(Received for publication, January 13, 1995; and in revised form, May 24, 1995)
From the
In this study, we have identified and partially characterized a
mouse T-lymphoma ryanodine receptor on a unique type of internal
vesicle which bands at the relatively light density of 1.07 g/ml.
Analysis of the binding of [ Immunoblot analyses using a monoclonal mouse
anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells
contain a 500-kDa polypeptide similar to the ryanodine receptor found
in skeletal muscle, cardiac muscle, and brain tissues. Double
immunofluorescence staining and laser confocal microscopic analysis
show that the ryanodine receptor is preferentially accumulated
underneath surface receptor-capped structures. T-lymphoma ryanodine
receptor was isolated (with an apparent sedimentation coefficient of
30 S) by extraction of the light density vesicles with
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
(CHAPS) in 1 M NaCl followed by sucrose gradient
centrifugation. Further analysis indicates that specific, high affinity
binding occurs between ankyrin and this 30 S lymphoma ryanodine
receptor (K Upon agonist stimulation, most cells release Ca Ryanodine receptors were
originally found in the sarcoplasmic reticulum of skeletal muscle (type
1 receptor, RYR1) and cardiac muscle (type 2 receptor,
RYR2)(3, 4) . The two forms of the ryanodine receptors
appear to be encoded by different genes(3, 4) .
Ca Non-muscle
cells, such as mouse T-lymphoma cells, may possess a specialized
sarcoplasmic reticulum-like organelle required for regulating internal
Ca In this paper, we have isolated and
partially characterized a mouse T-lymphoma ryanodine receptor from a
type of internal vesicle which bands at the relatively light density of
1.07 g/ml. Immunoblot analyses using a monoclonal mouse anti-ryanodine
receptor antibody indicate that mouse T-lymphoma cells contain a
500-kDa polypeptide similar to the ryanodine receptor found in skeletal
muscle, cardiac muscle, and brain tissues. Most importantly, we have
determined that ankyrin (a membrane-associated cytoskeletal protein)
binds specifically to the mouse T-lymphoma ryanodine receptor, and that
this binding significantly inhibits ryanodine binding to ryanodine
receptor and blocks ryanodine-mediated inhibition of internal
Ca
In order to characterize the lymphocyte ryanodine
receptor, we have carried out the following subcellular localization
experiments. First, mouse T-lymphoma cells were homogenized by nitrogen
cavitation, and vesicle populations were fractionated by discontinuous
sucrose density gradient centrifugation (i.e. 0%, 15%, 25%,
35%, 40%, and 50% sucrose) (Table 1). Enzyme marker analyses (13) indicate that the major organelle membranes (e.g. Golgi membrane, endoplasmic reticulum, lysosomal membranes, and
other large particles (e.g. membrane-bound ribosomes)) are
located at the 25-35% sucrose interface (fraction C), the
35-45% sucrose interface (fraction D), and the 40-50%
sucrose interface (fraction E) (Table 1). Mouse T-lymphoma
ryanodine binding sites, however, are preferentially located in the
light density vesicle fraction at the 15-25% sucrose
interface-fraction B (Table 1).
In this paper we have focused
on the ryanodine binding sites detected in the light density vesicles
(fraction B with a density of approximately 1.07 g/ml) (Table 1).
The fact that the mouse T-lymphoma IP
Figure 1:
Binding of
[
In nonexcitable cells, most extracellular signals activate
Ca We have also found that the light density vesicles display
Ca
Figure 6:
Effect of ankyrin on
Some
studies also indicate that cADPR does not act as a direct endogenous
activator of Ca
Figure 2:
Isolation of 30 S lymphoma ryanodine
receptor. A, lymphoma light density vesicles were solubilized
in a solution containing 1 M NaCl solution, 0.25%
CHAPS/phosphatidylcholine, and protease inhibitors. Solubilized
proteins were centrifuged on 5-30% linear sucrose density
gradients in a Beckman SW50 for 16 h at 100,000
Figure 3:
Immunoblot analyses of ryanodine receptor. A, skeletal muscle membranes incubated with monoclonal
anti-ryanodine receptor antibody (RYR.1) antibody. B, cardiac
muscle membranes incubated with monoclonal anti-ryanodine receptor
antibody (RYR.1) antibody. C, brain membranes incubated with
monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. D, lymphoma 30 S molecule incubated with monoclonal
anti-ryanodine receptor antibody (RYR.1) antibody. (Similar results
were observed when lymphoma light density vesicles were incubated with
monoclonal anti-ryanodine receptor antibody (RYR.1) antibody (data not
shown).) (As a control, normal mouse IgG was used. No staining was
detected on these samples (data not shown).) E, lymphoma total
cellular proteins were incubated with monoclonal anti-ryanodine
receptor antibody (RYR.1) antibody. (As a control, normal mouse IgG was
used. No staining was detected on these samples (data not shown).) F, Coomassie-stained lymphoma total cellular proteins. (The
protein concentration used in these experiments was approximately 100
µg/gel lane.)
In Fig. 4, we have shown that if the cells were prefixed with
2% formaldehyde, without being permeabilized by ethanol treatment, no
anti-ryanodine receptor-mediated staining can be detected (Fig. 4A). This finding suggests that ryanodine
receptor is not located on the surface membrane of mouse T-lymphoma
cells. When cells (in the absence of any external ligand challenge)
were fixed with 2% formaldehyde followed by ethanol permeabilization
and anti-ryanodine receptor-mediated staining, intracellular ryanodine
receptors are found to be localized around the periphery of the cell
and the perinucleus region of the cytoplasm (Fig. 4B).
Importantly, if cells were treated with an external ligand such as
concanavalin A (ConA), the intracellular ryanodine receptors (Fig. 4, D and F) become preferentially
associated with surface receptor-patched (Fig. 4C) and
-capped (Fig. 4E) structures. Since ConA is known to be
involved in T cell-mediated lymphocyte activation and
mitogenesis(35) , these results suggest that ryanodine receptor
could be critically important for internal Ca
Figure 4:
Double immunofluorescence staining of
ryanodine receptor- and surface ConA-patched/capped structures. A, staining of ryanodine receptor on the surface of cells
(without any ConA treatment or ethanol permeabilization) using
rhodamine-labeled mouse monoclonal anti-ryanodine receptor antibody
(note that no label was detected). B, staining of
intracellular ryanodine receptor of cells (without any ConA treatment
but with ethanol permeabilization) using rhodamine-labeled mouse
monoclonal anti-ryanodine receptor antibody. C, surface
ConA-patched structures induced by fluorescein-labeled ConA. D, intracellular ryanodine receptor staining of cells (with
ConA treatment and ethanol permeabilization) using rhodamine-labeled
mouse monoclonal anti-ryanodine receptor antibody in the same
ConA-patched cells shown in C. E, surface ConA-capped
structures induced by fluorescein-labeled ConA. F,
intracellular ryanodine receptor staining of cells (with ConA treatment
and ethanol permeabilization) using rhodamine-labeled mouse monoclonal
anti-ryanodine receptor antibody in the same ConA-capped cells shown in E.
In order to determine the
binding interaction between lymphoma ryanodine receptor and the
cytoskeletal proteins such as ankyrin, we have incubated lymphoma 30 S
ryanodine receptor-coated nitrocellulose sheets with
Figure 5:
Binding of
Most
importantly, the binding of ankyrin (but not fodrin) to the ryanodine
receptor in the light density vesicles also significantly inhibits
ryanodine binding (Table 3) and blocks the inhibitory effect of
ryanodine on internal Ca
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]ryanodine to these
internal vesicles reveals the presence of a single, low affinity
binding site with a dissociation constant (K
) of 200 nM. The second
messenger, cyclic ADP-ribose, was found to increase the binding
affinity of [
H]ryanodine to its vesicle receptor
at least 5-fold (K
40 nM).
In addition, cADP-ribose appears to be a potent activator of internal
Ca
release in T-lymphoma cells and is capable of
overriding ryanodine-mediated inhibition of internal Ca
release.
= 0.075 nM).
Most importantly, the binding of ankyrin to the light density vesicles
significantly blocks ryanodine binding and ryanodine-mediated
inhibition of internal Ca
release. These findings
suggest that the cytoskeleton plays a pivotal role in the regulation of
ryanodine receptor-mediated internal Ca
release
during lymphocyte activation.
sequestered in internal storage sites through Ca
release channels, identified as the ryanodine receptor
(RYR)
(
)(1) or the inositol
1,4,5-trisphosphate (IP
) receptor(2) . These two
Ca
channels are homotetrameric megaproteins (subunit
molecular mass of 500 kDa for the ryanodine receptor and 270 kDa for
the IP
receptor) which share extensive homology in their
C-terminal portion where the majority of the transmembrane segments are
predicted to be located(2) .
release from the sarcoplasmic reticulum through
these receptors plays a central role in regulating the contraction of
skeletal and cardiac muscle fibers. In vivo, the
Ca
release channel in skeletal muscle is activated by
direct physical coupling to the voltage sensor in the transverse tubule (5, 6) while the cardiac Ca
channel
is activated by Ca
influx through plasma
membrane-associated Ca
channels(7, 8) . Recently, a third ryanodine
receptor gene (type 3 receptor, RYR3) has been detected in brain
tissues(9) . Since this new isoform of RYR is also found in
smooth muscle tissue and several types of non-muscle
cells(4, 9, 10) , the concept that ryanodine
receptors are muscle-specific Ca
channels must be
changed. Furthermore, it has been suggested that activation of
ryanodine receptors by a second messenger (e.g. cyclic
ADP-ribose (cADPR)) plays a critical role in intracellular
Ca
signaling occurring during agonist-induced cell
activation in non-muscle cells(11, 12) .
release. It has been shown that IP
is
involved in stimulating ``light density vesicles'' in mouse
T-lymphoma cells to release Ca
(13) . Recent
cloning and cDNA sequence analysis show that the brain type of
ryanodine receptor (RYR3) transcript is expressed in human Jurkat
T-lymphocyte cells(14) . However, direct biochemical evidence
for the existence of a non-muscle ryanodine receptor (with a separate
type of intracellular Ca
-release channel) in lymphoid
cells has not been reported.
release. These results strongly suggest that the
interaction between ryanodine receptor and ankyrin may play an
important role in the regulation of internal Ca
release during lymphocyte activation.
Reagents
[H]Ryanodine
(specific activity 54.7 Ci/mmol) was purchased from DuPont NEN.
CaCl
(specific activity 5-30 Ci/g) was
obtained from ICN. cADPR was purchased from Amersham.
Cell Culture
The mouse T-lymphoma BW 5147 cell
line (an AKR/J lymphoma line) was grown at 37 °C in 5%
CO, 95% air using Dulbecco's modified Eagle's
medium supplemented with 10% heatinactivated horse serum (Life
Technologies, Inc.), 1% penicillin, and 1% streptomycin.
Cellular Fractionation
The cells (suspended in 50
ml of ice-cold buffer consisting of 15 mM KCl, 1.5 mM Mg(OAc), 1 mM dithiothreitol, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride (PMSF), and 10 mM HEPES (pH 7.0)) were disrupted by
nitrogen cavitation in an Artisan homogenizer (Artisan Industries,
Inc., Waltham, MA) held at 0 °C using a pressure of 600 p.s.i. for
15 min. After disruption, one-tenth volume of 700 mM KCl, 40
mM Mg(OAc)
, 1 mM dithiothreitol, 400
mM HEPES (pH 7.0), 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1 mM PMSF was added, and nuclei were removed by
centrifugation at 500
g
for 4 min. The
resulting supernatant was layered on a discontinuous sucrose gradient
consisting of 0%, 15%, 25%, 35%, 40%, 50% sucrose (w/w) in a buffer
containing 10 mM HEPES (pH 7.0), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgCl
, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. The gradient
was centrifuged in a Beckman SW28 rotor at 25,000 rpm for 16 h as
described previously(13) . The membranous materials located in
various sucrose layers were collected for further biochemical analyses
including enzyme marker assays, ryanodine binding assays,
Ca
flux measurement, and immunoblotting techniques as
described below.
Enzyme Marker
Assays
Na/K
-ATPase activity
was used as a specific enzyme marker for plasma
membrane(15, 16) . NADPH-dependent cytochrome c reductase and Sulfatase C activities were used as independent
markers of endoplasmic reticulum(17, 18) .
Galactosyltransferase activity, which was used as a Golgi marker, was
assayed according to the procedures described previously(19) .
-N-Acylglucosaminidase was used as a lysosomal
marker(20, 21) . Protein concentrations were
determined using the Bio-Rad Protein Assay Kit.
Double Immunofluorescence Staining
Mouse
T-lymphoma cells were washed with Dulbecco's modified
Eagle's medium and resuspended in the same medium.
Fluorescein-conjugated concanavalin A (FITC-Con A) (20 µg/ml) was
added directly to the cell suspension at 0 °C for 30 min. Cells
were washed with Dulbecco's modified Eagle's medium three
times followed by incubation at 37 °C for 15 min to induce patched
or capped structures. Cells were then fixed in 2% paraformaldehyde
containing 0.1 M phosphate buffer (pH 7.2). In some cases,
cells were fixed in 2% paraformaldehyde without pretreatment with
fluorescein isothiocyanate-ConA. Subsequently, fixed cells were either
rendered permeable by 90% ethanol or without any ethanol
permeabilization followed by staining with an affinity-purified
monoclonal mouse anti-ryanodine receptor antibody (RYR.1, it was raised
against the C terminus cytoplasmic domain of the ryanodine receptor).
These samples were then labeled with rhodamine-conjugated goat
anti-mouse IgG. To detect nonspecific antibody binding, cells were
incubated with normal mouse serum followed by rhodamine-conjugated goat
anti-mouse IgG. No staining was observed in such control samples.
Fluorescence-labeled samples were examined with a confocal laser
scanning microscope (MultiProbe 2001 Inverted CLSM system, Molecular
Dynamics).SDS-PAGE and Autoradiographic
Analyses
Electrophoresis was conducted using a 5% polyacrylamide
SDS-PAGE slab gel and the discontinuous buffer system described by
Laemmli(22) . For autoradiographic analysis, all gels were
vacuum-dried and exposed to Kodak x-ray film (X-Omat XAR-5).Preparation of Ankyrin
Human erythrocyte ankyrin
was purified by the procedure of Bennett and Stenbuck(23) .Ryanodine Binding Assays
Lymphoma light density
vesicles (isolated from the 15-25% sucrose interface according to
the procedures described above) were incubated in a solution containing
20 mM Tris-HEPES (pH 7.2), 150 mM KCl, 1 mM CaCl, 0.2 mM PMSF, 1 mM iodoacetamide, 5 µg
of aprotinin, 5 µg of leupeptin, and 10 nM [H]ryanodine in the presence of various
concentrations of unlabeled ryanodine (ranging from 50 nM-100
µM) for 16 h at 4 °C. Total Ca
concentration in the solution was monitored by atomic absorption
spectroscopy using a Perkin-Elmer model 5000 spectrophotometer equipped
with a Perkin-Elmer HGA500 graphite furnace. A computer program, taking
into account pH, Ca
, and K
, was used
to calculate the concentration of EGTA needed to adjust the free
Ca
concentration. In some cases, either cyclic
ADP-ribose (cADPR) (1-3 µM) or cytoskeletal proteins (e.g. ankyrin (10 µg/ml) or fodrin (10 µg/ml)) was
also included in the ryanodine binding solution. The binding reaction
was terminated by adding a solution containing 150 mM NaCl, 20
mM Tris-HEPES (pH 7.2), 1 mM MgCl
, and 1
mM EGTA and filtrating through a Millipore filter (HAWP, 0.45
µm). The filter-associated radioactivity was analyzed by liquid
scintillation counting. The results were expressed as ``specific
binding'' in which the nonspecific binding (defined as the
radioactivity associated with samples and the filter in the presence of
10
M unlabeled ryanodine) was subtracted
from the total [
H]ryanodine binding. The
background represents approximately 20% of the total
[
H]ryanodine binding activity.
Isolation of 30 S Ryanodine Receptor
Membranes (e.g. mouse lymphoma light density vesicles, rabbit skeletal
muscle sarcoplasmic reticulum, rat cardiac muscle sarcoplasmic
reticulum, and rat brain membrane vesicles) were solubilized in a
solution containing 1 M NaCl solution, 20 mM MOPS,
0.25% CHAPS/phosphatidylcholine (10:1), and protease inhibitors, a
modified procedure from a previous report (24) . Solubilized
proteins were centrifuged on 5-30% linear sucrose density
gradients in a Beckman SW50 for 16 h at 100,000 g
. Gradient fractions were analyzed by
postlabeling with [
H]ryanodine which involves an
incubation of various gradient fractions in a solution containing 20
mM Tris-HEPES (pH 7.2), 150 mM KCl, 1 mM CaCl, 0.2 mM PMSF, 1 mM iodoacetamide, 5 µg
of aprotinin, 5 µg of leupeptin, and 10 nM [
H]ryanodine in the presence of various
concentrations of unlabeled ryanodine (ranging from 50 nM-100
µM) for 16 h at 4 °C. To determine
[
H]ryanodine binding, the individual fraction was
applied onto a small (1-ml) Sephadex G-50 column equilibrated in a
solution containing 1 M NaCl, 20 mM MOPS, 0.25%
CHAPS/phosphatidylcholine, and protease inhibitors. The Sephadex G-50
eluent (i.e. [
H]ryanodine-bound
material) was collected by low speed centrifugation (500
g
), and the radioactivity was analyzed by liquid
scintillation counting.
Ankyrin Binding Assay
Ankyrin was labeled with
NaI using IODOBEADS.
I-Ankyrin (50 ng) was
incubated with a nitrocellulose sheet coated with purified ryanodine
receptors (30 S molecule obtained from lymphoma light density vesicles,
skeletal muscle sarcoplasmic reticulum, cardiac muscle sarcoplasmic
reticulum, or brain membrane vesicles according to the procedures
described above) in the presence of various concentrations of unlabeled
ankyrin (ranging from 0.01 nM-1.00 µM) in a
binding solution containing 20 mM Tris-HCl (pH 7.4), 150
mM NaCl, 0.05% Triton X-100, and 0.1% bovine serum albumin for
16 h at 4 °C. Following incubation, the nitrocellulose sheets were
washed 5 times with the same binding solution and counted in a
counter. The results were expressed as specific binding in which the
background level of binding was subtracted. As a control, we removed
ryanodine receptors by incubating the 30 S peak fraction with
anti-ryanodine receptor antibody-conjugated Sephadex beads. No specific
ankyrin binding was detected in these ryanodine receptor-free samples.
Ca
Flux Measurement in Light
Density Vesicles
Ca
fluxes were studied in a reaction mixture containing 20 mM Tris-HEPES (pH 7.2), 150 mM KCl, 0.2 mM PMSF, 1
mM iodoacetamide, 5 µg of aprotinin and 5 µg of
leupeptin, 1 mM
CaCl
in the presence
or absence of various concentrations of ryanodine (10-100
µM) overnight. In some cases, either cyclic ADP-ribose
(cADPR) (1-3 µM) or ankyrin (10 µg/ml) was also
included in the reaction medium in which the free Ca
was preadjusted to 0.1 µM. The Ca
efflux was quenched by the addition of 5 mM
LaCl
. The amount of Ca
released from the
light density vesicles was determined by a filtration method using
Millipore filters (HAWP, 0.45 µm) and washing with a buffer
containing 120 mM KCl and 20 mM Tris-HEPES (pH 7.2).
The filter-associated radioactivity was analyzed by liquid
scintillation counting.
Immunoblotting Techniques
Protein samples
including purified lymphoma 30 S molecule, lymphoma light density
vesicles (collected from 15-25% sucrose interface), lymphoma
total cellular proteins, rabbit skeletal muscle membranes, rat cardiac
muscle membranes, and rat brain membrane vesicles were analyzed by a 5%
polyacrylamide gel electrophoresis followed by transferring to
nitrocellulose sheets. Ryanodine receptor-related immunoreactive
proteins were detected by an enhanced chemiluminescence method using a
1:600 dilution of an affinity-purified monoclonal mouse anti-ryanodine
receptor antibody (RYR.1) (1 mg/ml) and a 1:7000 dilution of
horseradish peroxidase-conjugated sheep anti-rat IgG (1 mg/ml)
(Amersham) followed by exposure to Kodak XAR-5 film. Normal mouse serum
was used as a control.
Subcellular Localization of the Ryanodine Receptor on
Ca
It is well known that CaStorage Vesicles in Mouse T-lymphoma
Cells
plays an
important role in the regulation of a large number of cellular
activities including lymphocyte receptor capping(25) . It has
been suggested that the increase in intracellular Ca
concentration following the addition of ligand (or agonist)
results from either Ca
release from intracellular
storage vesicles or exchange with extracellular
Ca
(2) . Previously, we have shown that an
IP
receptor-gated Ca
channel is involved
in the internal Ca
release in mouse T-lymphoma
cells(13) . A recent study reports that lymphocytes contain a
brain-type ryanodine receptor transcript which appears to be involved
in T-cell function(14) . However, the biochemical properties
and regulatory aspects of this ryanodine receptor have not been
determined.
receptor is also
found in this light density vesicle fraction (13) indicates
that either the two types of receptors are located in different vesicle
populations which share similar isopycnic (density) points, or both the
ryanodine receptor and the IP
receptor are located on the
same vesicles. The relationship between the ryanodine receptor and the
IP
receptor in mouse T-lymphoma cells is currently under
investigation.
Ryanodine Binding Properties and Effects of Cyclic
ADP-Ribose (cADPR) on the Ryanodine-sensitive Ca
The affinity of
[Channel
H]ryanodine for the light density vesicles was
measured in binding inhibition experiments using increasing
concentrations of unlabeled ryanodine. The displacement curve reveals
the presence of a single low affinity ryanodine binding site with a
dissociation constant (K
) of 200 ±
10 nM (Fig. 1). This K
is
similar to that for the low affinity sites found on skeletal muscle
sarcoplasmic reticulum (4, 26, 27) and
reported to be associated with inhibition of Ca
channel activity(4, 28) . The high affinity
ryanodine binding site (K
2-5
nM) locks ryanodine receptors into an open state upon binding
of ryanodine in both skeletal (1, 4) and cardiac (1, 4) muscles. This high affinity ryanodine binding
site was not detected in resting (unstimulated) mouse T-lymphoma cells.
H]ryanodine to light density vesicles. Light
density vesicle membranes (collected from 15-25% sucrose
interface) were incubated with [
H]ryanodine in
the presence of various concentrations of unlabeled ryanodine in
binding buffer as described under ``Materials and Methods.''
, without any treatment;
, with 1 µM cADPR
treatment. In Fig. 1, the results were expressed as specific
binding. Specifically, the concentration of
[
H]ryanodine was held constant, and increasing
amounts of unlabeled ryanodine (ranging from 10
M to 10
M) were added to
adjust the total ryanodine concentration. Nonspecific binding was
defined as the [
H]ryanodine binding occurring in
the presence of 10
M unlabeled ryanodine.
The amount of nonspecific binding did not exceed 20% of total
[
H]ryanodine binding at any unlabeled ryanodine
concentration below 10
M and was subtracted
from the total [
H]ryanodine binding at all
ryanodine concentrations (10
M-10
M) to give the
specific binding. The maximal level of
[
H]ryanodine binding to lymphoma light density
vesicles is approximately 30 pmol/mg of protein. K was
measured as 50% inhibition of specific
[
H]ryanodine binding. The numbers shown in this
figure were the averages of triplicate determinants in three
experiments, which varied by less than 5% (n =
9).
release from internal stores through the
production of the second messenger, IP
(2) . Recent
studies on Ca
mobilization, however, suggest that
second messengers other than IP
may be involved in the
regulation of intracellular Ca
concentration. A
likely candidate for such as second messenger is cyclic ADP-ribose
(cADPR), a metabolite of NAD, which has been shown to be a potent
activator of internal Ca
release in sea urchins (29, 30) and a modulator of the ryanodine-sensitive
Ca
release in both cardiac and brain
microsomes(31) . Activation of ryanodine receptors by cADPR is
modulated by calmodulin which serves as an inhibitor for internal
Ca
release(32) . In this study, we have found
that cyclic ADP-ribose (cADPR) increases the binding affinity of
[
H]ryanodine to its receptor at least 5-fold (K
40 nM) (Fig. 1).
It is possible that low affinity ryanodine binding sites are normally
expressed in the resting (unstimulated) lymphoid cells. These low
affinity ryanodine binding sites may then be converted into higher
affinity ryanodine binding sites when a second messenger, such as
cADPR, is formed during lymphocyte activation by agonists. It has been
shown that both cADPR and its breakdown product, ADP-ribose, are
capable of influencing the Ca
release
channel(33) . In this study we cannot rule out the possibility
that cADPR and/or ADP-ribose are responsible for the increase of the K
of [
H]ryanodine
binding.
release activities (Table 2) which can be
selectively inhibited by ryanodine. These findings suggest that (i) the
mouse T-lymphoma cells' light density vesicles contain a
ryanodine-sensitive Ca
channel (Tables I, II, and Fig. 6, panel 1), (ii) the cADPR is a potent activator
of internal Ca
release; and (iii) cADPR-mediated
activation is not inhibited by ryanodine (Table 2). Presently,
the mode of action of cADPR in regulating Ca
channel
activity is not fully established. It is speculated that cADPR displays
stimulatory effects on both ryanodine-sensitive and
ryanodine-insensitive Ca
channel activities.
Inhibition of cADPR-stimulated ryanodine-sensitive
Ca
channels by ryanodine may be
overridden by the extensive cADPR stimulation on ryanodine-insensitive Ca
channels under our assay conditions. This hypothesis is currently under
investigation in our laboratory. It is noteworthy that a similar
overriding of ryanodine inhibition of internal Ca
release by cADPR has also been reported to occur in cardiac
muscle sarcoplasmic reticulum and brain vesicles(31) .
Ca
release in lymphoma light density
vesicles. Panel 1A, untreated vesicles; panel 1B,
ryanodine-treated vesicles; panel 2A, ankyrin-treated
vesicles; panel 2B, ankyrin-treated vesicles followed by
ryanodine treatment.
release activity associated with the
skeletal or cardiac muscle ryanodine
receptor(33, 34) . Structurally, the skeletal muscle
ryanodine receptor (RYR1) and the cardiac ryanodine receptor (RYR2) are
different from the brain and lymphoma ryanodine receptor (RYR3). It is
possible that: (i) cADPR displays high affinity binding with RYR3 but
has a low binding affinity with RYR1 and RYR2 or (ii) the binding
affinity for cADPR to RYR3 may be higher than that in RYR1 and RYR1.
The effective interaction between cADPR and RYR3 (but not RYR1/RYR2)
may activate Ca
release channels only in
RYR3-containing tissues and cells. A systematic analysis of cADPR
binding to the three types of ryanodine receptors (i.e. RYR1,
RYR2, and RYR3) is needed to test this hypothesis.
Immunological and Structural Analyses of Mouse T-lymphoma
Ryanodine Receptor
Extraction of the light density vesicles with
CHAPS in 1 M NaCl, followed by sucrose gradient centrifugation
and postlabeling with [H]ryanodine, yields a
single peak with a sedimentation coefficient of approximately 30 S,
similar to the skeletal muscle ryanodine receptor (Fig. 2A). SDS-PAGE analysis followed by silver
staining of the 30 S peak material reveals a single polypeptide
(molecular mass of
500 kDa) (Fig. 2B). Using
immunoblotting techniques, we have found that the monoclonal
anti-ryanodine receptor antibody (RYR.1) recognizes a single
polypeptide (with a molecular mass of
500 kDa) present among all
lymphoma cellular proteins (Fig. 3, E and F).
This antibody also binds the ryanodine receptor (Fig. 3D) isolated from skeletal muscle (Fig. 3A), cardiac muscle (Fig. 3B),
brain tissues (Fig. 3C), and the lymphoma 30 S peak
fraction (Fig. 3D). These findings clearly indicate
that mouse T-lymphoma cells do, in fact, contain ryanodine receptors.
g
. Gradient fractions were analyzed by
postlabeling with [
H]ryanodine as described under
``Materials and Methods.'' As a protein standard, skeletal
muscle ryanodine receptor (with a sedimentation value of 30 S) was used
to determine the s value of the
[
H]ryanodine binding peak. B,
polypeptide pattern revealed by SDS-PAGE analysis followed by silver
staining of the 30 S peak material.
release
at the onset of receptor capping and lymphocyte activation.
Regulation of Ryanodine Receptor by the Cytoskeletal
Protein, Ankyrin
Cytoskeletal proteins are included in the array
of cellular elements which underlie and interact with cellular
membranes (plasma membrane and/or internal vesicle membranes). It is
believed that the formation of a direct connection between the
cytoskeleton and certain membrane proteins is one of the earliest
events to occur during signal transduction(36) . In fact, a
number of cytoskeletal proteins, which are analogs of the major
erythrocyte membrane skeleton proteins (e.g. ankyrin, fodrin
(a spectrin-like protein), and band 4.1-like protein), are found to be
involved in transmitting regulatory signals during cell activation by
agonists(37) . Ankyrin is a cytoskeletal protein known to be
linked to certain membrane proteins, such as erythrocyte band 3 (37) and lymphocyte
GP85(CD44)(38, 39, 40, 41, 42) ,
and to spectrin/fodrin-actin-associated microfilaments. In addition,
ankyrin has been found to bind to the IP receptor in brain
tissue (43) and lymphoma cells(13, 44) , and
the binding of ankyrin to the IP
receptor modulates
IP
binding and internal Ca
release(13, 44) .
I-ankyrin in the presence of various concentrations of
unlabeled ankyrin (Fig. 5A). Our data indicate that
specific, high affinity binding occurs between ankyrin and this
lymphoma 30 S ryanodine receptor (K
= 0.075 nM) (Fig. 5A). In
addition, we have shown that the binding of
I-ankyrin to
various 30 S ryanodine receptors isolated from skeletal muscle (Fig. 5B, a), cardiac muscle (Fig. 5B, c), and brain tissue (Fig. 5B, e) is also specific because 100-fold
excess soluble unlabeled ankyrin can successfully compete with
I-ankyrin binding to these ryanodine receptors (Fig. 5B, b, d, and f).
These data suggest that the amino acid sequence of the ankyrin binding
domain on various ryanodine receptors must be highly conserved among
different cell types and tissues. Recently, we have shown that an
11-amino-acid synthetic peptide derived from sequence comparisons
between two ankyrin binding proteins (CD44 and IP
receptor)
inhibit the binding of ankyrin to mouse lymphoma IP
receptor(44) . Preliminary data indicate that this
11-amino acid synthetic peptide (containing the ankyrin binding domain)
also blocks ankyrin binding to purified lymphoma 30 S ryanodine
receptor with an apparent inhibition constant (K
) of 0.25 nM.
I-ankyrin to
various purified 30 S ryanodine receptors. A, purified
lymphoma 30 S ryanodine receptor was incubated with
I-ankyrin in the presence of various concentrations of
unlabeled ankyrin in binding buffer as described under ``Materials
and Methods.'' K was measured as 50% inhibition of
specific
I-ankyrin binding in which the background level
of nonspecific binding (i.e. radioactivity detected in the
presence of 100 nM unlabeled ankyrin) was subtracted. B, purified 30 S ryanodine receptor isolated from skeletal
muscle (a and b), cardiac muscle (c and d), and brain tissue (e and f) was incubated
with
I-ankyrin in the absence (a, c,
and e) and the presence (b, d, and f) of a 100-fold excess amount of unlabeled ankyrin in binding
buffer.
release (Fig. 6, panel 2). Therefore, it is possible that ankyrin binding
competes or overlaps with ryanodine binding sites. This may explain
ankyrin's overriding effect on ryanodine-mediated inhibition of
internal Ca
release in these vesicles (Fig. 6, panel 2). Alternatively, ankyrin binding may cause some
conformational change in the regulatory domain(s) of ryanodine receptor
resulting in a decrease in ryanodine binding as well as a reduction in
the potency of ryanodine inhibition of internal Ca
release. The fact that ankyrin also modulates IP
binding and IP
-mediated Ca
release
in mouse T-lymphoma cells (13, 44) strongly suggests
that ankyrin is one of the important molecules regulating
Ca
signaling. Currently, we are using in vitro mutagenesis and deletion mutation techniques to further define the
ankyrin binding domain(s) on the mouse T-lymphoma ryanodine receptor.
In conclusion, our findings support the notion that mouse T-lymphoma
cells contain ryanodine receptors which function as a second
intracellular Ca
release channel along with the
IP
receptors. Most importantly, we believe that both cADPR
and the cytoskeletal protein, ankyrin, play a pivotal role in the
regulation of ryanodine-sensitive channel-mediated internal
Ca
release during lymphocyte activation.
,
inositol 1,4,5-trisphosphate; cADPR, cADP-ribose; PMSF,
phenylmethylsulfonyl fluoride; ConA, concanavalin A; PAGE,
polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic
acid; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid.
We gratefully acknowledge the assistance of Dr. Gerard
J. Bourguignon in the preparation of this manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.