Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24127-24129
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rhodopsin Kinase Inhibition by
Recoverin
FUNCTION OF RECOVERIN MYRISTOYLATION (*)
(Received for publication, August 10, 1995)
Peter
D.
Calvert
(1), (2), (§),
Vadim
A.
Klenchin
(1), (§),
M.
Deric
Bownds
(1) (2) (3)(¶)From the
(1)R. M. Bock Laboratories,
(2)Neuroscience Training Program and
(3)Department of Zoology, University of Wisconsin,
Madison, Wisconsin 53706
ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS AND DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
Recoverin is a Ca
-binding protein that may
play a role in vertebrate photoreceptor light adaptation by imparting
Ca sensitivity to rhodopsin kinase. It is
heterogeneously acylated (mostly myristoylated) at its amino-terminal
glycine. Recent studies have shown that recoverin myristoylation is
necessary for its Ca-dependent membrane association
and cooperative Ca binding. We have addressed several
issues concerning the role of recoverin myristoylation with respect to
inhibition of rhodopsin kinase. We find that 1) myristoylation of
recoverin is not necessary for inhibition of rhodopsin kinase, 2)
myristoylation of recoverin induces a cooperative
Ca-dependence for rhodopsin kinase inhibition, and 3)
each Ca-binding site on the nonmyristoylated
recoverin partially inhibits rhodopsin kinase. The available data
suggest that the functions of recoverin myristoylation in the living
rod are to induce a sharp Ca dependence of rhodopsin
kinase inhibition and to bring this dependence into the rod's
physiological Ca concentration range.
INTRODUCTION
In the vertebrate photoreceptor recoverin may provide a
Ca-dependent feedback system involved in light
adaptation by binding Ca in the dark, when
Ca levels are high, and releasing it when
Ca levels drop upon
illumination(1, 2, 3, 4, 5) .
Ca-recoverin inhibits RK, (
)and release of
this inhibition would accelerate the inactivation of Rho* when
Ca levels drop. In a previous study (4) we
have demonstrated that Ca bound recoverin acts
through a direct interaction with RK to decrease its catalytic activity
(see also (2) and (5) ).
Recoverin belongs to a
family of proteins post-translationally modified by the covalent
addition of myristate to their amino-terminal glycine
residues(3) . Myristoylation of recoverin is necessary for its
Ca-dependent association with photoreceptor
membranes(6, 7, 8) . This membrane binding
serves to bring inhibition of RK by recoverin into the physiological
Ca range under in vivo conditions (4, 6) . Recently it was demonstrated that the
myristoylation of recoverin induces cooperative Ca binding to recoverin(10) . A previous study has asserted
that only one Ca-binding site on recoverin is
involved in the inhibition of Rho* phosphorylation, and that the other
is involved in membrane binding(8) . We have studied recoverin
myristoylation with regard to inhibition of RK in order to further
define its function and have addressed the following issues. 1) Is
myristoylation of recoverin necessary for RK inhibition? 2) Is
myristoylation of recoverin necessary for the inhibition of RK to be
cooperative with respect to Ca concentration? 3) Do
both Ca-binding sites on recoverin act to promote the
inhibition of RK?
EXPERIMENTAL PROCEDURES
Materials and
Solutions
[
-
P]ATP was purchased from
DuPont NEN, 1 M CaCl
solution from BDH,
dibromo-BAPTA from Molecular Probes, and bis-Tris propane from
Calbiochem. Other chemicals were obtained from Sigma. The standard
buffer used in all experiments contained 110 mM potassium
gluconate, 20 mM HEPES, pH 7.5, and 2 mM
MgCl.
ROS Preparation and RK Extraction
Bovine ROS were
purified under infrared illumination as described
elsewhere(11) . The same method of bovine ROS isolation was
used for RK extracts, but all sucrose solutions were prepared in 10
mM Tris, 5 mM MgCl, pH 7.5, and the
procedure was performed in room light. RK was extracted as previously
described(12) .
Expression and Purification of Recombinant Bovine
Recoverin
Recombinant bovine myristoylated and nonmyristoylated
recoverins were produced using a bacterial expression system (7) that was a generous gift from Dr. J. Hurley. For the
myristoylated form of recoverin, the extent of myristoylation was
determined by electrospray ionization mass spectrometry and found to be
greater than 95%. Both forms of recoverin were purified by a published
procedure (13) and stored at -70 °C. The
concentrations of each form of recoverin were determined by absorbance
at 280 nm using a molar extinction coefficient of 36,400(4) .
Calcium Buffering
A set of 4 
stock
solutions with different CaCl concentrations and a fixed
dibromro-BAPTA concentration in standard buffer were prepared as
described in Klenchin et al.(4) (1 = 3
mM dibromo-BAPTA).
RK Activity Assay
RK activity was measured as
described previously(4) . Briefly, urea-treated ROS membranes
were used as substrate (10 µM rhodopsin, final
concentration). Urea-treated ROS membranes, a ROS extract containing
RK, a CaCl/dibromo-BAPTA stock solution, and recoverin if
necessary were mixed in 15 µl, final volume, in the dark, the
suspension was illuminated bleaching all Rho, and 5 µl of
[-P]ATP (0.2-0.4 mM) were
added. After 1-2 min the reaction was stopped by addition of 100
mM EDTA, 100 mM KF, pH 7.5. P
incorporation was measured by filtration of samples through
nitrocellulose filters.
RESULTS AND DISCUSSION
Only the Ca bound form of recoverin is able
to inhibit RK. The potency of inhibition of RK by myristoylated and
nonmyristoylated recoverin can thus be compared when all
Ca-binding sites are occupied. The dependence of the
inhibition of RK on the concentration of myristoylated and
nonmyristoylated recoverin at saturating Ca levels is
shown in Fig. 1. Myristoylated and nonmyristoylated recoverin (filled and open symbols, respectively) are
practically the same with respect to their inhibition of RK activity.
Both recombinant forms of recoverin show a K of inhibition of
about 3.5 µM, the same as the native bovine
protein(4) , and at sufficiently high concentration are able to
fully suppress RK activity (I
96%, Fig. 1).
Thus, myristoylation of recoverin does not affect the ability of
recoverin to bind and inhibit RK. Several laboratories have reported
that myristoylation of recoverin is necessary for its
Ca-dependent association with
membranes(6, 7, 8) , suggesting that it might
be involved in recoverin inhibition of RK. Our data provide direct
evidence against this idea and are consistent with previous conclusions (4, 8) that the association of recoverin with
membranes is not necessary for its inhibitory activity toward RK.
Figure 1:
Myristoylated and
nonmyristoylated recoverin inhibit RK equally. Serial dilutions of
myristoylated and nonmyristoylated recoverins (filled and open symbols, respectively) were made, and their ability to
inhibit RK activity was determined using Ca buffered
to 25 µM. RK extracts were mixed with urea-treated,
dark-adapted photoreceptor membranes (10 µM Rho),
recoverin was added to the indicated concentrations, rhodopsin was
fully bleached, and [P]ATP was added to initiate
the reaction (50-100 µM, final concentration). After
2 min the reaction was stopped by the addition of EDTA/KF quench
solution. Data are plotted as a percent of maximal inhibition of
phosphate incorporation in the absence of recoverin. Each symbol represents a separate determination. The line is a
hyperbolic fit of all of the data where the K = 3.5
µM recoverin and I =
96%.
Myristoylation of recoverin has been shown to radically change the
way recoverin binds Ca. From direct Ca binding measurements Ames et al. (10) conclude
that nonmyristoylated recoverin has two independent
Ca-binding sites with affinities of 110 nM and 6.9 µM, whereas the presence of the myristoyl
group results in cooperative Ca binding with an
apparent K
= 17 µM. If this is
the case and if the hypothesis proposed earlier by us is correct (4) (namely, that binding of recoverin to RK is necessary and
sufficient for the RK inhibition and is a strict function of
Ca), then the difference in Ca binding by recoverin should be observed in the Ca dependence of RK inhibition for the respective forms of
recoverin. Indeed, the Ca dependence of RK inhibition
is strikingly different for myristoylated and nonmyristoylated
recoverin (Fig. 2). Fitting the data with the Hill equation
consistently shows a Hill coefficient (n) less than 1 and K of 0.35 µM for the nonmyristoylated
recoverin, while the best fit for the myristoylated form gives n = 1.9 and a much higher K (5.3 µM; Fig. 2). Thus, myristoylated recombinant recoverin behaves much
as the native form with respect to positive cooperativity with n 2 and a K in the micromolar range(4) .
Figure 2:
Myristoylation of recoverin induces
cooperative Ca-dependent inhibition of RK.
Myristoylated (
) or nonmyristoylated (
) recoverin (15
µM) was mixed with an extract containing RK, urea-treated
photoreceptor membranes (10 µM Rho), and
Ca-dibromo-BAPTA to yield the indicated free
Ca. The experiment then proceeded as in Fig. 1. Data were normalized to maximal phosphate incorporation
and are the means ± S.D. of six and four experiments for
myristoylated and nonmyristoylated recoverin, respectively. The line through data for nonmyristoylated recoverin is a fit
using the equation given under ``Results and Discussion''
where a = b = 0.35, K = 138 nM Ca and K = 896 nM Ca.
The line through the data for myristoylated recoverin is fit
using the Hill equation.
Nonmyristoylated recoverin inhibits RK activity with apparent
negative cooperativity (n 0.8). This data, considered
with the direct Ca binding study(10) ,
suggests an independent and additive action of the two
Ca-binding sites on nonmyristoylated recoverin with
respect to RK inhibition rather than true negative cooperativity. The
data for nonmyristoylated recoverin can be fit with the following
equation
where P is the proportion of maximal phosphate
incorporation by RK activity, P, at the
indicated Ca concentration, a and b are amplitude factors that reflect the contribution of the two
Ca-binding sites, and K and K are constants that reflect the Ca range of RK inhibition upon the occupancy of these sites (see
legend to Fig. 2). If the occupancy of only the first site is
sufficient for RK binding and inhibition, then in experiments of the
sort shown in Fig. 2one would expect to find an apparent K slightly less than the K for the high
affinity Ca binding site (110 nM) (10) . Similarly, if both sites have to be occupied by
Ca to observe full inhibition, the observed K would be close to the K of the lower affinity
site, in the micromolar range(10) . In both cases, the Hill
coefficient should equal 1. None of these predictions is seen
experimentally, indicating that occupation of each
Ca-binding site on recoverin leads to a partial
inhibition of RK activity.
Our data and conclusions differ, in part,
from those of Kawamura et al.(8) . Based on
Ca titration experiments analogous to the ones shown
in Fig. 2, they also conclude that there is essentially no
difference in the inhibition of RK by myristoylated and
nonmyristoylated recoverin. Such experiments are not sufficient to
prove this point. What is required are recoverin titration experiments
of the sort shown in Fig. 1. Our data on the Ca dependence of RK inhibition do unequivocally show a difference
between myristoylated and nonmyristoylated recoverin. Closer
examination of the data of Kawamura et al. reveals a
qualitatively similar difference for the two forms of recoverin ((8) , Fig. 2). Possible errors in Ca buffering (see (4) for discussion) and large error bars
may have led the authors to ignore this small difference. Kawamura et al. take their data, that there is no difference in
Ca-dependent inhibition of RK by myristoylated and
nonmyristoylated recoverin and that nonmyristoylated recoverin does not
bind to membranes, to suggest that there are two distinct
Ca-binding sites on recoverin. One of these sites is
responsible for the inhibition of RK, and the other is responsible for
membrane association. Our data (Fig. 2) rule out this model by
showing that occupation of each Ca-binding site on
recoverin leads to a partial inhibition of RK activity.
The
influence of recoverin myristoylation on its
Ca-dependent inhibition of RK has also been recently
addressed by Chen et al. (5) who report that
Ca titration curves of recoverin inhibition by
nonmyristoylated and myristoylated recoverins have the same apparent K of 3 µM free Ca and also that
myristoylated recoverin is a much more potent inhibitor of RK than the
nonacylated form (apparent K is 0.8 µMversus 8 µM recoverin at saturated Ca).
These results are entirely at variance with our own. We do not attempt
to explain this, but note that our findings are consistent with all
previous studies of Ca/recoverin/RK
interaction(1, 2, 4, 10) . It has
been shown that nonmyristoylated recoverin binds Ca better (10) and that Ca binding to
recoverin is sufficient for binding to and inhibition of
RK(1, 2, 4, 5) . It follows then
that the Ca dependence of nonmyristoylated recoverin
inhibition of RK should have a K lower than that for
myristoylated recoverin. This is exactly what we observe (Fig. 2). Also, similar results have been obtained by Kawamura et al.(8) (see above).
Most of the recoverin
found in the retina is acylated by myristate or closely related fatty
acids(3) . Thus, recombinant myristoylated recoverin should be
similar to the native protein. We find this to be the case both with
respect to Ca titration of RK inhibition and
recoverin titration at saturated Ca (4; present
report). The high half-saturating recoverin concentration that we find
for the recombinant protein (3.5 µM) is consistent
with what we and others observe for native bovine recoverin (5-7
µM(2) and 3.4 µM(4) ) and
for the frog protein (7.5 µM)(1) ; this
contrasts with the K of 0.8 µM found by Chen et al.(5) . Our finding that the myristoyl group has
little or no effect on recoverin interaction with RK (Fig. 1) is
consistent with our observation that RK inhibition by
Ca-recoverin is not sensitive to relatively high
detergent concentrations (as high as 0.4% Tween 80), but
Ca/myristoyl-dependent binding of recoverin to
membranes is diminished by Tween 80. This suggests that
hydrophobic interaction is not of crucial importance for recoverin
binding to RK.
It is interesting to note that myristoylation of
recoverin has opposing consequences on recoverin function. On one hand,
it induces cooperative Ca binding, a feature that
allows efficient detection of changes in Ca concentrations in photoreceptors. On the other hand, it
significantly increases the Ca range over which
inhibition of RK by recoverin occurs in vitro (Fig. 2),
bringing it far from the reported in vivo range of free
Ca concentrations (200-600 nM in the
darkness and much lower in bright
light)(14, 15, 16, 17) . In a
previous report we point out that a K for recoverin inhibition
of RK in the micromolar free Ca range under dilute, in vitro conditions is expected, because
Ca-dependent membrane association of recoverin
effectively reduces this K to about 270 nM Ca under more concentrated in vivo conditions(4) . This effect arises because
Ca-bound myristoylated recoverin binds to membranes
and no longer participates in free solution equilibrium. Thus, a higher
total concentration of Ca-recoverin results.
From
the available data to date we propose two roles for recoverin
myristoylation relevant to photoreceptor physiology. First, it imparts
a sharp calcium sensitivity range, allowing it to act as more of a
``switch'' in sensing Ca; second, it
induces Ca-dependent association of recoverin with
photoreceptor membranes, allowing recoverin to act in the physiological
Ca range of the photoreceptor.
FOOTNOTES
- *
- This work was supported by National Institutes of
Health Grant EY-00463 (to M. D. B.). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore by hereby marked
``advertisement'' in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
- §
- These authors contributed equally to the
manuscript.
- ¶
- To whom correspondence and
reprint requests should be addressed: 327 Molecular Biology, 1525
Linden Dr., Madison, WI 53706. Tel.: 608-262-4380; Fax: 608-262-4570.
- ()
- The abbreviations used are: RK, rhodopsin
kinase; Rho, rhodopsin; Rho*, catalytically active rhodopsin;
dibromo-BAPTA,
5,5`-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`
tetraacetic acid; ROS, rod photoreceptor outer segment; bis-Tris,
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
- ()
- P. D. Calvert and M. D. Bownds, unpublished
observation.
ACKNOWLEDGEMENTS
We thank J. B. Hurley for the kind gift of E. coli strains expressing recombinant recoverin and V. Y. Arshavsky for
comments on the manuscript.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
