, immunoblot analysis using
antibodies against RK (64 kDa), arrestin (48 kDa),
-transducin (38
kDa), and phosducin (33 kDa). Lane1, 20 µg of
retinal extract; lane2, 20 µg of flow-through
proteins from the C14:0-Rv column; lane3, 0.1 µg
of proteins eluted by EGTA from the C14:0-Rv column; lane4, 20 µg of flow-through proteins from the NA-Rv
column; lane5, 0.1 µg of proteins eluted by EGTA
from the NA-Rv column. A calmodulin column and a bovine serum albumin
column were used as controls. RK binds only to Rv columns, while
tubulin binds to both Rv and calmodulin columns (data not
shown).
Immobilized Rv as an Affinity Matrix for RK
Purification
The NA-Rv column nearly quantitatively depleted RK
from retinal extracts. Bound RK eluted when Ca was
removed by washing the column with buffer containing EGTA. Based on
this Ca
-dependent interaction, we developed a method
to purify RK from crude retinal extracts as shown in Fig. 2A. The NA-Rv column was chosen rather than a
C14:0-Rv column because it did not bind IRBP and it appears to have a
higher capacity for RK. It appears that purified retinal RK is
farnesylated since it has the same mobility as recombinant farnesylated
RK (data not shown) and has a mobility distinct from RKC558S, a
non-farnesylated form of RK (Inglese et al., 1992). The yield
of RK purified by this scheme from retinal extracts ranges from 10 to
20 µg/50 retinas. Using a baculovirus expression system for RK, we
can typically purify 500 µg of expressed RK from 1 liter of Sf9
cell cultures.
(
)
Purified RK Reconstituted with Urea-stripped
ROS Catalyzes High Gain Phosphorylation That Is Inhibited by
Rv
To determine if the effect of Rv on rhodopsin phosphorylation
involves RK and to determine whether this effect requires any soluble
protein in addition to RK, we reconstituted urea-stripped ROS
membranes, purified recombinant RK (Fig. 2B), and
either C14:0-Rv or NA-Rv. Purified recombinant RK, when reconstituted
with urea-stripped ROS membranes, catalyzed the light-dependent
incorporation of as many as several hundred phosphates into the
rhodopsin pool for every rhodopsin that was photolyzed. This indicates
that nonphotolyzed rhodopsin is being phosphorylated in response to
light. This high gain phosphorylation can be seen in Fig. 3. The
extent of high gain phosphorylation increased as RK concentration was
raised. High gain phosphorylation was most prominent when only a small
fraction of rhodopsin was bleached (Fig. 4). In the presence of
Rv and Ca, the high gain phosphorylation appears to
be quenched ( Fig. 5and Fig. 3and Fig. 4). When
the effect of recoverin was titrated with free Ca
buffered by 2.5 mM Br
-BAPTA, both NA-Rv and
C14:0-Rv had an EC
for free Ca
of 3
µM, but the presence of a covalently attached myristoyl
residue appears to introduce cooperativity to the inhibitory effect (Fig. 6). The Hill coefficient (n
) of the
Ca
effect for NA-Rv is 0.7, and the n
for C14:0-Rv is 1.5. The covalently attached myristoyl residue
also enhances the inhibitory effect of Rv. The IC
for
C14:0Rv is 0.8 µM, and the IC
for NA-Rv is 8
µM at saturating Ca
concentration (Fig. 7).
, RK, and urea-stripped ROS membranes in the
assay were 500 µM, 100 µM, 200 nM,
and 20 µM, respectively. The different bleaching level was
obtained by controlling the length of the test flashes. The rate of
bleaching was measured to be 4%/s. Similar results were obtained from
three triplicate experiments.
concentration on the inhibitory effect of Rv on RK activity. Rhodopsin
phosphorylation was performed as described under ``Experimental
Procedures.'' The free Ca
concentration was
controlled by buffering with 2.5 mM Br
-BAPTA and
was verified by a Ca
-sensitive electrode (Orion)
calibrated using commercially available standards (World Precision
Instruments, Inc.). The effect of Ca
on the amount of
phosphate incorporated per bleached rhodopsin (*R) is shown
when no Rv (opentriangles), 18 µM NA-Rv (opencircles), or 7 µM C14:0-Rv (closedcircles) was present. The final
concentrations of ATP, RK, and urea-stripped ROS membranes were 500
µM, 200 nM, and 12.8 µM,
respectively. Similar results were obtained from two triplicate
experiments.
, and urea-stripped ROS membranes were 500, 100,
and 20 µM, respectively. RK was present at either 180 or
320 nM. Similar results were obtained from two triplicate
experiments.
Ca
Our results using immobilized recoverin demonstrate a
direct and specific interaction between Rv and RK, but they do not
address the importance of the Rv/RK interaction for the inhibitory
effect of Rv on rhodopsin phosphorylation. It is conceivable that
inhibition of rhodopsin phosphorylation may involve additional
interactions of Rv, perhaps with rhodopsin (Dizhoor et al.,
1991) or with phospholipids (Zozulya and Stryer, 1992; Dizhoor et
al., 1993). To investigate the mechanism of recoverin's
action, we sought to determine whether Rv interacts with other members
of the G-protein-coupled receptor kinase family such as
-dependent Rv/RK Interaction Is
Required for Inhibition of Rhodopsin Phosphorylation by
Rv
-adrenergic receptor kinase 1 (Inglese et al., 1993).
-Adrenergic receptor kinase 1, like RK, phosphorylates rhodopsin
in a light-dependent fashion in vitro (Benovic et
al., 1986). Fig. 8A shows that recombinant
-adrenergic receptor kinase 1 does not bind to immobilized
recoverin. As would be expected from the data in Fig. 8A, the phosphorylation of photoactivated
rhodopsin by
-adrenergic receptor kinase 1 is not inhibited by Rv (Fig. 8B). These results were obtained in the presence
of the bovine brain G
, a cofactor required for
maximal
-adrenergic receptor kinase 1 activity, but similar
results were also obtained in the absence of G
(data not shown). These results show that the interaction between
Rv and RK is essential for the Ca
-dependent
inhibition of rhodopsin phosphorylation.
-adrenergic receptor kinase 1. A,
-adrenergic receptor kinase 1 (
ARK1)
does not bind Rv. Sf9 cell extracts containing recombinant
-adrenergic receptor kinase 1 were passed through immobilized
NA-Rv, and the binding of
-adrenergic receptor kinase 1 was
examined by 12% SDS-PAGE followed by Coomassie Blue staining (lanes
1-3) and by immunoblotting using an antibody specific to
-adrenergic receptor kinase 1 (lanes 4-6). Lanes 1 and 3, 10 µg of Sf9 extracts containing
recombinant
-adrenergic receptor kinase 1; lanes 2 and 4, 10 µg of flow-through fraction; lanes 3 and 6, EGTA eluate. Since we overdeveloped the immunoblot to
determine if traces of
-adrenergic receptor kinase 1 bound to
NA-Rv, the lower bands shown underneath
-adrenergic receptor
kinase 1 in lanes 4 and 5 are background. B,
Rv does not inhibit the phosphorylation of rhodopsin catalyzed by
-adrenergic receptor kinase 1. Hatchedbars represent RK activities, and solidbars represent
-adrenergic receptor kinase 1 activities. Relative
activities are expressed as percentages of each kinase activity at 100
µM CaCl
when no Rv was present. Similar
results were obtained in three separate
experiments.
Ca
Our results demonstrate for the first time that
there is a direct Ca-dependent Interaction of Rv
with RK
-dependent interaction between Rv
and RK ( Fig. 1and Fig. 2A). The Rv/RK
interaction is highly specific because Rv does not interact with other
members of the G-protein-coupled receptor kinase family such as
-adrenergic receptor kinase 1 (Fig. 8A). By
developing a reconstituted rhodopsin phosphorylation system, we have
shown that RK, purified using immobilized Rv as an affinity matrix, is
active and catalyzes the incorporation of as many as several hundred
phosphates/photolyzed rhodopsin into the rhodopsin pool (high gain
phosphorylation). Since Rv inhibits rhodopsin phosphorylation in this
reconstituted system, no other soluble factor is required for the
inhibitory effect of Rv.
-dependent binding of IRBP to Rv
requires N-terminal myristoylation of Rv. It has been reported that
IRBP binds a variety of fatty acids (Bazan et al., 1985). This
suggests that IRBP binds to the N-terminal myristoyl residue of Rv that
becomes exposed when Rv binds Ca
(Dizhoor et
al., 1993), as depicted by the calcium-myristoyl protein switch
model. However, it is unlikely that IRBP interacts with Rv within the
retina because IRBP resides in the extracellular interphotoreceptor
matrix (Bunt-Milam and Saari, 1983), whereas Rv resides primarily
within photoreceptors. The binding of tubulin appears weaker than the
binding of RK and electrostatic in nature because it can be disrupted
by high ionic strength (Fig. 2). We have not attempted to
address whether or not the Rv/tubulin interaction is physiologically
relevant.
Contribution of the Rv/RK Interaction to the Inhibitory
Effect of Rv on Rhodopsin Phosphorylation
A direct interaction
between Rv and RK was suggested by a previous report in which Rv
coeluted with a 67-kDa protein, presumably RK, during gel filtration
chromatography (Gorodovikova and Phillipov, 1993). Our findings provide
direct evidence that Rv interacts with RK in a
Ca-dependent manner. We also found that C14:0-Rv is a
better inhibitor than NA-Rv. This suggests that the interaction of Rv
with ROS membranes enhances the inhibitory effect. The observation that
-adrenergic receptor kinase 1, which does not bind Rv, is not
inhibited by Rv suggests that the Rv/RK interaction is necessary for
the inhibitory effect of recoverin.
Ca
It has
been reported that S-modulin and Rv inhibit rhodopsin phosphorylation,
with KDependence
= 100 nM free
Ca
(Kawamura et al., 1993). More recently,
the Ca
dependence was reported to be significantly
higher (Klenchin et al., 1994). These previous studies used
either EGTA or BAPTA to buffer Ca
. Both EGTA and
BAPTA do not buffer well above 1 µM because of their
relatively high affinity for Ca
. We buffered free
Ca
with Br
-BAPTA (K
= 1.6 µM for Ca
)
(Tsien, 1980) in our experiments. The EC
for the Rv effect
on rhodopsin phosphorylation is 3 µM for both NA-Rv and
C14:0-Rv. Interestingly, the presence of a covalently attached
myristoyl residue does not affect the EC
, but introduces
an apparent cooperativity. A similar effect of myristoylation on the
affinity of Rv for Ca
has recently been reported
(Ames et al., 1995).
affinity
for C14:0-Rv and the Ca
dependence of the inhibitory
effect of Rv require Ca
concentrations significantly
higher than the bulk intracellular free Ca
levels
detected by dye measurements in vertebrate photoreceptors (Gray-Keller
and Detwiler, 1994). What accounts for this apparent discrepancy? One
possibility is that Rv functions in a local environment, e.g. close to the plasma membrane, where the local free Ca
concentration may be different from the bulk free Ca
concentration. Another possible explanation may be that membranes
stabilize the Ca
-bound form of Rv by binding the
myristoyl group. According to a concerted allosteric model that was
adapted to the calcium-myristoyl protein switch mechanism (Zozulya and
Stryer, 1992) to interpret the difference in Ca
binding to NA-Rv and C14:0-Rv, the presence of a membrane
environment increases the affinity of C14:0-Rv for Ca
(Ames et al., 1995). It will be necessary to show an
increase in Ca
affinity for Rv in the presence of
membranes and/or RK to experimentally support this idea. Alternatively,
Rv may require other proteins absent from our reconstituted system to
operate in the physiological range of free Ca
concentration.
High Gain Phosphorylation
It was previously
reported that light activation of one rhodopsin molecule stimulates the
phosphorylation of hundreds of nonphotolyzed rhodopsins in
electropermeabilized frog ROS preparations (Binder et al.,
1990). This high gain phosphorylation was attributed to the intactness
of isolated ROS structure preserved in the preparations used. The
kinase responsible for high gain phosphorylation was not identified,
and more recently, it was suggested that protein kinase C could be
responsible for high gain phosphorylation (Newton and Williams, 1993).
Our results, however, demonstrate that highly purified RK itself can
produce high gain phosphorylation when reconstituted with urea-stripped
ROS membranes. The extent of high gain phosphorylation is proportional
to the amount of RK present (Fig. 3). The requirement for the
structure intactness reported by Binder et al.(1990) may
reflect dilution of RK when ROS are homogenized. It has also been
reported that RK can phosphorylate a synthetic peptide derived from the
C-terminal cytoplasmic loop of rhodopsin when RK is stimulated by a
truncated form of photolyzed rhodopsin (Palczewski et al.,
1991).-dependent Rv/RK
interaction may represent a model protein/protein interaction that
exists between the members of the newly identified neuronal specific
Ca
-binding protein family represented by Rv and the
G-protein-coupled receptor kinase family represented by RK.
-BAPTA,
5,5`-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid; GCAP, guanylyl cyclase-activating protein; VILIP, visinin-like
protein; NA-Rv, nonacylated recoverin; C1420-Rv, myristoylated
recoverin.
We thank A. M. Dizhoor, T. Neubert, J. C. Saari, K.
Palczewski, V. A. Klenchin, M. Erickson, and V. Slepak for valuable
discussions. We thank S. Kumar for protein sequencing and amino acid
analysis, A. Taylor for determining the mass of recombinant recoverin,
and G. Irons for tissue culture assistance. We also thank K. Palczewski
for arrestin and RK antibodies, S. Yarfitz for transducin
-antibody, and R.-W. Lee for phosducin antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.