Specific Substitutions at Amino Acid 256 of the
Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transport ATPase
Mediate Resistance to Thapsigargin in Thapsigargin-resistant Hamster
Cells*
Myounghee
Yu
,
Lilin
Zhang§,
Arun K.
Rishi
,
Mohammed
Khadeer
,
Giuseppe
Inesi§, and
Arif
Hussain
§¶
From the
Division of Oncology, Department of
Medicine, Greenebaum Cancer Center and § Department of
Biological Chemistry, School of Medicine, University of Maryland,
Baltimore, Maryland 21201, and the ¶ Baltimore Veterans Affairs
Medical Center, Baltimore, Maryland 21201
 |
ABSTRACT |
High levels of resistance to thapsigargin (TG), a
specific inhibitor of intracellular Ca2+ transport
ATPases (SERCAs), can be developed in culture by stepwise exposure of
mammalian cells to increasing concentrations of TG. We have identified,
in two independently selected TG-resistant hamster cell lines of
different lineages, mutant forms of SERCA. In the TG-resistant Chinese
hamster lung fibroblast cell line DC-3F/TG, a T
C change at
nucleotide 766 introduces a Phe256
Leu alteration
within the first cytosolic loop of the SERCA. In contrast, in the
TG-resistant Syrian hamster smooth muscle cell line DDT/TG
4µM, a T
C change at nucleotide 767 introduces a
Phe256
Ser mutation at that position. When these
specific mutations are introduced into a wild-type full-length avian
SERCA1 cDNA, transfection experiments reveal that Ca2+
transport function and ATP hydrolytic activity are not altered by such
mutations. However, a 4-5-fold resistance to TG inhibition of
Ca2+ transport function occurs upon the introduction of
either the Phe256
Leu or the Phe256
Ser
mutation into wild-type SERCA1. These specific mutations also render
the hydrolytic activity of the ATPase resistant to inhibition by TG.
Our results not only implicate amino acid 256 in TG-SERCA interactions,
but also demonstrate that specific mutations within SERCA can mediate
resistance to TG.
 |
INTRODUCTION |
The sarcoplasmic/endoplasmic reticulum Ca2+
transport ATPases
(SERCAs)1 are intracellular
Ca2+ pumps that play a central role in Ca2+
homeostasis. Several inhibitors of the SERCAs have been described, of
which thapsigargin (TG) is the most potent and specific (1). By
inhibiting SERCA function, TG depletes intracellular Ca2+
stores, resulting in inhibition of cell proliferation. However, high
levels of resistance to TG inhibition of cell proliferation can be
developed (2, 3). Several mechanisms that contribute to the increased
production of SERCA protein become operative upon the selection of
TG-resistant cells.2 In
addition, resistance to TG can be associated with overexpression of the
multidrug resistance transporter P-glycoprotein (Pgp) (2).2
Although increased expression of SERCA or Pgp may contribute to TG
resistance, our previous studies suggest that a SERCA(s) that is
directly resistant to TG inhibition can also be selected for during
development of the TG-resistant phenotype (3). To determine whether
altered forms of SERCA do in fact occur during TG selection and
potentially contribute to TG resistance, we have begun to study the
SERCAs from the TG-resistant cells.
Although TG affects SERCA function rather globally, the sites of
interaction between TG and SERCA have not been clearly defined. Chimeric recombinations between SERCA1 and
Na+,K+-ATPase demonstrate that TG does not bind
within the catalytic domain (i.e. the large cytosolic loop)
of the ATPase (5). More recently, studies suggest that binding to TG
may occur within the M3 transmembrane domain of SERCA (6). Hence, our
initial efforts have been to analyze the 5
ends of SERCA, which
encompass the M3 domain, from the TG-resistant cell lines. In this
report, we demonstrate that substitutions at amino acid (aa) position 256 occur within the SERCAs obtained from two independently derived TG-resistant cell lines. The mutant ATPases contain either a
Phe256
Leu or a Phe256
Ser change.
Moreover, when either of these two specific mutations are introduced
into wild-type (wt) SERCA1, both Ca2+ transport and ATP
hydrolytic activities of the resulting ATPase become resistant to
inhibition by TG. No mutations occur within the M3 domain
(i.e. the putative TG binding site) of the TG-resistant cell
derived SERCAs. This is the first demonstration of naturally occurring
mutations within SERCA during development of the TG-resistant phenotype, and our results suggest that Phe256, which lies
within the first cytosolic loop just upstream of the M3 domain,
represents a " hot spot" for mutations upon TG selection, and is
potentially involved in TG-SERCA interactions.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
The Chinese hamster lung fibroblast cell line
DC-3F has been described previously (7). The TG-resistant cell line
DC-3F/TG was derived from DC-3F cells by stepwise selection in TG, with the final maintenance concentration being 2 µM TG (2).
Both DC-3F and DC-3F/TG cells were maintained in
-minimum essential media (Sigma) supplemented with 5% fetal calf serum (Gemini
Bio-Products, Inc.) plus 50 µg/ml gentamicin (Life Technologies,
Inc.). The Syrian hamster smooth muscle cell line
DDT1-MF2 (8) was maintained in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
4% enriched calf serum (Gemini Bio-Products, Inc.) plus 50 µg/ml
gentamicin. The DDT/TG 4µM cells were derived from
DDT1-MF2 cells by a series of stepwise
selections with TG, the final maintenance concentration of TG being 4 µM.2 Clonal sublines were derived by limiting
dilution.
Cloning and Sequencing of cDNA Fragments--
Based on the
published rat SERCA2a cDNA sequence (9), 5
and 3
degenerate
primers, designated 2.1 and 2.2, respectively, were designed between
nucleotide (nt) 1-18 and nt 976-992, respectively (Table I). Total
RNA isolated from DC-3F cells was reverse-transcribed (RT) using
oligo(dT)12-18 primer, the double-stranded cDNA amplified by polymerase chain reaction (PCR) using primers 2.1 and 2.2, and the resulting 992-bp PCR product cloned into pBluescript SK+. Nitrocellulose replica filters were screened as per
Grunstein's method (10) using radiolabeled avian SERCA cDNA probe
(11). The sequences of five positive clones were determined using the dideoxy chain termination method. Based on these sequence data, hamster-specific 5
forward primer (designated 2.3, corresponding to nt
104-121) and 3
reverse primer (designated 2.4, corresponding to nt
936-953) (Table I) were used to RT-PCR amplify 850-bp 5
end SERCA2
cDNA fragments from DC-3F/TG, DDT1-MF2, and
DDT/TG 4µM cells. The PCR products were separately
ligated into pBluescript SK+, several independent clones
from each ligation were selected, and their sequence obtained by
dideoxy sequencing using T7 and T3 primers.
Allele-specific Oligonucleotide (ASO)-based PCR--
Two
antisense primers (designated 2.16 and 2.17) that are complementary to
the DC-3F SERCA2 cDNA sequence between nt 766 and 782 were
designed. Primers 2.16 and 2.17 are identical except at nt position 766 (Table I). Primer 2.16 is complementary to wt SERCA2 cDNA encoding
Phe256, while primer 2.17 is complementary to mutant SERCA2
cDNA encoding Leu256. Each antisense primer (2.16 or
2.17) was used in combination with the sense primer 2.3 (Table I) to
amplify SERCA2 cDNA using RT-PCR from DC-3F or DC-3F/TG cells (12).
The PCR parameters consisted of denaturation at 94 °C for 1 min,
annealing at 60 °C for 1 min, extension at 72 °C for 1.5 min for
40 cycles. Two other 17-mer primers (2.27 and 2.28) (Table
I) that are complementary to the
DDT1-MF2 SERCA2 cDNA sequence between nt
767 and 783, were used in combination with primer 2.3, to RT-PCR
amplify SERCA2 from DDT1-MF2 and DDT/TG
4µM cells under the above conditions. Primer 2.27 is
complementary to wt SERCA2 cDNA encoding Phe256, and
primer 2.28 is complementary to mutant SERCA2 cDNA encoding Ser256.
In Vitro Mutagenesis by PCR--
Overlap extension PCR (13) was
used to introduce mutations at amino acid 256 in the avian fast muscle
SERCA1 cDNA (11). With appropriately designed primers, 720-bp PCR
products, containing the desired mutations within the body of the
amplified products as well as appropriate restriction site overhangs,
were obtained using SERCA1 cDNA template. The PCR products were
exchanged with the corresponding fragments from the wt SERCA1 cDNA,
and sequenced to ensure fidelity of the PCR amplification step. The
resulting pCDL-SR
296 plasmids, containing the wt or mutant SERCA
cDNAs, were transfected as described previously (5, 14). As
described by Zhang et al. (15), a c-myc tag at
the 3
terminus allowed assessment of SERCA1 expression after
transfection.
Transfection, Microsome Preparation, and
Immunodetection--
Transient transfection into monkey kidney COS1
cells was carried out using the DEAE-dextran method as detailed
previously (5, 14). For each experiment, 20 plates (150 × 25 mm)
of logarithmically growing COS1 cells were used. Preparation of
microsomes from the transfected cells was as described by Zhang
et al. (15). The monoclonal antibody 9E10 to the
c-myc epitope (16) was used to detect SERCA1 expression in
the transfected COS1 cells via Western blotting.
Functional Assays--
Ca2+ transport activity of
the microsomal fractions was determined as described previously (3).
ATPase activity was measured by the amount of Pi released
upon the addition of ATP to the microsomal fractions (15, 17). Both
Ca2+ transport and ATPase activities were normalized by the
amount of expressed SERCA1 protein in the microsomal fractions obtained by Western blotting.
 |
RESULTS |
Identification of Amino Acid Substitutions at aa 256 of SERCA in
the TG-resistant Cell Lines--
Although cDNAs encoding the
different isoforms of SERCA have been cloned from many species, the
hamster SERCAs have yet to be cloned. We therefore used 5
and 3
degenerate oligonucleotide primers, corresponding to the published rat
SERCA2a cDNA sequence, to clone a ~1-kilobase pair 5
end SERCA2
cDNA from Chinese hamster DC-3F cells. Six different clones,
obtained from two independent RT-PCR reactions, have been sequenced
(data not shown). Based on these sequence data, hamster-specific 5
and
3
oligonucleotide primers were used to clone 850-bp 5
end cDNA
fragments of SERCA2 from the wt DDT1-MF2 cells,
as well as the TG-resistant DC-3F/TG and DDT/TG 4µM
cells. To obtain sequence information of the translation start site
region of hamster SERCA2, two additional primers were used to clone a
440-bp overlapping cDNA fragment encompassing the SERCA translation
start site from the hamster cells. In the sequenced 5
end of the
molecule, the hamster and rat SERCA2 are identical at the protein
level, although several nt changes between the two are noted.
Nucleotide changes in SERCA are also noted to occur among different
strains within the same species (i.e. between DC-3F and
DDT1-MF2 SERCA).
Sequencing several independent clones from DC-3F/TG cells (which were
obtained via separate RT-PCR reactions) reveals that the sequences of
the clones are identical to that of wt DC-3F cells except at nt 766 (Table II). A T
C change at nt 766 occurs in five out of the eight clones, which predicts for a
Phe256
Leu change at the protein level. Since the
clones were obtained from separate RT-PCR reactions, it is unlikely
that this change represents a PCR artifact (see also below). That some
clones have the wt phenotype and others the mutant suggests that the
DC-3F/TG cell lines might represent a mixed population of cells, with
some cells expressing the wt and others the mutant SERCA. The
possibility also exists that the resistant cells represent a
homogeneous population, with the SERCA allele being heterozygous with
respect to nt 766 (i.e. aa 256). This is suggested by the
fact that in clonally derived DC-3F/TG cell lines, both wt
and mutant SERCA are expressed (see below).
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Table II
Results of sequencing the 5 ends of the SERCA2 clones from
wild-type and TG-resistant hamster cells
The 5 end cDNA fragments of SERCA2 were obtained from wild-type
(wt) and resistant cells via RT-PCR and cloned into pBluescript SK+ as described under "Experimental Procedures." The
resulting positive clones were analyzed by dideoxy sequencing using
T7 and T3 primers. The data were confirmed by at least
two independent RT-PCRs and sequencing.
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With respect to the DDT/TG 4µM cell line, 12 independent
SERCA clones, obtained from three separate RT-PCR reactions, have been
sequenced. Five of these clones have a T
C change at nt 767 (instead of at nt 766), predicting for a Phe256
Ser
mutation (Table II). Since the DDT/TG 4µM cell line
represents a clonal population, the presence of both wt and mutant
clones suggests that, similar to DC-3F/TG cells, the SERCA allele is heterozygous in DDT/TG 4µM cells with respect to nt 767. The M3 domain, which is the putative TG binding site, lies between nt 781 and 843 (i.e. from aa 261 to aa 283). It should be
pointed out that in neither of the above TG-resistant cell lines is a mutation observed within this region, i.e. between nt 781 and 843 (data not shown).
ASO-based PCR Analysis--
To further clarify the observed
changes at nt 766 and 767, ASO-based PCR assays were performed. Shown
in Fig. 1A are the wt and
mutant hamster SERCA2 sequence between nt positions 766 and 782. Also
shown in parentheses are the reverse primers 2.16 and 2.17 that were
used in the PCR assay. Note the T
C change at nt 766 between the wt
and mutant sequence, which are otherwise identical. The 2.16 and 2.17 reverse primers are identical except for the single nt change at their
3
ends, which distinguishes primer 2.16 from primer 2.17 (Fig.
1A, Table I). Since DC-3F cells express only wt SERCA, the
primer pair 2.3 and 2.16 (2.3 is the forward primer) (Table I) should
amplify, under appropriate conditions of annealing and extension, a
679-bp product from reverse-transcribed DC-3F RNA (Fig. 1B,
lane 2). On the other hand, the primer pair 2.3 and 2.17 amplifies only mutant SERCA2, and hence this pair does not give rise to
any PCR product under similar conditions when reverse-transcribed
DC-3F RNA is used as template (Fig. 1B, lane 3).
In contrast, DC-3F/TG cells express both wt and mutant SERCA. Hence,
either primer pair (i.e. 2.3 and 2.16 for wt, or 2.3 and
2.17 for mutant) should amplify a 679-bp fragment when RNA from
DC-3F/TG cells is used as template (Fig. 1B, lanes
5 and 6).

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Fig. 1.
Allele-specific primer-based PCR assay of
DC-3F/TG cells. A, the wt and mutant sequence of SERCA2
cDNA between the nt positions 766 and 782 are depicted, along with
the antisense primers 2.16 and 2.17 in parentheses. Note the
T C change at position 766 between the wt and mutant sequence.
B, PCR products obtained from reverse-transcribed total RNA
are run on 2% agarose gels. DC-3F, lanes 1-3: lane
1, primer pair 2.3 and 2.4; lane 2, primer pair 2.3 and
2.16; lane 3, primer pair 2.3 and 2.17. DC-3F/TG,
lanes 4-6: lane 4, primer pair 2.3 and 2.4;
lane 5, primer pair 2.3 and 2.16; lane 6, primer
pair 2.3 and 2.17. Note that the forward primer 2.3 (nt 104-121) plus
the reverse primer 2.4 (nt 936-953) amplify an 850-bp fragment from
either cell line, primer 2.3 plus reverse primer 2.16 (nt 766-782)
amplify a 679-bp fragment from DC-3F cells, while both primer pairs 2.3 plus 2.16 and 2.3 plus 2.17 amplify a 679-bp fragment from DC-3F/TG
cells. C, three independent clonal sublines were derived, by
limiting dilution, from the DC-3F/TG mass population of cells depicted in lanes 4-6 of B. Lanes 1 and
2, subclone 1 (lane 1, primers 2.3 plus 2.16;
lane 2, primers 2.3 plus 2.17); lanes 3 and
4, subclone 2 (lane 3, primers 2.3 plus 2.16;
lane 4, primers 2.3 plus 2.17); lanes 5 and
6, subclone 3 (lane 5, primers 2.3 plus 2.16;
lane 6, primers 2.3 plus 2.17); lanes 7 and
8, parental DC-3F cells (lane 7, primers 2.3 plus
2.16; lane 8, primers 2.3 plus 2.17). In all three
subclones, SERCA is mutated at nt 766 (lanes 2,
4, and 6). The presence of wt SERCA (lanes
1, 3, and 5) in each subclone demonstrates
that the SERCA allele is heterozygous with respect to the
mutation.
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The DC-3F/TG cell line shown in Fig. 1B represents a mass
population. Three independent clonal sublines have been derived from
the DC-3F/TG mass population by limiting dilution. Analysis of the
clonal lines demonstrates that in each cell line both wt and mutant
SERCA are expressed (Fig. 1C, lanes 1 and
2, clone 1; lanes 3 and 4, clone 2;
lanes 5 and 6, clone 3; lanes 7 and 8,
wt DC-3F). Since each clonal population was presumably derived from a
single cell, the above data are consistent with the SERCA allele being
heterozygous with respect to the Phe256
Leu mutation in
the DC-3F/TG cells.
Fig. 2 shows the results of a typical
ASO-based PCR assay performed on DDT/TG 4µM cells. In
Fig. 2A are shown the two antisense primers 2.27 and 2.28, which are identical to each other except at nt 767. Under appropriate
conditions of annealing, primer 2.27 hybridizes to the complementary
DNA strand containing T at nt 767 (which predicts for
Phe256), while primer 2.28 hybridizes to the complementary
strand containing C at nt 767 (which predicts for Ser256).
That the primer pair 2.3 and 2.27 amplifies a 680-bp PCR product from
both DDT1-MF2 cells (Fig. 2B,
lane 8) and DDT/TG 4µM cells (Fig.
2B, lane 5) demonstrates that the wt
Phe256 SERCA exists in both drug-sensitive and
drug-resistant cells. The primer pair 2.3 and 2.28 amplifies a 680-bp
fragment from DDT/TG 4µM cells (Fig. 2B,
lane 6) but not from wt DDT1-MF2
cells (Fig. 2B, lane 9). These data confirm and
extend the initial sequencing data with respect to the heterozygous
Phe256
Ser change in DDT/TG 4µM cells.
The specificity of the above primers is demonstrated by the data in
Fig. 2B, lanes 2 and 3. Lane
2 shows the presence of a 679-bp fragment when reverse-transcribed RNA from DDT/TG 4µM cells is amplified with the primer
pair 2.3 plus 2.16. Primer 2.16 is identical to primer 2.27, except the former extends from nt 766 to 782, while the latter extends from 767 to
783. Importantly, the primer pair 2.3 plus 2.17 (which recognizes
Leu256; Fig. 1) fails to amplify any PCR product from
DDT/TG 4µM cells (Fig. 2B, lane 3),
thus confirming the ability of the above primers in recognizing only
specific nt changes.

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Fig. 2.
Allele-specific primer-based PCR assay of
DDT/TG 4µM cells. A, the wt and mutant
sequence of SERCA2 cDNA between nt position 767 and 783 are
depicted, along with the antisense primers 2.27 and 2.28 in
parentheses. Note the T C change at position 767 between
the wt and mutant sequence. B, PCR products obtained from
reverse-transcribed total RNA are run on 2% agarose gels. DDT/TG
4µM cells, lanes 1-6: lane 1,
primers 2.3 and 2.4; lane 2, primers 2.3 and 2.16;
lane 3, primers 2.3 and 2.17; lane 4, primers 2.3 and 2.4; lane 5, primers 2.3 and 2.27; lane 6,
primers 2.3 and 2.28. DDT1-MF2 cells,
lanes 7-9: lane 7, primers 2.3 and 2.4;
lane 8, primers 2.3 and 2.27; lane 9, primers 2.3 and 2.28. Note that the primer pair 2.3 and 2.17 (which recognizes
Leu256) does not give rise to a PCR product (lane
3).
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Functional Analysis of the Phe256
Leu and
Phe256
Ser Mutations--
To determine whether the
observed mutations do in fact contribute to TG resistance, the
appropriate substitutions within codon 256 have been introduced by
PCR-based in vitro mutagenesis in a wild-type full-length
avian SERCA1-encoding cDNA (11). The resulting SERCA-containing
expression plasmids have been transfected into COS1 cells. Western blot
analysis demonstrates that introduction of the above mutations at aa
256 do not affect the expression of the transfected mutant SERCAs, when
compared with transfected wt SERCA, in COS1 cells (Fig.
3). Furthermore, measurement of Ca2+ transport and ATPase activities of the respective
microsomal fractions demonstrate that neither the Phe256
Leu nor the Phe256
Ser change substantially alters
either the Ca2+ transport function or the ATPase activity
of the mutants when compared with wt SERCA (Table
III).

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Fig. 3.
Immunodetection of SERCA1 expression.
The microsomal fractions derived from transfected COS1 cells were
analyzed by Western blot as described under "Experimental
Procedures." The monoclonal antibody 9E10 was used to detect the
c-myc tag at the C terminus of SERCA1. Lane 1,
wt; lane 2, Leu256; lane 3,
Ser256.
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Upon measuring Ca2+ transport activity as a function of TG
concentration, each aa substitution renders COS1 microsomal fraction 45Ca2+ uptake 4-fold resistant to inhibition by
TG (Fig. 4). Moreover, each mutation
results in a 5-fold increase in resistance to TG in terms of hydrolysis
of ATP by the mutant SERCA (Fig. 5).

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Fig. 4.
Thapsigargin inhibition of
ATP-dependent Ca2+ uptake activity by
microsomal fractions obtained from SERCA1-transfected COS1 cells.
Microsomes were isolated from wt ( ), Leu256 ( ), and
Ser256 ( ) transfected COS1 cells.
ATP-dependent Ca2+ uptake was determined either
in the absence or presence of TG by the accumulation of
45Ca2+ in microsomal vesicles separated from
the reaction mixture by filtration (0.45-µm Millipore filters), as
described previously (3). Ca2+ uptake activity is expressed
relative to the activity obtained in the absence of TG. Each point
represents the average of two independent measurements, and standard
errors (S.E.) are indicated as bars.
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Fig. 5.
Thapsigargin inhibition of
Ca2+-dependent ATPase activity by microsomal
fractions obtained from SERCA1-transfected COS1 cells. ATPase
activities in the microsomal fractions obtained from wt ( ),
Leu256 ( ), and Ser256 ( ) transfected COS1
cells were measured by determination of Pi in the presence
or absence of TG as described by Zhang et al. (14). The
Ca2+-dependent activity was calculated by
subtracting the Ca2+-independent ATPase activity from the
total ATPase activities. ATPase activities are expressed relative to
the activity obtained in the absence of TG. Each point represents the
average of two independent measurements, and standard errors (S.E.) are
indicated as bars.
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It is of note that, although the above mutations lead to essentially
equivalent levels of resistance to TG with respect to both
Ca2+ transport (Fig. 4) and ATP hydrolytic activities (Fig.
5), the absolute concentrations of TG required to inhibit
Ca2+ transport function appear to be somewhat lower than
that required to inhibit the ATPase activity of both wt and mutant
SERCAs (compare Figs. 4 and 5). The apparent discrepancy in the
absolute concentrations of TG between Figs. 4 and 5 is in part due to
the very nature of the two assays, i.e. Ca2+
transport function is determined by the amount of
45Ca2+ retained by microsomal fractions, while
ATPase function is assessed spectrophotometrically. Nevertheless, these
data clearly demonstrate that certain mutations at aa 256 can modify
SERCA's transport and catalytic properties with respect to the
enzyme's response to its specific inhibitor TG.
 |
DISCUSSION |
In this report, we identified mutant forms of SERCA that can
confer resistance to TG inhibition. To our knowledge, this is the first
demonstration of naturally occurring mutations within SERCA that occur
upon long term exposure of mammalian cells to SERCA's highly specific
inhibitor TG. Although wt cells in culture are exquisitely sensitive to
SERCA pump inhibition, high levels of resistance to TG can be developed
by long term selection so that the selected cells continue to
proliferate in TG (2, 3).2 Previously, we reported several
adaptive changes that the TG-selected cells employ to maintain growth
in TG (2, 3).2 Among these is overexpression of the
multi-drug resistance transporter Pgp (2), as well as overproduction of
SERCA protein by a multiplicity of mechanisms including gene
amplification, transcriptional up-regulation, and post-transcriptional
alterations.2
Previous in situ studies, as well as analysis of
Ca2+ transport activity following isolation of microsomal
fractions from TG-resistant cells, suggested qualitative changes that
directly contribute to TG resistance might also occur within these
ATPase (3). Our present studies clearly demonstrate that aa 256 is
susceptible to specific mutations (Table II, Figs. 1 and 2). That cell
lines of two different lineages, selected independently for TG
resistance, give rise to two different mutations at the same location
suggests that this aa is particularly susceptible to mutations. The
data also suggest that aa 256 might be closely linked with TG-SERCA interactions.
Since the M3 domain is the putative TG binding site, our initial
efforts have been to analyze regions of SERCA encompassing this domain.
If any mutations occur within SERCA in the TG-selected cells, they
would be expected to occur within the TG binding pocket. Such mutations
could potentially alter SERCA's affinity for TG, thus rendering the
ATPase resistant to inhibition by TG. For instance, antifolates like
methotrexate (MTX) inhibit dihydrofolate reductase (DHFR) (18).
However, specific mutations in DHFR (e.g. Leu 22
Phe or Leu 22
Arg) within the binding pocket of MTX
can alter MTX's affinity for DHFR, resulting in a MTX-resistant
phenotype (19, 20).
Interestingly, no mutations in the TG-resistant cell lines have been
found within the M3 domain upon natural drug selection (data not
shown). However, Phe256, which is only 4 aa upstream from
where the M3 domain begins (4), is mutable under our conditions with
drug selection. Although isoform-specific changes distinguish the
different isoforms of SERCA within and across species, at the protein
level and with respect to the overall topology, the various SERCAs have
a high degree of homology to each other. The M3 domain is highly
conserved, but not identical, between SERCA1 and SERCA2 (4).
Interestingly, the 14 aa within the first cytosolic loop that are
immediately upstream of the M3 domain (i.e. aa 247-260) are
identical between SERCA1 and SERCA2 across diverse species (4), and are
also similar to but not identical with the corresponding region in SERCA3 (4). Although our initial observations in terms of mutations at
aa 256 are with hamster SERCA2, the fact that introduction of the
observed mutations in a different isoform from a different species
(i.e. avian SERCA1) renders the resulting mutant enzymes resistant to TG (Figs. 4 and 5) suggests that the aa 256 position is
particularly important in TG-SERCA interactions. Interestingly, we have
only observed heterozygous changes at aa 256 in the TG-resistant cells (Figs. 1 and 2). Although no mutations are observed within the actual M3 domain in our resistant cell lines, previous studies have suggested that this region is involved in TG-SERCA
interactions (5, 6).
Since the relative resistance to TG by the documented mutations at aa
256 is only 4-5-fold (Fig. 4), we cannot rule out the possibility that
additional qualitative changes within other regions of SERCA could also
occur, and potentially contribute to the overall TG resistance
phenotype. In summary, our studies show that the SERCAs exhibit a
variety of responses, including point mutations, that enable cells to
survive in the presence of their highly selective and potent inhibitor
TG. Thus, the SERCAs follow the same paradigm exhibited by enzymes like
DHFR that are subjected to selective pressures by their specific
inhibitors such as the antifolates.
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FOOTNOTES |
*
This work was supported in part by a merit review award from
the Medical Research Services of the Department of Veterans Affairs (to
A. H.), a Department of Veterans Affairs career development award
(to A. H.), a grant-in-aid from the American Heart Association, Maryland Division (to A. H.), and National Institutes of Health Grant PO1HL27867.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Greenebaum Cancer
Center, University of Maryland, 655 W. Baltimore St., Bressler Research Building, Rm. 9-047, Baltimore, MD 21201.
1
The abbreviations used are: SERCA,
sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase;
TG, thapsigargin; Pgp, P-glycoprotein; PCR, polymerase chain reaction;
RT-PCR, reverse transcription-polymerase chain reaction; nt,
nucleotide(s); bp, base pair(s); aa, amino acid(s); wt, wild-type; ASO,
allele-specific oligonucleotide; MTX, methotrexate; DHFR, dihydrofolate
reductase.
2
A. K. Rishi, M. Yu, J.-J. Tsai-Wu, C. P. Belani, J. A. Fontana, D. L. Baker, M. Periasamy, and A. Hussain, submitted for publication.
 |
REFERENCES |
-
Sagara, Y.,
and Inesi, G.
(1991)
J. Biol. Chem.
266,
13503-13506[Abstract/Free Full Text]
-
Gutheil, J. C.,
Hart, S. R.,
Belani, C. P.,
Melera, P. W.,
Hussain, A.
(1994)
J. Biol. Chem.
269,
7976-7981[Abstract/Free Full Text]
-
Hussain, A.,
Garnett, C.,
Klein, M. G.,
Tsai-Wu, J. J.,
Schneider, M. F.,
Inesi, G.
(1995)
J. Biol. Chem.
270,
12140-12146[Abstract/Free Full Text]
-
Inesi, G.,
and Kirtley, M. R.
(1992)
J. Bioenerg. Biomembr.
24,
271-283[Medline]
[Order article via Infotrieve]
-
Sumbilla, C.,
Lu, L.,
Lewis, D. E.,
Inesi, G.,
Ishii, T.,
Takeyasu, K.,
Feng, Y.,
Fambrough, D. M.
(1993)
J. Biol. Chem.
268,
21185-21192[Abstract/Free Full Text]
-
Norregaard, A.,
Vilsen, B.,
and Andersen, J. P.
(1994)
J. Biol. Chem.
269,
26598-26601[Abstract/Free Full Text]
-
Biedler, J. L.,
and Riehn, H.
(1970)
Cancer Res.
30,
1174-1184[Medline]
[Order article via Infotrieve]
-
Smith, R. G.,
Syms, A. J.,
and Norris, J. S.
(1984)
J. Steroid Biochem.
20,
227-281
-
Gunteski-Hamblin, A.-M.,
Greeb, J.,
and Shull, G.
(1988)
J. Biol. Chem.
263,
15032-15040[Abstract/Free Full Text]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T. E.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Scpring Harbor, NY
-
Karin, N. J.,
Kaprielian, Z.,
and Fambrough, D. M.
(1989)
Mol. Cell. Biol.
9,
1978-1986[Medline]
[Order article via Infotrieve]
-
Rishi, A.,
Hatzis, D.,
McAlmon, K.,
and Floros, J.
(1992)
Am. J. Physiol.
262,
L566-L573[Abstract/Free Full Text]
-
Ho, S. N.,
Hunt, H. D.,
Horton, R. M.,
Pollen, J. K.,
Pease, L. R.
(1989)
Gene (Amst.)
77,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
-
Hussain, A.,
Lewis, D. E.,
Sumbilla, C.,
Lai, L.,
Melera, P. W.,
Inesi, G.
(1992)
Arch. Biochem. Biophys.
296,
539-546[Medline]
[Order article via Infotrieve]
-
Zhang, Z.,
Sumbilla, C.,
Lewis, D.,
Summers, S.,
Klein, M. G.,
Inesi, G.
(1995)
J. Biol. Chem.
270,
16283-16290[Abstract/Free Full Text]
-
Evan, G. I.,
Lewis, G. K.,
Ramsay, G.,
and Bishop, J. M.
(1985)
Mol. Cell. Biol.
5,
3610-3616[Medline]
[Order article via Infotrieve]
-
Lanzetta, P. A.,
Alvarez, L. J.,
Reinsch, P. S.,
Candia, O. A.
(1979)
Anal. Biochem.
100,
95-97[Medline]
[Order article via Infotrieve]
-
Melera, P. W.
(1991)
in
Seminars in Cancer Biology (Biedler, J. L., ed), pp. 245-255, W. B. Saunders, Philadelphia
-
Yu, M.,
and Melera, P. W.
(1993)
Cancer Res.
53,
6031-6035[Abstract]
-
Simonsen, C. C.,
and Levinson, A. D.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
2495-2499[Abstract]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.