(Received for publication, May 17, 1995; and in revised form, August 30, 1995)
From the
Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A >500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.
Sorcin was initially identified as a 22-kDa protein in cultured
cells selected for resistance to natural product cancer drugs, such as
vincristine, adriamycin, and actinomycin D, i.e. multidrug-resistant
cells(1, 2, 3, 4, 5) . One
of the major mechanisms of resistance in these cells is mediated by
overexpression of the membrane-bound drug transporter,
P-glycoprotein(6) . Molecular cloning studies demonstrated that
the sorcin and P-glycoprotein genes are tightly linked and that both
may be amplified during the acquisition of multidrug resistance.
However, while P-glycoprotein overexpression correlates with resistance
development, increased sorcin expression is not obligatory, and its
abundance does not correlate with the degree of
resistance(4, 5, 6, 7, 8, 9) .
Complementary DNA for sorcin has been isolated from hamster (2) and human (10) multidrug-resistant cells, which
amplify the sorcin gene. The highly conserved sequence, with 95%
homology between hamster and human sorcin, predicts a 22-kDa protein
with four putative Ca-binding domains, two with
strong homology to calmodulin ``EF hand'' motifs(2) .
In a classification of Ca
-binding proteins based on
sequence, sorcin was placed among members of the closely related
calpain and sarcoplasmic Ca
-binding protein
subfamilies (11) . Direct
Ca
binding to sorcin has been demonstrated by in vitro assays(3) , and Ca
affinity in the
µM range has been determined by fluorescence
spectroscopy(12) . Sorcin also contains two putative
recognition sites for protein kinase A, and phosphorylation of sorcin
in drug-resistant cell-free extracts or of purified sorcin in the
presence of the catalytic subunit of protein kinase A has been
observed(4, 5, 13) . Although initially
characterized as a soluble protein, our recent studies have shown that
sorcin undergoes Ca
-mediated translocation from
soluble to cellular membrane sites(12) . Despite these
biochemical data suggesting a role for sorcin in Ca
handling, a function for sorcin in multidrug resistance or in
P-glycoprotein activity remains
speculative(2, 3, 4, 5) .
In the
present study, we began to characterize the expression of sorcin in
normal tissues and gain some insight into its function. We found that
sorcin is widely expressed in mammalian tissues, including the heart,
where it was localized to cardiac sarcoplasmic reticulum (SR). ()Co-immunoprecipitation studies revealed an association
between sorcin and the ryanodine receptor (RyR), the calcium release
channel located in muscle SR at the junction of transverse tubules and
SR terminal cisternae(14) . Sorcin transfectants in DC-3F
Chinese hamster lung fibroblasts were generated to study a possible
role for sorcin in intracellular calcium transport. These cells were
characterized immunocytochemically for sorcin and ryanodine receptor
expression as well as functionally, by digital
Ca
-imaging for their response to caffeine, a
potentiator of Ca
release from the SR through
RyR/Ca
release channels(14, 15) .
Metabolic labeling with 100 µCi/ml
[S]methionine (DuPont NEN) in methionine-free
medium was accomplished by overnight incubation of monolayer cells at
37 °C. Labeled or unlabeled cardiac myocytes or cultured Chinese
hamster cells and sublines were lysed in 20 mM sodium
phosphate buffer (pH 7.4) containing 0.15 M NaCl, 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride by needle
extrusion. Where noted for some experiments lysis buffers also
contained 1% 2-mercaptoethanol. Cross-linking of proteins was carried
out by treating cell lysates with 1 mM
dithiobis(succinimidylpropionate) (Pierce) in dimethyl sulfoxide for 30
min at room temperature, followed by quenching with 1 M Tris-HCl (pH 7.4), as suggested by the manufacturer. All
procedures involving use of animal tissues were undertaken in
accordance with institutional guidelines.
DC-3F, DC-3F/VCRd-5L, transfected DC-3F cells and other controls were grown on two-chamber glass slides (Lab-Tek Chamber Slide System, VWR Scientific, Rochester, NY) or glass coverslips. The cells were rinsed and fixed as described above for heart tissue. Cells were incubated for 12-18 h in 0.1 M Tris-buffered saline containing 0.1% bovine serum albumin and either a 1:1000 dilution of the rabbit antiserum against sorcin C terminus peptide or a 1:500 dilution of GP561, a polyclonal antiserum raised in guinea pig against purified rabbit brain ryanodine receptor. The cells were then processed with the ABC method described above, and the bound peroxidase was visualized by light microscopy.
Figure 1: Distribution of sorcin among mouse tissues. Western blot analysis of homogenates of liver (lane 1), small intestine (lane 2), heart (lane 3), brain (lane 4), lung (lane 5), skeletal muscle (lane 6), spleen (lane 7), kidney (lane 8), and DC-3F/VCRd-5L cells (lane 9) with antiserum raised against the sorcin C terminus peptide is shown. Samples containing 60 µg of protein from tissues or 2 µg of DC-3F/VCRd-5L cell protein were heated at 100 °C for 2 min before electrophoresis on 13% acrylamide gels.
Figure 2: Specificity of antiserum to sorcin C terminus peptide. Western blot analysis of 60 µg of mouse heart homogenate (lanes 1 and 3) and 2 µg of DC-3F/VCRd-5L cell protein (lanes 2 and 4) per lane with peptide antisera (lanes 1 and 2) or with antisera preadsorbed with C terminus peptide (lanes 3 and 4). Samples were treated as described in Fig. 1. The minor band at 35 kDa was present in some tissue (see Fig. 1, lane 6) and cultured cell samples.
Figure 3:
Northern blot analysis of rodent tissue
RNA with sorcin cp6 cDNA probe. Lanes 1, 4, and 5 contain 10 µg of total RNA from rat heart, rat spleen, and
mouse heart, respectively. Lanes 2 and 3 contain 2
µg of poly (A) RNA from mouse spleen and heart,
respectively. Sizes of the transcripts are identical to those detected
in DC-3F/VCRd-5L cells(18) .
Figure 4: Immunofluorescent labeling of rat cardiac myocytes with sorcin antiserum. Cells were labeled with rabbit antibody raised against sorcin C terminus peptide with rhodamine-conjugated goat anti-rabbit secondary antiserum (right panel) and double-labeled with mouse monoclonal antibody directed against sarcomeric myosin heavy chain with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antiserum (left panel). Labeling with sorcin N terminus peptide antibody was identical in distribution to that seen in the right panel (not shown).
Figure 5: Localization of sorcin to rat ventricular SR by immunoelectron microscopy. Sections were labeled with unadsorbed or preadsorbed antibody directed against the sorcin N terminus peptide. A, section shows intense labeling of SR (arrow) near transverse tubule (*) and mitochondrion (m) with unadsorbed antibody. B, section labeled as in panel A shows intense peroxidase reaction product in the SR (arrow) near the plasmalemma of the myocyte and nearby mitochondrion (m). Plasmalemma (arrowheads) and mitochondrial membranes are also peroxidase-positive near points of apposition with the labeled SR. Other portions of the plasmalemma (small arrows) appear markedly less electron dense. C, section collected from tissue processed as in panels A and B but at depths in the tissue having limited access to immunoreagents. No peroxidase product is associated with the SR (arrow) near the transverse tubule (*) or mitochondrion (m), as was the case when preadsorbed antibody was used for labeling. Bars, 0.5 µm.
Figure 6:
Immunoprecipitation of
[S]methionine-labeled proteins with sorcin and
RyR antibodies. Mouse (lane 1) and rat (lane 2)
cardiac myocyte proteins were immunoprecipitated with antibody raised
against sorcin N terminus peptide; rat cardiac myocyte proteins were
immunoprecipitated with antibody to RyR (C3-33) (lane 3) (arrow at left indicates 565-kDa RyR protein);
DC-3F/VCRd-5L cell proteins were immunoprecipitated with RyR antibody (lane 4), antibody to sorcin N terminus peptide (lane
5), and antiserum to sorcin C terminus peptide (lane 6);
and cardiac fibroblasts were immunoprecipitated with RyR antibody
(C3-33) (lane 7). Aliquots of homogenates containing 200
µg of protein (at equivalent specific radioactivities) were
analyzed by immunoprecipitation on 5% acrylamide gels. The >500-kDa
band present in lanes 1 and 2 was also detected when
myocyte lysates were treated with dithiobis(succinimidylpropionate)
cross-linking agent before
immunoprecipitation.
To address the question of whether the >500-kDa band represented aggregated material or a true association between sorcin and the RyR, additional immunoprecipitation experiments were carried out with the use of RyR-specific antibody. Immunoprecipitates of proteins from rat cardiac myocytes were solubilized in Laemmli buffer containing 2-mercaptoethanol and examined by Western blot analysis with sorcin antiserum, resulting in display of a prominent 22-kDa protein (Fig. 7, lane 1). Interestingly, no sorcin was detected when the myocytes were initially lysed in the presence of a reducing agent (Fig. 7, lane 2), suggesting a disulfide-linkage between sorcin and the RyR complex. RyR antibody did not recognize sorcin, nor did sorcin antibodies recognize RyR, by Western blot analysis.
Figure 7: Immunoprecipitation/immunoblot analysis of sorcin in unlabeled rat cardiac myocytes. Aliquots of cell homogenates containing 100 µg of protein were immunoprecipitated by RyR antibody (C3-33) (lane 1), RyR antibody (with myocytes lysed in the presence of 2-mercaptoethanol) (lane 2), sorcin C terminus peptide antiserum (lane 3), and preimmune sorcin antiserum (lane 4). Antigen-antibody complexes were solubilized by heating in Laemmli buffer containing 2-mercaptoethanol (21) at 100 °C for 2 min, and products were subjected to gel electrophoresis on 13% acrylamide gels before analysis by Western blot with antiserum raised against sorcin C terminus peptide. The arrow indicates 22-kDa sorcin. Sorcin was also detected if myocyte proteins were cross-linked before immunoprecipitation. The 45-50-kDa proteins in the figure are observed in all lanes, including the preimmune sorcin antiserum samples (lane 4), suggesting lack of specificity.
Figure 8: Immunocytochemical labeling of Chinese hamster cells with sorcin antiserum or GP561 RyR antibody. Panels A and B show DC-3F/VCRd-5L cells, panels C and D show DC-3F cells, and panels E and F show DC-3F/sor.3 sorcin transfectants. Panels A, C, and E show labeling with sorcin C terminus peptide antiserum, and panels B, D, and F show labeling with RyR antibody. Bar, 100 µm.
Figure 9:
Effect of 10 mM caffeine on
intracellular Ca by digital fluorescent
Ca
imaging with fura-2/AM indicator. False color
images show levels of intracellular Ca
in
sham-transfected DC-3F cells (DC-3F/neo) of about 100 nM in
the absence (A) or presence (B) of 10 mM caffeine. Caffeine stimulates an increase in intracellular
Ca
in DC-3F/sor.3 sorcin-transfected cells from about
100 nM (C) to 240 nM (D).
Sorcin-overproducing DC-3F/VCRd-5L cells also respond to 10 mM
caffeine with a rise in intracellular Ca
from about
100 nM (A`), to about 250 nM (B`),
followed by a gradual decay toward base line in 4-8 min (C`, D`). More than 90% of sorcin-transfected or
drug-resistant cells in each field were caffeine-responsive. The rainbow spectrum on the right correlates with
Ca
concentration as calculated from in vitro calibration methods described under ``Materials and
Methods.'' Addition of EGTA to extracellular solutions during
caffeine perfusion did not affect these
results.
The time
frame of effects of caffeine on intracellular Ca levels is shown in Fig. 10. Responsive cells showed peak
Ca
levels within 1 min after application of caffeine,
and levels gradually returned to base line over the course of 2-8
min. Removal of extracellular Ca
by the addition of 5
mM EGTA to the Hepes-buffered Krebs-Henseleit medium did not
affect the response to caffeine, confirming that caffeine induced
cytosolic Ca
increase by releasing Ca
from intracellular stores and not by Ca
influx
from the extracellular medium.
Figure 10:
Time course of the effect of 10 mM caffeine on free intracellular Ca. Change in
intracellular Ca
levels with time in DC-3F/sor.3 (solid circles) and in DC-3F or sham-transfected DC-3F
(DC-3F/neo) (open circles) and low level sorcin DC-3F/AD X
cells (open triangles) is shown. No change in intracellular
free Ca
was measured for DC-3F, DC-3F/neo, or
DC-3F/AD X cells over the course of the experiments. Values represent
the average of 10 determinations with ±S.E. represented by error bars; there were at least 50 cells/microscope
field.
Sorcin was initially identified as a protein differentially expressed during the acquisition of multidrug resistance(1, 2, 3, 4, 5) . Although the mechanism underlying sorcin's enhanced expression in these cells has been determined, its pattern of expression and function in normal tissues have not previously been characterized. With the use of immunological reagents, we now demonstrate that 22-kDa sorcin is widely expressed in mammalian tissues and is highly conserved among mammalian species. Antibodies raised against hamster sorcin peptide sequences recognized the 22-kDa protein in a number of other species, including mouse, rat (this paper), and human(4, 5) . Higher molecular weight immunoreactive bands are observed in some tissue and cultured cell samples. Their identity has not been determined; however, they may represent different forms of sorcin or sorcin dimers. A related 28-kDa protein, grancalcin, has been shown to exist in dimeric form, possibly through covalent linkage stable to thiol reagents(33) .
The primary sequence of sorcin gives
some clues as to its potential function. The two EF hand
Ca-binding domains are homologous with those in a
number of Ca
-binding proteins, including calpain and
calmodulin(2) , and sorcin has been shown to bind
Ca
by in vitro assays(3, 12) . Immunoelectron microscopy
directly localized sorcin to the SR, at or near transverse tubules,
suggesting that sorcin might participate in SR-mediated intracellular
Ca
regulation.
Biochemical studies presented here
suggest that sorcin interacts directly with the cardiac RyR. Sorcin
antisera immunoprecipitated a >500-kDa species from cardiac myocytes
that co-migrated on gels with the protein immunoprecipitated by
antibody raised against the 565-kDa RyR receptor. This finding suggests
that sorcin is part of a complex, and an association between sorcin and
the cardiac RyR/SR Ca release channel was confirmed
by co-immunoprecipitation of 22-kDa sorcin from cardiac myocytes with
RyR-specific antibody. Immunoblot analysis of isolated SR protein with
antibody to RyR and to sorcin revealed the presence of the 565-kDa RyR
and of 22-kDa sorcin in a location protected from proteolysis by
proteinase K (not shown), supporting the possibility of an
intramembrane interaction between sorcin and the RyR complex.
Sorcin
transfectants, generated to study a function for the
Ca-binding protein, were unexpectedly found to be
RyR-positive. DC-3F/VCRd-5L cells, which overproduce sorcin by an
entirely different mechanism, also displayed RyR immunoreactivity. The
species accounting for the immunoreactivity in the drug-resistant cells
may be the
110-kDa protein immunoprecipitated by RyR antibody (Fig. 6, lane 4) (also identified by Western blot with
RyR antibody in other experiments), although the apparent absence of
the 565-kDa RyR as an immunoprecipitated protein in those cells could
be associated with differences between C3-33 and GP561 RyR
antibodies, assay sensitivity, or low expression of RyRs.
Increase in RyR immunoreactivity in sorcin transfectants and in DC-3F/VCRd-5L cells suggested that sorcin influences the abundance of RyR protein. Given the demonstrated biochemical association between the two proteins, it is conceivable that sorcin directly stabilizes the RyR protein and retards its degradation, a mechanism that seems more plausible than a transcriptional effect on RyR gene expression. Clearly, sorcin alone is insufficient to promote the accumulation of the RyR in all cell types, since the former protein is much more widely expressed than the latter. However, in the appropriate cellular environment, such as sarcomeric muscle, the levels of sorcin may influence the abundance of co-expressed RyR. Thus, it will be of interest to compare the profiles of sorcin and RyR expression during myogenic differentiation, both in vivo and in cell culture model systems, such as the C2C12 mouse myoblast cell line(34) , where our preliminary studies suggest a marked accumulation of sorcin during the process of differentiation into myotubes. It should be pointed out, with regard to a possible role for sorcin in multidrug-resistant cells, that forced expression of sorcin in Chinese hamster lung cells did not, on its own, confer the multidrug resistance phenotype.
Both DC-3F/VCRd-5L and DC-3F/sor.3 cells, with increased
levels of sorcin and RyR protein, demonstrated a characteristic
property of the RyR/Ca release channel, i.e. caffeine-induced intracellular Ca
release. The
temporal profile of Ca
movement exhibited by the
sorcin-transfected fibroblasts in response to caffeine paralleled that
observed in similarly treated cardiac myocytes. These results suggest
that nonexcitable cells may serve as a useful model to study some
aspects of intracellular Ca
movement normally
associated with excitable tissues, analogous to the recent use of such
cells for the study of contractile protein function(35) . The
ultimate goal of such analyses is to produce new information about
cardiac or muscle cell function and diagnose dysfunction.
Although
these data suggest an important interaction between sorcin and the RyR,
they do not indicate whether interaction between sorcin and RyR is
direct or through a third or intermediary protein. The data also do not
address whether sorcin modulates any gating parameters of the
Ca release channel. Detailed functional studies using
expression systems in which sorcin and/or RyR abundance can be
manipulated and Ca
movement determined will be
required to investigate the latter possibility.