From the
Cyclic ADP-ribose (cADPR) is emerging as an endogenous regulator
of Ca
Cyclic ADP-ribose (cADPR)
Another notable feature
of the cADPR-sensitive Ca
Lytechinus pictus eggs were used
for the microinjection experiments. The procedures for microinjection
by pressure and for measuring the Ca
Similar to its effects on cADPR, CaM can also potentiate the
Ca
CaM also appears to be
responsible for regulating cADPR-sensitivity in intact sea urchin eggs.
To monitor intracellular Ca
The simplest model for the Ca
The modulator mechanism described apparently is
operative in live cells since W7, an antagonist of CaM, can selectively
inhibit the cADPR-sensitive Ca
We thank Gale Strasbourg for providing the wheat germ
calmodulin.
Note Added in Proof-Bovine serum albumin
(six different types from Sigma, Boeringer Mannheim and United States
Biochemicals) and egg albumin (Sigma), do not potentiate the calcium
releasing activity of cADPR even at 1 mg/ml, further demonstrating the
specificity of CaM.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-induced Ca
release (CICR),
and we have recently demonstrated that its action is mediated by
calmodulin (CaM) (Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E.,
and Walseth, T. F. (1994) Nature 370, 307-309). In this
study we show by immunoblot analyses that the protein factor in sea
urchin eggs responsible for conferring cADPR sensitivity to egg
microsomes was CaM. This was further supported by the fact that bovine
CaM was equally effective as the egg factor. In contrast, plant CaM was
only partially active even at 10-20-fold higher concentrations.
This exquisite specificity was also shown by binding studies using
I-labeled bovine CaM. The effectiveness of various CaMs
(bovine > spinach > wheat germ) in competing for the binding
sites was identical to their potency in conferring cADPR sensitivity to
the microsomes. A comparison between bovine and wheat germ CaM in
competing for the sites suggests only 10-14% of the total binding
was crucial for the activity. Depending on the CaM concentration, the
sensitivity of the microsomes to cADPR could be changed by several
orders of magnitude. The requirement for CaM could be alleviated by
raising the divalent cation concentration with Sr
.
Results showed that CaM, cADPR, and caffeine all act synergistically to
increase the divalent cation sensitivity of the CICR mechanism. The
combined action of any of the three agonists was sufficient to
sensitize the mechanism so much that even the nanomolar concentration
of ambient Ca
was enough to activate the release.
Unlike the CICR mechanism, the microsomal inositol
1,4,5-trisphosphate-sensitive Ca
release showed no
dependence on CaM. Using an antagonist of CaM, W7, it was demonstrated
that the cADPR- but not the inositol 1,4,5-trisphosphate-dependent
release mechanism could be blocked in live sea urchin eggs. These
results indicate cADPR can function as a physiological modulator of
CICR and, together with CaM, can alter the sensitivity of the release
mechanism to divalent cation by several orders of magnitude.
(
)
was discovered
as a metabolite of NAD
which has Ca
releasing activity in an invertebrate cell, the sea urchin egg
(1, 2, 3) . The generality of its function is
shown by the fact that amphibian neurons and a variety of mammalian
cells have since been found to be also responsive to cADPR (reviewed in
Ref. 4). That the Ca
release mechanism activated by
cADPR is independent of the inositol 1,4,5-trisphosphate
(IP
) system is indicated by its insensitivity to heparin,
an antagonist of the IP
receptor
(5) . Conversely,
8-amino-cADPR blocks specifically the action of cADPR without affecting
the IP
-sensitive Ca
release
(6) .
Also, microsomes desensitized to high concentrations of either cADPR or
IP
can still respond, respectively, to the heterologous
activator
(5) . Specific binding of cADPR to the microsomal
receptor is, likewise, unaffected by IP
and heparin
(7) . It is increasingly clear that the cADPR-dependent
Ca
release mechanism resembles the
Ca
-induced Ca
release (CICR) in its
pharmacology. Thus, agonists of CICR, such as Ca
and
caffeine, potentiate cADPR-dependent Ca
release
(8, 9) . Antagonists of CICR, such as ruthenium red and
procaine, selectively block only the Ca
release
induced by cADPR but not that induced by IP
(8, 9) . Finally, ryanodine, an effector of CICR,
can release Ca
from the cADPR-sensitive stores and
desensitize them selectively to cADPR but not to IP
(8, 9) . Therefore, the cADPR-sensitive
Ca
release mechanism is pharmacologically
indistinguishable from the CICR mechanism.
release that differentiates
it from the IP
mechanism is its requirement for a soluble
protein factor
(1) . Separation of sea urchin egg microsomes
from soluble protein components results in loss of cADPR responsiveness
(1, 10) . Purification and characterizations of the
cADPR-conferring factor indicate it is calmodulin (CaM)
(10) .
The cADPR sensitivity-conferring activity of CaM does not appear to be
mediated through enzymatic means, but is more likely through direct
interaction with the Ca
release mechanism
(10) . In this study, we show that the requirement for CaM is
highly specific, with plant CaM being 10-20-fold less effective
than either sea urchin or mammalian CaM. Analyses indicate CaM acts
synergistically with cADPR to increase the sensitivity of the release
mechanism to divalent cations. In addition to being a second messenger
for Ca
mobilization, these results suggest a new role
for cADPR as a modulator of the Ca
sensitivity of
CICR.
Fractionation of Egg Microsomes and Microinjection of
Intact Eggs
Homogenates of sea urchin egg
( Strongylocentrotus purpuratus) were prepared as described
previously
(7) . Frozen egg homogenates (25%) were thawed and
incubated at 17 °C for 20-40 min. Egg homogenates (2 ml for
each gradient) were layered on 10 ml of 25% Percoll prepared as
described previously
(10) . After centrifugation for 30 min at
25,000 rpm in a Beckman Ti50 rotor at 10 °C, the microsomes were
collected by inserting a syringe needle directly into the band of
vesicles on the upper part of the gradient. Microsomes collected from
each gradient (1 ml) were diluted to 5 ml with a medium containing
250 m
M N-methylglucamine, 250 m
M potassium
gluconate, 20 m
M Hepes, 1 m
M MgCl
, 2
units/ml creatine kinase, 8 m
M phosphocreatine, 0.5 m
M ATP, and 1.5-3 µ
M Fluo 3, pH 7.2, adjusted with
acetic acid (GluIM). The final microsomal protein concentrations were
about 0.6 mg/ml. Ca
release activity was tested after
incubating the microsomes with or without CaM (1-60 µg/ml) at
17° C for about 2 h.
changes in the
injected eggs using Fluo 3 were as described previously
(11) .
Samples were dissolved in the injection buffer containing 0.5
M KCl, 50 µ
M EGTA, 10 m
M Hepes, pH 6.7.
Purification of the cADPR Sensitivity-conferring
Factor
The supernatants from the Percoll density gradient
centrifugation described above were collected and used as the source
for the cADPR sensitivity-conferring factor. The supernatant (20 ml)
was dialyzed against 2 liters of 20 m
M Hepes, pH 7.2,
overnight and centrifuged at 50,000 rpm for 1 h at 4 °C using a
Beckman Ti50 rotor. The supernatant was collected and concentrated to
11 ml using Centriprep filters with M3,000 cutoff
(Amicon, Beverly, MA). The concentrate was chromatographed on a DEAE
5PW column (Waters, Milford, MA) using a linear gradient of 0 to 2
M potassium acetate, pH 7.2, at a flow rate of 0.7 ml/min. The
cADPR sensitivity-conferring activity was assayed by incubating 50
µl of each fraction with 0.55 ml of purified microsomes and
challenging with 200 n
M cADPR. All of the activity was eluted
at about 1.3
M potassium acetate. The active fractions were
combined (
2 ml), concentrated to 0.3 ml, and finally purified on a
300 SW gel-filtration column (Waters) by elution with 0.2
M potassium acetate, pH 7.2, at 0.7 ml/min.
Calmodulin Binding
Egg microsomes were purified by
Percoll density gradient centrifugation as described above. Microsomes
collected from each gradient were diluted to 2.5 ml with GluIM
(composition listed above) and 0.2-ml aliquots (1.2 mg protein/ml)
were incubated for 2 h at 17 °C with 23,000-30,000 cpm of
I-CaM (bovine, 69-88 µCi/µg) in the
presence (1-60 µg/ml) or absence of unlabeled CaM. After
incubation, the microsomes were pelleted by centrifugation at 4 °C
for 10 min at 100,000 rpm in a Beckman TLA 100.1 rotor. The supernatant
was discarded, and the pellets were counted in a
-counter.
Immunoblot
The procedure used was similar to that
described by Hulen et al. (12) . CaM samples were
chromatographed on SDS-polyacrylamide gel electrophoresis and
transferred to Immobilon-P (Millipore, Bedford, MA). The membrane was
fixed for 45 min at room temperature with 0.2% glutaraldehyde. After
rinsing, the membrane was incubated for 1 h at 37 °C with a
monoclonal antibody against CaM (clone 6D4; Sigma). The antibody was
diluted 2,000 times with 1% milk powder. After rinsing, the membrane
was incubated with a secondary antibody labeled with horseradish
peroxidase (Amersham Corp.) and developed using the ECL reagents and
procedure supplied by Amersham.
Materials and Miscellaneous Methods
cADPR was
synthesized by incubation of NADwith ADP-ribosyl
cyclase purified from Aplysia ovotestis as described
previously
(13) . Fluo 3 was from Molecular Probes (Eugene, OR).
Bovine and spinach CaM, activator-deficient 3`,5`-cyclic nucleotide
phosphodiesterase, caffeine, and ryanodine were from Sigma. Wheat germ
CaM was a generous gift from Dr. Gale Strasbourg, Michigan State
University, East Lansing, MI.
I-Labeled bovine CaM was
from DuPont NEN. Concentrations of CaM were determined using the
Coomassie reagent from Bio-Rad with bovine serum albumin as the
standard.
Specificity of Calmodulin in Conferring cADPR
Sensitivity to Microsomes
We have previously shown that the loss
of cADPR responsiveness of sea urchin egg microsomes purified by
Percoll density gradient can be restored by a soluble protein factor
(10) . That the purified factor is CaM is indicated by the
immunoblot shown in the inset of Fig. 1. On
SDS-polyacrylamide gel electrophoresis, the factor ( SU,
inset of Fig. 1) appeared as a protein of about 17 kDa
( lane 1b), identical to that of authentic bovine CaM ( lane
2b). It was recognized by a monoclonal antibody against CaM
( lane 1a) to a similar extent as bovine CaM ( lane
2a). The specificity of the monoclonal antibody is shown by its
lack of reactivity to bovine serum albumin included in the samples as a
control. The authentic bovine CaM could substitute for the sea urchin
egg factor (SU) in conferring cADPR to the egg microsomes
(Fig. 1). In the absence of either the egg factor or bovine CaM,
purified egg microsomes did not response to cADPR (250 n
M).
Both the egg factor and bovine CaM were equally effective in
reconstituting the Carelease induced by cADPR (250
n
M) and both had half-maximal concentrations of about 2
µg/ml when assayed with the same preparation of egg microsomes.
Figure 1:
Identification
of the cADPR sensitivity-conferring factor in egg extracts as CaM.
S. purpuratus egg microsomes were purified by Percoll density
gradient centrifugation and incubated with bovine CaM or the purified
conferring factor ( SU) at the indicated concentrations.
Carelease activity was induced by 250 n
M cADPR. The inset shows immunoblots of SU ( lane
1a) and bovine CaM ( lane 2a). Lanes 1b and
2b are the respective blots stained for protein. Bovine serum
albumin ( BSA) was included in the samples as an internal
control to demonstrate the specificity of the monoclonal antibody
used.
Although animal CaMs such as bovine can substitute for sea urchin
CaM, plant CaMs are much less effective. Fig. 2shows that
microsomes treated with up to 30 µg/ml of wheat germ CaM remained
unresponsive to cADPR. For comparison, bovine CaM was used as a control
in the same preparation of egg microsomes, which produced close to a
maximal effect at 2 µg/ml. The inset of
Fig. 2
summarizes the results from three different preparations of
microsomes. Spinach CaM at 37-73 µg/ml could produce only
about half of the effect of bovine CaM at 2-5 µg/ml. Wheat
germ CaM at 30 µg/ml was essentially ineffective.
Figure 2:
Difference in effectiveness of animal and
plant CaM in conferring cADPR sensitivity. Egg microsomes were
incubated with either bovine, wheat germ, or spinach CaM at the
indicated concentrations. Carelease activity was
induced by 250 n
M or 200-300 n
M ( inset) cADPR. The error bars shown in the
inset represent the S.E. of the number of determinations
( N).
The
concentration-response curves for bovine and spinach CaM are shown in
Fig. 3A. For bovine CaM, maximal response was seen at
about 3-5 µg/ml. Spinach CaM could support only about
one-third of the Carelease even at the highest
concentration of 73 µg/ml tested with the same preparation of
microsomes. Similar and dramatic differences between bovine and spinach
CaM were seen when the concentrations of the CaM were, respectively,
held constant while the concentration of cADPR was varied as shown in
Fig. 3B. Microsomes treated with 10 µg/ml bovine CaM
produced close to maximal Ca
release when challenged
with about 150 n
M cADPR. The same preparation of microsomes
treated with 37 µg/ml spinach CaM produced no response at 150
n
M cADPR and only about half-maximal release when challenged
with up to 400 n
M cADPR. These results clearly show that the
Ca
release mechanism activated by cADPR exhibits
exquisite specificity toward animal CaM. As will be discussed later,
the sequences of bovine and spinach CaM differ by only 15 amino acids.
This specificity is rather unique as bovine cyclic nucleotide
phosphodiesterase shows relatively little preference between animal and
plant CaM. The half-maximal concentrations of CaM for activating the
phosphodiesterase were measured to be 0.30 ± 0.06 µg/ml
(±S.E., n = 37) for bovine, 0.50 ± 0.07
µg/ml ( n = 39) for spinach, and 0.42 ± 0.1
µg/ml ( n = 16) for wheat germ. Also, with
sufficient concentrations of the plant CaM, 100% activation of the
phosphodiesterase was achieved. This was not the case for the cADPR
sensitivity-conferring activity as shown in Fig. 3, maximal
Ca
release was not achieved even at the highest plant
CaM used.
Figure 3:
Concentration dependence of CaM in
conferring cADPR sensitivity. A, egg microsomes were incubated
with either bovine or spinach CaM at the indicated concentrations.
Carelease activity was induced by 200 n
M cADPR. B, egg microsomes were incubated with either 10
µg/ml bovine or 37 µg/ml spinach CaM and challenged with the
indicated concentrations of cADPR.
Previous results suggest the cADPR sensitivity-conferring
activity of CaM is not mediated by enzymatic reactions but is likely to
be through direct interaction between CaM and the Carelease mechanism
(10) . This is supported by the
measurements of CaM binding to the microsomes shown in Fig. 4.
The measurement was done under identical experimental conditions as the
Ca
release assays. The ambient Ca
concentrations were generally in the range of 20-40 n
M (9) and thus, the binding observed did not require high
concentrations of Ca
. The binding is specific since
increasing the concentration of unlabeled bovine CaM progressively
decreased the total binding of the
I-labeled bovine CaM.
The residual radioactivity observed in the presence of 20 µg/ml of
unlabeled CaM was mainly due to trapping of the label in the microsomal
pellets. Scatchard analysis of the binding data is shown in the inset.
Binding appears to follow single component kinetics with a dissociation
constant ( K
) of 128 n
M and a
density ( B
) of 10.7 pmol/mg. It is well
documented that the ryanodine receptor has especially high affinity for
CaM and the binding occurs even in the absence of Ca
(14) .
Figure 4:
Binding of I-labeled bovine
CaM to egg microsomes. The binding was measured in the presence of the
indicated concentrations of unlabeled bovine CaM as described under
``Experimental Procedures.'' The inset shows the
Scatchard analysis of the binding data.
The effectiveness of various CaMs in competing
for the binding of I-labeled bovine CaM to egg microsomes
is shown in Fig. 5. The bovine CaM was the most effective competitor
and, at 20 and 60 µg/ml, reduced the total binding to 30% and
23.4%, respectively. The residual 23% binding was nonspecific and
represented mainly the trapping of the label in the microsomal pellets.
Plant CaMs were less effective. The residual binding was significantly
higher in the presence of either spinach ( p < 0.009, t test) or wheat germ ( p < 2
10
) than bovine CaM at the same concentrations. The
effectiveness sequence of bovine > spinach > wheat germ was
identical to the cADPR-sensitivity conferring activity as depicted in
the inset of Fig. 2. Since wheat germ CaM was essentially
inactive in conferring the activity, the difference between the
residual binding in the presence of wheat germ and bovine CaM may
represent the crucial CaM binding sites for conferring cADPR
sensitivity. This amounted to about 10-14% of the total CaM
binding or 1.1-1.5 pmol/mg using the B
value of Fig. 4.
Mechanism of the cADPR Sensitivity-conferring Activity of
Calmodulin
Fig. 6
shows the remarkably strong dependence
of the microsomal cADPR sensitivity on calmodulin. In the absence of
calmodulin, the microsomes would not release Caeven
when challenge with as high a concentration of cADPR as 200
µ
M. In the presence of a low concentration of calmodulin,
the cADPR-responsiveness of the microsomes was restored only at high
concentrations of cADPR. At saturating concentrations of calmodulin,
the microsomes responded maximally to about 0.1 µ
M of
cADPR. Therefore, depending on how much calmodulin is present, the
microsomal responsiveness to cADPR can be varied by more than 3-4
orders of magnitude.
Figure 6:
Sensitization of the Ca
release mechanism to cADPR by CaM. Percoll density purified microsomes
were incubated with the indicated concentrations of bovine CaM. The
Ca
release was induced by various concentrations of
cADPR as indicated.
The biological effects of CaM generally are
related to Ca. We, therefore, investigated the
relationship between divalent cations and the microsomal cADPR
sensitivity. The results are shown in Fig. 7. Sr
was used instead of Ca
since it minimally
interfered with Fluo 3, which was used for monitoring Ca
release from microsomes. In the absence of CaM, cADPR as high as
1 µ
M produced no Ca
release
(Fig. 7 a). Fig. 7 b shows that adding 40
µ
M Sr
greatly sensitized the release
system such that cADPR as low as 13 n
M produces significant
Ca
release, and 75 n
M cADPR produced close
to a maximal response. Another way of interpreting these results is
that the Ca
sensitivity of the release mechanism is
increased by the presence of cADPR. In the absence of cADPR, addition
of 40 µ
M of Sr
did not produce any
Ca
release (Fig. 7 b). In the presence
of cADPR, the release mechanism is sensitized to divalent cations and
the same concentration of Sr
could now induce a large
Ca
release (Fig. 7 a).
Figure 7:
Effects of strontium on the
cADPR-dependent Ca release. Microsomes were purified
by Percoll density centrifugation and incubated without CaM. Strontium
( Sr) and cADPR were added to the final concentrations
indicated.
This is shown
more clearly in Fig. 8 A. In the absence of CaM, adding
less than 100 µ
M Srto microsomes did
not produce any Ca
release. Above 100 µ
M Sr
, significant Ca
release was
eventually induced. In the presence of calmodulin, the dose-response
curve is shifted about 2-5-fold to the left. A low concentration
of cADPR alone was more effective in sensitizing the release mechanism
and produces a larger shift of the dose-response curve to the left. At
25 n
M, cADPR was too low to induce Ca
release even in the presence of CaM ( cf.
Fig. 3B). The sensitivity of the release mechanism to
Sr
was increased more than 2 orders of magnitude in
the presence of both cADPR and CaM, and only 10-20 µ
M Sr
was sufficient to produce maximal
Ca
release. The dose-response curve could, in fact,
be shifted further to the left if higher concentrations of cADPR were
used (data not shown). Therefore, cADPR and CaM can act synergistically
to increase the sensitivity of the Ca
release system
to divalent cations.
Figure 8:
Sensitization of
Ca-induced Ca
release by CaM,
cADPR, and caffeine. Percoll density purified microsomes were incubated
without (- CaM) or with (+ CaM) 0.6
µ
M (10 µg/ml) bovine CaM. CICR was activated using the
indicated concentrations of strontium in the presence of 25 µ
M cADPR (+ cADPR) or caffeine (1
m
M).
It is generally accepted that caffeine also
acts by sensitizing CICR to Ca. This is shown in
Fig. 8B. A low concentration of caffeine (1
m
M), which was not sufficient to release Ca
on its own ( cf. Fig. 9), could sensitize the release
mechanism to Sr
to an extent similar to that of CaM.
Together, CaM and caffeine could increase the sensitivity even further.
With both caffeine and cADPR present, the Ca
release
mechanism was so much sensitized that maximal Ca
release was induced without any added divalent cation. These
results indicate that CaM, cADPR and caffeine all act synergistically
to increase the Ca
sensitivity of CICR. When two of
these sensitizers are present in sufficient concentrations, even
ambient concentrations of Ca
, which generally are in
the nanomolar range, are sufficient to activate maximal release.
release activity of caffeine and ryanodine, which
are modulators of CICR. In the absence of CaM, at least 10-11
m
M of caffeine was needed to induce minimal Ca
release. In the presence of CaM, 8-9 m
M of
caffeine was enough to produce maximal Ca
release.
Similar sensitizing effects of CaM were also seen with ryanodine as
shown in Fig. 9 B. Compared with the large sensitization
of CaM on cADPR (Fig. 6), its stimulating effects on these two
artificial modulators of CICR were rather minimal.
Figure 9:
Sensitization of the Ca
release mechanism to caffeine and ryanodine by CaM. Percoll
density-purified microsomes were incubated without or with 7 µg/ml
bovine CaM. The Ca
release was induced by various
concentrations of caffeine or ryanodine as
indicated.
Although CaM can
sensitize all four known modulators of CICR, Ca,
cADPR, caffeine, and ryanodine, it has no effect on the
IP
-dependent Ca
system as shown in Fig.
10. In the presence of CaM, about 200 n
M of cADPR was
sufficient to induce maximal Ca
release. In the
absence of CaM, 200 n
M of cADPR produced no release. In
contrast, the same preparation of microsomes responded equally well to
IP
with or without CaM. The sensitizing effect of CaM is,
therefore, specific for the CICR system.
changes, eggs were
pre-loaded with the Ca
indicator Fluo 3, and the
change in its fluorescence was expressed as a ratio between initial
fluorescence ( F
) and that at various
times afterward ( F). Fig. 11shows that microinjection
of cADPR into an unfertilized egg elicited Ca
release
resulting in about a 10-fold increase in the Fluo 3 fluorescence ratio
( F/ F
). To inhibit the endogenous
CaM, the antagonist, W7, was used; W7 has previously been shown to
block CaM from sensitizing the cADPR-induced Ca
release in egg homogenates
(10) . In the presence of W7,
injection of similar concentrations of cADPR did not produce much
change in Fluo 3 fluorescence. The inset of
Fig. 11
summarizes the results. Of the 12 eggs injected with 313
± 50 n
M (mean ± S.E.) of cADPR in the presence
of 80-120 µ
M W7, 10 showed very little
Ca
change, while two had a normal response. The
average increase in Fluo 3 fluorescence ratio
( F/ F
- 1) was 1.69
± 0.82 for the 12 eggs. In the absence of W7, the increase in
the ratio value ( F/ F
- 1)
of 5.70 ± 1.09 was measured for the nine eggs injected with 337
± 64 n
M cADPR, more than 3-fold higher than in the
presence of W7. In contrast, IP
-induced changes in the
fluorescence ratio were independent of W7 (80-120
µ
M). The increase of the ratio of 5.17 ± 0.78
measured after injection of 456 ± 62 n
M IP
to eight eggs in the presence of W7 was identical to the ratio of
5.18 ± 1.03 measured in six eggs injected with 318 ± 55
n
M of IP
in the absence of W7. These results
indicate cADPR sensitivity in live cells can be regulated by CaM.
Figure 11:
Inhibition of cADPR-induced
Ca release in intact sea urchin eggs by a CaM
antagonist. Methods for microinjection are described under
``Experimental Procedures.'' Intracellular Ca
changes were monitored with Fluo 3. For normalization,
fluorescence intensity at various times ( F) was divided by the
initial fluorescence ( F). The CaM antagonist, W7, was added to
a final concentration of 120 µ
M (+ W7). When
indicated, cADPR was microinjected into L. pictus eggs to a
final concentration of 270 n
M (+ W7) or 350
n
M (- W7). The inset shows the average
increase in the fluorescence ratio ( F/ F - 1)
induced by microinjection of cADPR or IP
in the absence
(- W7) or presence (+ W7) of W7
(80-120 µ
M). The numbers inside the data bars indicate the number of eggs injected, and the error bars indicate the S.E.
release
action of cADPR is through activation of a ligand-gated Ca
channel. This would be analogous to that described for the
IP
receptor (reviewed in Ref. 15). However, unlike the
IP
receptor, the cADPR-gated channel appears to have close
to an absolute requirement for CaM. In its absence, cADPR as high as
200 µ
M cannot activate the channel. The fact that
caffeine, a known Ca
sensitizer of CICR and
Sr
, can substitute for CaM suggests it may also act
by sensitizing the release channel to Ca
. This was
directly demonstrated by showing that both CaM and cADPR can indeed
potentiate the Sr
-induced Ca
release in a manner similar to caffeine. That all three agonists
act synergistically to increase the Ca
sensitivity
indicates the central role of Ca
in the activation of
the Ca
release mechanism. These results are
consistent with cADPR functioning as a modulator of CICR. There appears
to be a certain limit on the maximal extent of sensitization that can
be achieved by cADPR alone. When the ambient Ca
concentration is below that level, Ca
release
cannot be activated by cADPR alone; the synergistic action by CaM or
caffeine is also needed. Raising the ambient divalent cation
concentration alleviates the additional requirement. This
interpretation postulates Ca
as the final activator
of the release channel, while cADPR and CaM play a modulator role by
increasing the Ca
sensitivity of the release system
to such an extent that ambient Ca
can activate. It is
generally believed that CICR is important in propagation of
Ca
waves and in amplifying the Ca
signals due to influx (reviewed in Ref. 4). The mechanism of how
the Ca
sensitivity of this important signaling
pathway can be regulated has not been described. Results described in
this study provide just such a regulatory mechanism. As directly shown
in Fig. 8, depending on the endogenous levels of cADPR and CaM,
the Ca
sensitivity of CICR can be shifted by several
orders of magnitude.
release in sea urchin
eggs without affecting that of IP
. The modulator model
depicted in Fig. 12 summarizes these results. Low concentrations of
cADPR could be present in cells at all times, which together with CaM
are sufficient to sensitize the Ca
release system. An
increase in intracellular Ca
concentration, either by
receptor activated influx or by Ca
mobilization
through the IP
pathway, can then activate further
Ca
release. This is, in fact, what was observed in
amphibian neurons, where cADPR was shown to potentiate the
Ca
influx elicited by membrane depolarization
(16) . The modulator function of cADPR, of course, does not
exclude the possibility that cADPR may also be a second messenger.
Indeed, it has previously been shown that ADP-ribosyl cyclase in sea
urchin eggs, the synthetic enzyme of cADPR, can be activated by a
cGMP-dependent mechanism
(17) . This has led to the proposal
that nitric oxide, through its stimulation of cGMP production, may
activate the cADPR-signaling mechanism
(18) . Alternatively, as
depicted in the messenger model in Fig. 12, binding of an
external ligand to its receptor could result in increased synthesis of
cADPR which, in the presence of endogenous CaM, can then activate
Ca
release from the internal stores. The main
difference between the modulator and the messenger models is that the
former emphasizes the major role of Ca
as the
activation signal, with the endogenous levels of cADPR being relatively
constant. These two models are not mutually exclusive. Depending on how
much calmodulin, cADPR, and Ca
are present in cells,
the signaling mechanism can operate anywhere between these two
extremes.
Figure 12:
Models depicting two modes of action for
cADPR. The activation signal in the Messenger Model is the
ligand-induced activation of the ADP-ribosyl cyclase ( C)
leading to an increase in cytosolic cADPR, while in the Modulator
Model, it is the agonist-gated influx or internal release of
Ca(not shown). In the Modulator Model, the
primary function of cADPR and CaM is to sensitize the Ca
release channel ( R) to
Ca
.
The CaM requirement of the cADPR-dependent Carelease described in this study is remarkable in its exquisite
specificity toward animal CaMs. Both sea urchin and bovine CaM are
equally effective while plant CaMs were much less so. The sequences of
bovine and spinach CaM show a difference of 15 out of a total of 148
amino acids, with 12 of them being conservative substitutions
(19, 20) . The three nonconservative changes from bovine
to spinach CaM are: Thr-26
Cys, Gly-96
Gln, and Thr-146
Met. The first two are within the two of the four Ca
binding sites of CaM, while the third change is also very close
to the fourth Ca
site. That these changes are
concentrated in the Ca
binding regions of CaM is
consistent with the Ca
sensitizing effect of CaM on
the release system as described in this study. The three apparently
crucial amino acids are identical between bovine and sea urchin CaM
(19, 21) , accounting for their equal potency in
conferring the cADPR sensitivity. Perhaps the most remarkable
specificity is that observed between spinach and wheat germ CaM. There
are only two differences in their sequences, a replacement of Asp-97 by
Asn and an insertion of an additional Asn at position 9
(21, 22) . That point mutations on CaM can dramatically
affect its regulation on the Ca
-dependent
K
channel has been well documented in Paramecium (reviewed in Ref. 23). Comparisons of the competition between
wheat germ and bovine CaM for the microsomal binding sites shown in
this study suggest that only 10-14% of the total binding sites
are crucial for cADPR-sensitive Ca
release. This
exquisite specificity of the cADPR-dependent mechanism for CaM could
potentially be exploited for the identification of the relevant CaM
binding sites.
-induced
Ca
release; IP
, inositol
1,4,5-trisphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.