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Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4379-4386
Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C
Protein and Regulates Its Intracellular Expression
By
Taizo Hoshino,
Jianxiang Wang,
Marcel P. Devetten,
Nobuhisa Iwata,
Sachiko Kajigaya,
Robert J. Wise,
Johnson M. Liu, and
Hagop Youssoufian
From the Department of Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX; the Hematology Branch, National Heart, Lung
and Blood Institute, Bethesda, MD; and the Hematology-Oncology
Division, Brigham and Women's Hospital, Boston, MA.
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ABSTRACT |
The FAC protein encoded by the gene defective in Fanconi anemia (FA)
complementation group C binds to at least three ubiquitous cytoplasmic
proteins in vitro. We used here the complete coding sequence of
FAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a
B-lymphocyte cDNA library. Binding was confirmed by
coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear,
lysates of transfected COS-1 cells, as well as from mouse liver
cytoplasmic extracts. Deletion mutants of FAC showed that residues
103-308 were required for interaction with GRP94, and a natural
splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the
rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the
cellular phenotype of FA. Our results demonstrate that GRP94 interacts
with FAC both in vitro and in vivo and regulates its intracellular
level in a cell culture model. In addition, the pathogenicity of the
IVS-4 splicing mutation in the FAC gene may be mediated in part
by its inability to bind to GRP94.
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INTRODUCTION |
THE AUTOSOMAL RECESSIVE disorder Fanconi
anemia (FA) frequently leads to bone marrow failure, leukemia, and
other cancers at an early age.1,2 Hence, it is regarded as
an important genetic model of preleukemia and cancer susceptibility.
Cellular manifestations of FA include chromosomal instability, enhanced sensitivity to bifunctional cross-linking agents (eg, mitomycin C
[MMC]) and to oxygen,3 cell cycle defects characterized
by delays in the G2 phase of the cell cycle (Seyschab et
al4 and the references therein) and a predisposition to
apoptosis.5-8 Although the molecular basis of these
abnormalities is not well understood, we and others have challenged
earlier views9 on the pathogenesis of FA and suggested that
it does not necessarily involve a primary defect in DNA
repair.10-12
At least eight distinct complementation groups of FA have been defined
by somatic cell fusions,13-16 three of which map to different chromosomal loci.13,17,18 The genes for
complementation groups A (FAA)19,20 and C
(FAC)21 have been cloned, and mutations consisting
of deletions, a splicing defect, as well as nonsense and missense
mutations have been identified (for examples, see Whitney et
al,22 Verlander et al,23 Gibson et
al,24 and Lo Ten Foe et al25). However, the
predicted polypeptide sequences of FAA and FAC have not shed light on
their biological functions. The 163-kD FAA protein contains a putative
nuclear localization signal,19,20 but otherwise it has
little homology to known proteins, including FAC. It may play a role in
DNA repair.26 Although fibroblasts from certain FA patients
have elevated rates of homologous recombination, the significance of
this observation for particular complementation groups and for the
proximal effects of FA proteins are unknown.27 The FAC
protein is considerably smaller (63 kD),10,11,21 and
cytoplasmic residence is obligatory for its ability to correct the
hypersensitivity of FA group C (FA-C) cells to bifunctional
alkylators.12
We have shown that at least three ubiquitous cytoplasmic proteins
interact with FAC.28 One of the interacting proteins is the
microsomal enzyme NADPH:cytochrome c (P-450) reductase. The amino-terminal region of FAC binds to the cytosolic, membrane-proximal domain of the reductase and attenuates its catalytic activity (H. Youssoufian et al, manuscript submitted). We postulate
that, in the absence of FAC, excess reactive metabolites of MMC and perhaps other compounds that are subject to bioreductive modification could damage DNA and compromise chromosomal stability.
In this study, we show that FAC binds directly to a member of the
glucose-regulated family of proteins, GRP94.29-31 This
abundant, stress-inducible glycoprotein with homology to the molecular
chaperone HSP90 is localized primarily in the lumen of the endoplasmic
reticulum (ER). There is also evidence for a transmembrane form of
GRP94 that spans the ER membrane and extends into the
cytoplasm.32-34 GRP94 has been shown to bind to both normal
and mutant proteins,32-36 including
p185erbB2 and a mutant form of herpes simplex
virus-1 glycoprotein B. GRP94 may act in tandem with GRP78 on partially
folded intermediates of the nascent Ig chains.35 It has
also been implicated in a variety of other cellular
processes,29 particularly intracellular Ca2+
homeostasis.37 This association with FAC establishes a
novel, albeit indirect, function for GRP94 in the maintenance of
chromosomal stability in mammalian cells.
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MATERIALS AND METHODS |
Cell culture.
NRK cells stably transfected with GRP78-promoter constructs (gift of Dr
Amy Lee, University of Southern California, Los
Angeles)38 driving a GRP94 ribozyme (NRK-Ribo)
and a vector control (NRK-Vect) were maintained in Dulbecco's Modified
Essential Medium (DMEM; GIBCO-BRL, Grand Island, NY) containing 10%
fetal bovine serum (FBS) and G418 (400 µg/mL). COS-1, HSC536 (FA-C
lymphoblastoid cells), and HSC536-FAC (HSC536 cells transfected with
wild-type FAC) were maintained as described
previously.11,12
Plasmid constructions and cDNA libraries.
Mammalian expression vectors encoding wild-type FAC cDNA and a
fusion cDNA of FAC with the constant region of the IgG1 heavy chain (FAC-Ig) have been described previously.28 As a
control for FAC-Ig, seven residues from the amino-terminal region of
FAC were fused in-frame with the IgG1 epitope in the vector pED6 to generate  FAC-Ig. Full-length FAC cDNA was also fused
in-frame downstream of the DNA-binding domain of GAL4 in the vector
pGBT (Clontech, Palo Alto, CA) to generate pGBT-FAC. Carboxy-terminal deletion mutants of FAC and an interstitial deletion that
removes exon 4 (the product of the IVS4+4 mutation, which can generate a 111-bp, in-frame deletion of exon 4)22 were derived by
polymerase chain reaction (PCR) and cloned into either
pGBT or pBD-GAL4Cam (Strategene, La Jolla, CA). Inserts in both
expression vectors were under the control of the ADH1 promoter. cDNA
libraries from human B-lymphocyte, fetal liver, bone marrow, and HeLa
cells cloned as fusion genes with the GAL4 transcriptional activation
domain in the vector pACT were obtained from a commercial source
(Clontech). A 2.4-kb EcoRI fragment obtained from pACT-GRP94
that contains the entire coding region of human GRP94 was cloned into
pcDNA3 (Invitrogen, San Diego, CA) for expression into COS-1 cells.
Human factor VIII cDNA in the vector pMT2 was used in cotransfection studies.
Yeast two-hybrid screen.
The yeast strain HF7c was transformed initially with pGBT-FAC by the
lithium acetate procedure (MATCHMAKER Two Hybrid System; Clontech) and
grown on synthetic minimal medium (SD) in the absence of Trp. As
expected, colonies appearing on the plate lacked transcriptional activity. Next, pooled colonies were transformed with cDNA libraries (200 µg) and selected on SD plates in the presence of 50 fmol/L 3-aminotriazole, but lacking Leu, Trp, and His. Colonies that grew in
the absence of these amino acids were tested for their ability to
activate the lacZ reporter gene. Plasmids recovered from such
LacZ+ colonies were used to transform Escherichia
coli, which were selected on M9 plates in the absence of Leu to
obtain activation-domain plasmids.
Analysis of yeast two-hybrid inserts.
The inserts obtained from the yeast two-hybrid screen were
characterized by DNA sequencing using Sequenase Version 2.0 and analyzed by the BLAST program of the National Center for Biotechnology Information database.
 -Galactosidase activity.
Yeast transformants were assayed for -galactosidase expression using
a filter assay.39 Briefly, yeast cells were transferred onto Whatman filters (Whatman, Maidstone, UK),
permeabilized in liquid nitrogen, and placed on Whatman No. 1 filter
papers that had been soaked in Z buffer (60 mmol/L NaHPO4,
40 mmol/L NaH2PO4, 10 mmol/L MgCl2,
50 mmol/L -mercaptoethanol) containing 1 µg/mL 5-bromo-4-chloro-3-indolyl--D-galactoside at 30°C. Color
developed between 5 minutes and 10 hours and was scored by visual
inspection.
Cell transfections.
COS-1 cells at 40% to 60% confluence in 100-mm dishes were
transfected with plasmid DNA at a concentration of 1 µg/mL by the diethylaminoethyl ether (DEAE)-dextran procedure as
before.28 Cells were then cultured in DMEM-10% FBS for 16 hours, detached with trypsin, and replated in fresh medium. NRK-Vect
and NRK-Ribo cells were transiently transfected with Lipofectamine
(GIBCO-BRL) for 5 hours with 20 µg of total plasmid DNA according to
the suggestions of the manufacturer. After 16 hours, 2 × 106 cells detached with trypsin were replated on 60-mm
dishes for ribozyme induction.
Immunoprecipitation and immunoblotting.
After 48 hours (for transfected COS-1 cells) or 72 hours (for
transiently transfected NRK cells), the monolayer was washed with cold
phosphate-buffered saline and detached by scraping, and
cytosolic extracts were prepared by incubation of the cell pellets in
hypotonic buffer (20 mmol/L HEPES, pH 7.9, 10 mmol/L NaCl, 10%
glycerol, 0.5 mmol/L EDTA, protease inhibitors). Nuclei were pelleted
by centrifugation at 600g for 5 minutes, and the supernatant
was centrifuged at 100,000g for 30 minutes to obtain a
cytosolic extract. Soluble liver extracts from C57BL/6 mice were
prepared by homogenization in ice-cold buffer (20 mmol/L HEPES, pH 7.9, 150 mmol/L NaCl) containing protease inhibitors. Nuclei and cellular
debris were pelleted by centrifugation at 600g for 10 minutes.
The final concentration of all of the extracts was adjusted to 20 mmol/L HEPES (pH 7.9), 50 mmol/L NaCl, 5% glycerol, 0.25 mmol/L EDTA,
and 0.1% Nonidet P-40. In some experiments, the concentration of NaCl
or EDTA was increased to 150 mmol/L and 2 mmol/L, respectively.
Extracts of transfected cells were incubated with the anti-FAC antibody
typically for 2 hours and with protein A-agarose beads for 1 hour at
4°C. Liver extracts were incubated with either anti-FAC antibody or
with a rat monoclonal antibody specific for GRP94 (StressGen
Biotechnologies Corp, Victoria, British Columbia, Canada), and
immunocomplexes were precipitated with a mixture of immobilized protein
A and protein G. Beads were washed three times with binding buffer over
30 minutes and boiled in 1× Laemmli buffer containing 200 mmol/L
dithiothreitol, and the proteins were resolved by denaturing sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to polyvinylidene difluoride
(PVDF) membranes (NEN, Boston, MA) by electroblotting. After blocking in TBST buffer (50 mmol/L Tris, pH 8.3, 150 mmol/L NaCl,
0.05% Tween-20) containing 5% nonfat milk, filters were incubated
sequentially with the primary antibodies (1 µg/mL) for 2 hours and
appropriate secondary antibodies conjugated to horseradish peroxidase
(HRP; GIBCO-BRL) for 45 minutes, which were then washed and visualized
by chemiluminescence (ECL; Dupont, Wilmington, DE).
Determination of factor VIII level.
COS-1 cells transfected with pMT2-factor VIII were incubated for 36 hours in DMEM-10% FBS containing aprotinin. The conditioned medium was
clarified of cellular debris by centrifugation at 600g for 5 minutes, and factor VIII antigen level was determined by enzyme-linked
immunosorbant assay using the monoclonal antibodies ESH2 (American
Diagnostica, Greenwich, CN) and factor VIII (Genetics Institute,
Cambridge, MA) for capture and detection, respectively. Antigen levels
were confirmed by the COATEST chromogenic assay (Chromogenix/DiaPharma,
Franklin, OH) using purified recombinant factor VIII as standard.
Determination of cell survival.
For measurements of cell survival, 2 × 105
exponentially growing cells were exposed to MMC (0 to 250 ng/mL; Sigma
Chemical Co, St Louis, MO) for 5 days, and viable cells were counted by trypan blue exclusion.
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RESULTS |
Identification of GRP94-FAC interaction by yeast two-hybrid assay.
To identify proteins that interact with FAC, we used the yeast
two-hybrid system,39 a genetic strategy that can identify in vivo protein-protein interactions by exploiting the ability of
fusion proteins to reconstitute a transcriptional activator. Plasmids
encoding FAC cDNA fused with the DNA-binding domain of GAL4
(pGBT-FAC) and libraries of cDNAs (human B-lymphocyte, bone marrow,
fetal liver, and HeLa cells) fused to the transcriptional activation
domain of GAL4 (in pACT) were used to cotransform HF7c yeast cells.
Double transformant colonies were screened and selected under His,
Trp-, and Leu-nutrient conditions. Viable colonies were
then assayed for LacZ expression. Using full-length FAC as bait, we identified 15 positive clones: 2 clones from the B-lymphocyte library, 3 from bone marrow, 4 from fetal liver, and 6 from HeLa cells.
Both clones from the B-lymphocyte library conferred FAC-dependent expression of LacZ in yeast cells
(Fig 1A). Sequence analysis of these clones
showed the full-length coding region of human GRP94 (a 782-amino acid
polypeptide with a 21-amino acid signal peptide), in-frame with the
GAL4 DNA-activation domain.
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| Fig 1.
Interaction of GRP94 with FAC by yeast two-hybrid
analysis. (A) Structure of full-length FAC and carboxy-terminal
truncated mutants (amino acid residues are residues indicated) and (B)
an in-frame protein product of the IVS4+4 mutation that deletes exon 4 sequences were fused in-frame, downstream of the DNA-binding domain
of GAL4 in the vector pGBT or pBDGAL4Cam. Transcriptional activation of
LacZ in yeast transformed with these constructs as well as with
pACT-GRP94 that contains the entire coding region of human GRP94 fused
to the GAL4 transcriptional activation domain was assessed by a filter
color assay. The rapidity and intensity of color development was scored
by visual inspection as shown:  , white color after 12 hours; +,
blue color after 1 to 12 hours; ++, dark blue color or blue color
after less than 1 hour.
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GRP94-binding domain of FAC.
To further delineate the amino acid sequences that are important for
the interaction of FAC with GRP94, we constructed carboxy-terminal truncation mutants of FAC and subcloned into pGBT9 to generate fusion
proteins with the DNA-binding domain of GAL4 (Fig 1A). Yeast cells were
cotransformed with FAC constructs in pGBT9 and full-length GRP94 in
pACT. Fusion polypeptides containing residues 1-467 or 1-308 of FAC
bound to GRP94, as indicated by the transcriptional activation of
LacZ. By contrast, a fusion protein containing FAC residues
1-103 did not bind GRP94. Thus, residues 103-308 of FAC interact with
GRP94.
A common natural mutation in FAC affects the splice donor
sequence of intron 4 (called IVS4+4 allele)22 and gives
rise to two different truncated products. A severe truncation of
carboxy-terminal sequences results by the partial removal of exon 4, use of a downstream cryptic splice donor site, and frameshift. The
second, and potentially more interesting, mutation gives rise to
complete in-frame deletion of exon 4 involving codons 111 to 148. Because the sequences removed by this mutation are included within the
GRP94 binding domain, we tested the possibility that the in-frame
protein generated by the IVS4+4 allele is defective in its interaction
with GRP94. Indeed, when this fragment was fused to the DNA-binding
domain of GAL4 and introduced into yeast cells with pACT-GRP94, there was no evidence of LacZ transactivation (Fig 1B). By contrast, wild-type FAC in pGBT9 cotransformed with pACT-GRP94 resulted in strong
transactivation. These results suggest that the pathogenesis of FA in
the setting of the IVS4+4 mutation may result, at least in part,
through a deficient interaction of the mutant FAC protein with GRP94.
FAC and GRP94 can be coimmunoprecipitated.
The endogenous expression of FAC in most mammalian cells is very
low.11 To look for an interaction between FAC and GRP94 in
mammalian cells and characterize binding parameters, we used two
complementary strategies. First, we overexpressed GRP94 in COS-1 cells
singly or by cotransfection with either wild-type human FAC or a
functional isoform fused to the heavy chain of human IgG1 (FAC-Ig).
After 48 hours, we performed sequential immunoprecipitation and
immunoblotting experiments. Because FAC is localized primarily in the
cytoplasm,10,11 we prepared cytoplasmic lysates from COS-1
cells transfected with pED6-FAC and immunoprecipitated with anti-FAC
antibodies and protein A-agarose. Immunocomplexes resolved by SDS-PAGE
were then immunoblotted and probed with a rat monoclonal antidody
specific for GRP94. Lysates containing FAC-Ig were immunoprecipitated directly with protein A-agarose. In both cases, the expected 94-kD GRP94 protein coimmunoprecipitated with FAC and FAC-Ig
(Fig 2A). Immunoblotting of the total
lysates showed abundant expression of the human GRP94 in cells
transfected with pcDNA3-GRP94. The anti-GRP94 antibody also
cross-reacted with a 90-kD species in both transfected and
nontransfected COS-1 cells, which probably represents the endogenous
monkey GRP94 (Fig 2B). FAC-GRP94 complexes were not detected in cells
transfected with the empty vector, with GRP94 alone, or with FAC-Ig.
Thus, it is possible that the human FAC fails to interact with the
monkey homologue. Stable complexes were formed in 50 mmol/L NaCl and in
the absence of EDTA, but these complexes were disrupted when the
concentration of NaCl and EDTA were raised to 150 mmol/L and 2 mmol/L,
respectively (data not shown). Similar immunoprecipitations from
mock-transfected or FAC-Ig-transfected cells showed no evidence of
interactions with GRP94. Moreover, under similar conditions, the 78-kD
heat shock protein (BiP; also called Ig heavy chain binding protein) did not coimmunoprecipitate with FAC (data not shown). These data demonstrate that functionally competent forms of FAC associate with
GRP94 in vivo. The sensitivity of FAC-GRP94 interaction to NaCl and
EDTA concentrations suggests several possibilities, including a
low-affinity interaction in vivo and stabilization of the complex by
divalent cations. Second, we attempted to obtain evidence for FAC-GRP94
interaction in normal tissues by sequential immunoprecipitation of
protein complexes and immunoblotting. As described previously, our
anti-FAC antibody recognizes the murine fac protein,40
which is expressed in higher amounts in the liver than in most other adult tissues (H. Youssoufian, unpublished observations).
Both anti-FAC and anti-GRP94 antibodies were capable of
immunoprecipitating GRP94 from cytoplasmic extracts
(Fig 3). The anti-GRP94 antibody precipitated a greater amount of GRP94 than the anti-FAC antibody. These observations demonstrate that fac-GRP94 complexes are normally present in the cytoplasm of mammalian cells.
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| Fig 2.
FAC binds to GRP94 in vivo in transfected cells. (A)
COS-1 cells transfected with the indicated constructs (vector, pED6; remainder as described in the Materials and Methods) were processed after 48 hours, and cytoplasmic extracts were prepared in a final concentration of 50 mmol/L NaCl. Extracts were immunoprecipitated with
either anti-FAC antiserum and protein A-agarose sequentially (+) or
with protein A-agarose () only, boiled, resolved by SDS-PAGE, and
probed for GRP94 expression by immunoblotting. (B) Analysis of
transfected COS-1 cell lysates directly by immunoblotting and probing
with anti-GRP94 antibody. The endogenous COS-1 GRP94 appears slightly
smaller than the transfected human GRP94 cDNA (shown as bent arrows).
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| Fig 3.
Interaction of murine fac with GRP94 in vivo. Liver
cytoplasmic extracts from C57B/6 mice were subjected to
immunoprecipitation with either polyclonal anti-FAC antibody or
monoclonal anti-GRP monoclonal antibody. The anti-FAC antibody can bind
efficiently protein A, whereas the rat anti-GRP94 antibody binds well
to protein G, but not to protein A. Thus, to increase the recovery of
immune complexes and minimize quantitative differences that may result from such interactions, immune complexes were precipitated with a
mixture of protein A-agarose and protein G-agarose. After
electrotransfer to PVDF membranes, the expression of GRP94 was detected
using anti-GRP94 antibody (1 µg/mL), HRP-conjugated goat antirat IgG, and EC. Lane 1, 50 µg extract analyzed directly without prior immunoprecipitation; lane 2, 250 µg extract incubated with protein A-agarose and protein G-agarose without primary antibodies; lane 3, 250 µg extract immunoprecipitated with anti-FAC antibody (4 µg in 250 µL extract) and subsequently with protein A-agarose and protein
G-agarose; 250 µg extract immunoprecipitated with anti-GRP94 (4 µg
in 250 µL extract) antibody and subsequently with protein A-agarose
and protein G-agarose. The positions of murine GRP94 and rabbit IgG
heavy chain are shown (arrow).
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Modulation of intracellular levels of FAC with GRP94.
Does the interaction of FAC with GRP94 have any functional consequence?
To assess this possibility, we took advantage of a cell line in which
the expression of GRP94 can be conditionally inhibited by a targeted
ribozyme that can cleave the GRP94 mRNA.38 The ribozyme is
driven by the stress-inducible GRP78 promoter. Under steady-state
conditions, the activity of the promoter is minimal; consequently, the
expression of the ribozyme is also limited. However, under stress
conditions, the activation of the promoter can induce high levels of
the ribozyme, which degrades the GRP94 mRNA. Rat NRK cells stably
expressing GRP78 promoter constructs were transiently transfected with
pED6-FAC. After 48 hours, cells were either untreated (steady-state) or
treated with 100 nmol/L MMC for 24 hours (stress), and total cell
lysates were assayed for GRP94 and FAC expression simultaneously.
Lysates of NRK-Vect cells probed simultaneously for GRP94 and FAC
(Fig 4A) and subsequently for -tubulin
as a loading control (Fig 4B) showed no differences in the levels of
FAC and GRP94 in the presence or absence of MMC. Although the lack of
an effect by MMC on FAC expression is consistent with similar previous
observations,11 the absence of induction in GRP94 levels
seemed incongruent. A likely explanation may be an incomplete induction
of the endogenous GRP94 promoter by MMC. This possibility is consistent
with previous observations that the GRP94 promoter is generally weaker
than the related GRP78 promoter.38 By contrast, MMC
treatment of NRK-Ribo cells that presumably induces the targeted
ribozyme lead to profound reductions in both the level of GRP94 and
of FAC. Indeed, after MMC treatment, the transfected FAC
was no longer detectable by immunoblotting. By contrast, there were no
significant changes in the expression of -tubulin, indicating that
the reduced levels of FAC and GRP94 do not result from a nonspecific
cytotoxic effect of MMC on these cells. These results demonstrate that
the intracellular level of FAC is closely coupled to the level of GRP94.
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| Fig 4.
Ribozyme-mediated inactivation of GRP94 causes severe
reduction in the level of FAC. (A) NRK-Vect and NRK-Ribo cells
transfected with pED6 (mock) or pED6-FAC (FAC) were either untreated or
treated with 100 nmol/L MMC for 24 hours. Cytosolic lysates were then assayed for expression of FAC and GRP94 simultaneously by
immunoblotting. (B) The same immunoblot as in Fig 3A was
reprobed with an antibody against -tubulin.
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GRP94 inactivation in vivo.
We also performed experiments to discount the possibility that reduced
level of GRP94 in ribozyme-activated cells results from a spurious
effect on vector-encoded cDNA. First, Northern analysis of cytoplasmic
RNA from NRK-Ribo cells transfected with pED6-FAC showed no differences
in the steady-state levels of FAC mRNA in MMC-treated or untreated
cells (data not shown). Thus, a transcriptional effect on FAC
expression would appear to be unlikely. Second, we identified another
molecular target of GRP94 that is regulated by protein-protein
interactions and analyzed its expression concomittantly with FAC. We
reasoned that such an independent assay for the intracellular function
of GRP94 could further minimize the possibility of an artifact caused
by MMC or the ribozyme on the observed variations in the levels of FAC. Previous studies have shown that secretion of the human coagulation protein factor VIII from transfected Chinese Hamster Kidney cells is
enhanced by antisense inhibition of GRP78.41 Because GRP78 and GRP94 can work in tandem,35 we tested the effect of
GRP94 levels on factor VIII secretion. In pilot studies, we
cotransfected pcDNA3-GRP94 and pMT2-factor VIII in COS-1 cells and
noted a significant reduction in the secretion of factor VIII with
GRP94 coexpression (data not shown). Next, we cotransfected NRK-Vect
and NRK-Ribo cells with pED6-FAC and pMT2-factor VIII. It should be
noted that pED6 and pMT2 are closely related vectors, and any potential
effect of the ribozyme on the vector is most likely comparable. After 24 hours, cells were exposed to 100 nmol/L MMC continuously for 36 hours and the conditioned media was assayed for factor VIII activity.
There was a significant increase in factor VIII activity in the media
of NRK-Ribo cells treated with MMC, but not in NRK-Vect cells
(Fig 5A). Third, an alternative possibility
is that FAC regulates the intracellular level of GRP94. In this case,
the level of GRP94 should be different in parental FA-C cells compared with complemented cells. Immunoblot analysis of total cellular lysates
from HSC536 and HSC536-FAC cells showed no significant variations in
the level of GRP94 (Fig 5B). There was also little variation in GRP94
levels in cells of other FA groups and in non-FA cells (data not
shown). Thus, the alternative possibility that FAC regulates the
expression of GRP94 is not supported by these data.
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| Fig 5.
GRP94 expression in vivo. (A) Enhanced factor VIII
secretion by inactivation of GRP94. NRK-Vect and NRK-Ribo cells
transfected with pMT2-factor VIII were analyzed for their ability to
secrete factor VIII in the presence or absence of MMC. (B) Invariant
GRP94 levels in parental and complemented FA-C cells. Lysates of
parental HSC536 and HSC536-FAC cells were analyzed by immunoblotting
with anti-GRP94 antibody. Each lane contained 10 µg protein as
determined by the Bradford assay.
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GRP94 inactivation causes hypersensitivity to MMC.
The hallmark of FA cells is their hypersensitivity to bifunctional
cross-linking agents. By conditional inactivation of FAC, we reasoned
that NRK-Ribo cells should display an enhanced sensitivity to MMC.
NRK-Vect and NRK-Ribo cells were treated with MMC and viable cells were
counted after 5 days. The results of two separate experiments are shown
(Fig 6). NRK-Ribo cells were significantly more sensitive to the cytotoxic effects of MMC than NRK-Vect cells. The
difference in the absolute values between the two experiments may
reflect unequal levels of ribozyme activation by MMC. These results
demonstrate that inactivation of GRP94 leads to a
cross-linker-sensitive phenotype similar to that of FA cells.
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| Fig 6.
GRP94 inactivation induces sensitivity to MMC. NRK-Vect
or NRK-Ribo cells treated with the indicated concentrations of MMC were
analyzed after 5 days for the presence of viable cells by trypan blue
staining. Two independent experiments are shown. Values represent the
mean of triplicates.
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DISCUSSION |
In this study, we demonstrate that FAC-GRP94 immunocomplexes can be
detected in mammalian cells, and residues 103-308 of FAC are essential
for this interaction. At first glance, the abundance of heat-shock
factors and their frequent isolation from yeast two-hybrid screens,
possibly through interactions with misfolded domains, might raise
concern that the interaction of FAC with GRP94 is of dubious biological
significance. However, both structural mapping studies and functional
evidence show that, in this instance, this possibility is unlikely.
First, we demonstrate that GRP94 can form protein complexes in normal
mouse liver cells. Relatively large amounts of extract were required
for this experiment, because the expression of both human FAC and
murine fac is low in most mammalian cells. Our ability to demonstrate
such an interaction in normal cells gives credence to the data from
heterologous systems. Second, the identification of a specific binding
domain on FAC makes it less likely that the observed interaction
results are artifactual. FAC-GRP94 complexes are stable at relatively
low salt concentrations, but dissociate in both higher salt
concentrations and in the presence of EDTA. These observations suggest
that the interaction of FAC with GRP94 may be transient or of
low-affinity and subject to regulation by divalent cations. Third, our
preliminary experiments fail to show an interaction between GRP94 and
FAA (H. Youssoufian, unpublished observation, November
1997), which also contains a large number of leucine
residues and other hydrophobic segments. Fourth, and most important,
the use of a powerful genetic system in mammalian cells demonstrates
that the FAC-GRP94 biochemical interactions are functionally relevant.
Ribozyme-mediated inactivation of GRP94 reduces the level of GRP94
protein, which in turn greatly reduces the level of immunoreactive FAC.
Concomittant with these reductions is an acquired sensitivity of the
host cells to MMC. Taken together, our data support the conclusion that
GRP94 most likely interacts with FAC in vivo and regulates its
intracellular level. A plausible interpretation might be that GRP94
prevents the premature degradation of FAC.
A wealth of biochemical data has defined a broad spectrum of biological
activities for GRP94.29-38 Consistent with its role in
antigenic recognition,29 GRP94 acts as a peptide acceptor and participates in the loading of MHC class I molecules. These peptides are usually bound tightly and can only be eluted by relatively harsh conditions, such as acid extraction.34 As a molecular chaperone, GRP94 also binds to a number of mutant and wild-type proteins,29-32,35,36 but the available evidence indicates
that such interactions are of considerably lower affinity. This also appears to be the case for the interaction between GRP94 and FAC. However, the abundance of GRP94 and the rapid turnover of FAC with a
half-life of approximately 45 minutes42 could make even low-affinity interactions physiologically tenable. Thus, it is plausible that any disruption of FAC-GRP94 interaction could lead to a
rapid degradation of FAC. This interpretation is consistent with our
inability to detect immunoreactive FAC after even partial inactivation
of GRP94. It is also similar to the interaction between p185erbB2 and GRP94,32 whereby
disruption of the complex by ansamycins leads to a dramatic reduction
in the half-life of p185erbB2 from 9 hours to 2 hours and hastens its degradation. The latter role of GRP94 is similar
to its postulated effect on FAC.
It is noteworthy that the 205-amino-acid central domain of FAC that
binds to GRP94 contains seven heptameric sequences that represent a
characteristic alternating array of hydrophobic residues and conform to
the general motif [Hy(W/X)HyXHyXHy]. This motif is thought to mediate
interactions between peptides and BiP.43 Given that BiP and
GRP94 act in tandem on folding peptides, it is possible that both
chaperones use similar motifs for protein interactions. One of these
heptamers (GLGYAPI) is located within the relatively small
(37 amino acids in length) exon 4 of FAC, suggesting that this site may
be particularly important for FAC-GRP94 interaction.
The subcellular localization of GRP94 is somewhat controversial.
Although a large fraction of GRP94 is thought to reside in the lumen of
the ER, there is also evidence that a fraction of this protein spans
the ER membrane.31 In the latter model, the domain
organization of GRP94 predicts a relatively short, amino-terminal intraluminal domain that contains two potential glycosylation sites, a
single transmembrane domain, and a carboxy-terminal cytoplasmic domain.
Given the known cytoplasmic location of FAC, we suggest that its
association is most likely with the membrane-bound isoform of GRP94. By
contrast, the intraluminal form of GRP94 is unlikely to interact with
FAC. The absence of a signal peptide in FAC and the inability of
N-glycanase to affect the mobility of FAC immunoprecipitates (H. Youssoufian, unpublished observation, November 1994)
make it highly unlikely that FAC passes through the ER. Thus, only a
fraction of GRP94 molecules would be expected to associate with FAC.
This is perhaps consistent with our immunoprecipitation results (Fig 3)
in which anti-GRP94 antibody precipitates a considerably larger
fraction of GRP94 than anti-FAC antibody. However, other explanations
(eg, a difference in the avidity of the two antibodies) may also apply.
Given the topological organization of GRP94, an additional prediction
of our data is that FAC associates with the carboxy-terminal domain of
GRP94. We plan to test this prediction and identify specific residues
on GRP94 that are critical for FAC-GRP94 interaction.
Does GRP94 simply serve as a scaffold for FAC? Although the answer is
not clear, a number of observations about the stress response of
mammalian cells as well as their vulnerability to DNA damage and
apoptosis open the possibility of a broader array of effects. GRP94 is
induced by interferon,44 which is increasingly implicated
in the pathogenesis of FA. For example, a recent study has demonstrated
that hematopoietic progenitor cells from fac-knockout mice have
an enhanced sensitivity to interferon- .8,45 In addition,
failure to signal a stress response by GRP94 may predispose lymphoid
cells to apoptosis.46 Whether this response is related to
the lower apoptotic threshhold of FA cells5-8 remains to be determined. It should be possible to dissect these molecular pathways by manipulating GRP94 levels in cells using molecular techniques (Little and Lee38 and this study) or drugs.32
At present, we are unable to explain completely several of our own
observations. For example, why should human FAC fail to interact with
the monkey homologue of GRP94? The latter gene has not been cloned and
its evolutionary relatedness to the human gene is unknown. Such
sequence conservation may be less than expected, because the human and
monkey proteins differ in size by approximately 4 kD (Fig 2). Also,
although our data demonstrate that the effect of MMC on the NRK-Ribo
cells is most likely mediated by induction of the ribozyme promoter, we
cannot exclude other mechanistic possibilities. In several models of
detoxification, both heat shock (a classic inducer of GRP94) and MMC
appear to regulate the expression of target genes, such as
DT-diaphorase, by modulating the activity of NF- B.47
Hence, there may be secondary effects of NF-B activation that could
play a role in this process. MMC can also induce heat-shock proteins in
nonmammalian systems, but its effect is not necessarily equivalent for
all such proteins.48 Thus, it is not surprising that MMC
induces the GRP78 promoter in the NRK-Ribo cells, but not the promoter
of GRP94. Nevertheless, additional experiments will be required to
characterize these phenomena.
In conclusion, our studies demonstrate that a central domain of FAC
binds to GRP94, which adds to our knowledge of the domain structure of
FAC. The carboxy-terminus of FAC is thought to bind to
cdc2,49 and the amino-terminus binds NADPH cytochrome P-450 reductase (Youssoufian et al, manuscript submitted). This
modular organization of FAC should facilitate future analysis of its
physical structure. Finally, the interaction between FAC and GRP94
suggests a novel approach to cancer chemotherapy. Disruption of this
complex in vivo may sensitize cancer cells to MMC and related agents.
| |
FOOTNOTES |
Submitted September 5, 1997;
accepted January 27, 1998.
Supported in part by grants from the National Institutes of Health
(HL52138 to H.Y. and PO1 HL42443 to R.J.W.) and the Aplastic Anemia
Foundation of America (to H.Y.).
Address reprint requests to Hagop Youssoufian, MD, Department of
Molecular and Human Genetics, Baylor College of Medicine, One Baylor
Plaza, Houston, TX 77030.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs Amy Lee and Randall J. Kaufman for their gifts of
cell lines and reagents, Sally Tetrault for technical assistance, and
Dr Neal Young for stimulating discussions.
| |
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Q. Pang, T. A. Christianson, W. Keeble, J. Diaz, G. R. Faulkner, C. Reifsteck, S. Olson, and G. C. Bagby
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I. Garcia-Higuera, Y. Kuang, J. Denham, and A. D. D'Andrea
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R. K. Reddy, J. Lu, and A. S. Lee
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F. A.E. Kruyt, T. Hoshino, J. M. Liu, P. Joseph, A. K. Jaiswal, and H. Youssoufian
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F. A.E. Kruyt and H. Youssoufian
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Abstract
Introduction
Abstract