THE JOURNAL OF BIOLOGICAL CHEMISTRY
3113131138, 2004
2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Appropriate Function of 11-Hydroxysteroid Dehydrogenase
Type 1 in the Endoplasmic Reticulum Lumen Is Dependent on
Its N-terminal Region Sharing Similar Topological
Determinants with 50-kDa Esterase*
Received for publication, December 15, 2003, and in revised form, April 28, 2004
Published, JBC Papers in Press, May 19, 2004, DOI 10.1074/jbc.M313666200
Christoph Frick, Atanas G. Atanasov, Peter Arnold, Juris Ozols, and Alex Odermatt
From the Division of Nephrology and Hypertension, Department of Clinical Research, University of Berne,
3010 Berne, Switzerland and the Biochemistry Department, University of Connecticut Health Center,
Farmington, Connecticut 06030
By interconverting glucocorticoids, 11 -hydroxy-
steroid dehydrogenase type 1 (11-HSD1) exerts an im-
portant pre-receptor function and is currently consid-
ered a promising therapeutic target. In addition, 11-
HSD1 plays a potential role in 7-ketocholesterol
metabolism. Here we investigated the role of the N-
terminal region on enzymatic activity and addressed the
relevance of 11-HSD1 orientation into the endoplasmic
reticulum (ER) lumen. Previous studies revealed that
the luminal orientation of 11-HSD1 and 50-kDa ester-
ase/arylacetamide deacetylase (E3) is determined by
their highly similar N-terminal transmembrane do-
mains. Substitution of Lys5 by Ser in 11-HSD1, but not
of the analogous Lys4 by Ile in E3, led to an inverted
topology in the ER membrane, indicating the existence
of a second topological determinant. Here we identified
Glu25/Glu26 in 11-HSD1 and Asp25 in E3 as the second
determinant for luminal orientation. Our results sug-
gest that the exact location of specific residues rather
than net charge distribution on either side of the helix is
critical for membrane topology. Analysis of charged res-
idues in the N-terminal domain revealed an essential
role of Lys35/Lys36 and Glu25/Glu26 on enzymatic activity,
suggesting that these residues are responsible for the
observed stabilizing effect of the N-terminal membrane
anchor on the catalytic domain of 11-HSD1. Moreover,
activity measurements in intact cells expressing wild-
type 11-HSD1, facing the ER lumen, or mutant K5S/
K6S, facing the cytoplasm, revealed that the luminal
orientation is essential for efficient oxidation of cortisol.
Furthermore, we demonstrate that 11-HSD1, but not
mutant K5S/K6S with cytoplasmic orientation, catalyzes
the oxoreduction of 7-ketocholesterol. 11-HSD1 and E3
constructs with cytosolic orientation of their catalytic
moiety should prove useful in future studies addressing
the physiological function of these proteins.
* This work was supported by grants from the Cloetta Research
Foundation and Swiss National Science Foundation Grant 3100AO-
100060. The costs of publication of this article were defrayed in part by
the payment of page charges. This 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: Division of Nephrol-
ogy and Hypertension, Dept. of Clinical Research, University of Berne,
Freiburgstrasse 15, 3010 Berne, Switzerland. Tel.: 41-31-632-9438;
Fax: 41-31-632-9444; E-mail: alex.odermatt@dkf1.unibe.ch.
This paper is available on line at http://www.jbc.org
Vol. 279, No. 30, Issue of July 23, pp.
Printed in U.S.A.
In humans, 11-HSD11 catalyzes the reduction of biologi-
cally inactive cortisone to active cortisol, thereby playing an
essential role in the local activation of the glucocorticoid recep-
tor. Recent animal experiments provided insight into the
pathophysiological role of 11-HSD1. Mice deficient of 11-
HSD1 were resistant to hyperglycemia induced by obesity or
stress (1), whereas transgenic mice overexpressing 11-HSD1
developed visceral obesity with insulin resistance and dyslipi-
demia. In addition, overexpression of 11-HSD1 in adipose
tissue caused salt-sensitive hypertension mediated by an acti-
vated renin-angiotensin system (2, 3). Experiments in obese
and diabetic mice treated with a specific 11-HSD1 inhibitor
showed reduced blood glucose levels and increased insulin sen-
sitivity (4, 5). Therefore, 11-HSD1 is currently considered a
promising drug target for the treatment of cognitive dysfunc-
tion in elderly men and patients with obesity and type 2 dia-
betes mellitus (6, 7). However, whether 11-HSD1 is indeed a
suitable target for therapeutic treatment of excessive glucocor-
ticoid actions remains to be tested.
Recently, we provided evidence that 11-HSD1 plays a role
in the rapid hepatic metabolism of 7-ketocholesterol (7KC) (8),
the major oxysterol in processed cholesterol-rich food and, after
27-hydroxycholesterol, in advanced atherosclerotic plaques (9
11). In rats treated with 7KC and the 11-HSD inhibitor car-
benoxolone, 7KC tended to accumulate in liver and plasma (8).
In addition, 11-HSD1 seems to catalyze the interconversion of
7-hydroxylated dehydroepiandrosterone metabolites (12) and
has a potential role in biotransformation by reducing reactive
ketones such as the potent tobacco carcinogen nicotine-derived
nitrosamine ketone, the anti-cancer drug oracin, as well as
metyrapone and its derivatives (1316).
Despite its importance, relatively little is known about the
molecular mechanisms by which 11-HSD1 exerts its physio-
logical function. 11-HSD1 belongs to the family of short-chain
dehydrogenases-reductases, characterized by a core domain
with conserved regions, including the Rossmann fold for bind-
ing of the cofactor NADP(H) and the Tyr-(Xaa)3-Lys motif in
the catalytic site, and less conserved N- and C-terminal se-
quences (17, 18). Several studies using 11-HSD1 constructs
with N-terminal deletions demonstrated that this part of the
enzyme is not only essential to anchor the enzyme to the ER
membrane but also for stability and catalytic activity (19 22).
The residues involved, however, were not identified.
1
The abbreviations used are: 11-HSD, 11-hydroxysteroid dehydro-
genase; 7KC, 7-ketocholesterol; E3, 50-kDa esterase/arylacetamide
deacetylase; EGFP, enhanced green-fluorescent protein; ER, endoplas-
mic reticulum; HEK, human embryonic kidney; SCAP, sterol regulatory
element-binding protein cleavage-activating protein.
31131
31132 Topology of 11-HSD1 and Esterase E3
We have shown previously (19, 23) that the single N-termi-
nal transmembrane helix is responsible for the luminal orien-
tation of the catalytic moiety of 11-HSD1. However, in con-
trast to 11-HSD2, whose cytoplasmic orientation is required
to tether the mineralocorticoid receptor to the ER membrane in
the absence of steroid and that prevents its occupation by
cortisol through conversion of cortisol to cortisone (24), the
physiological role of the luminal orientation of 11-HSD1 re-
mained unclear. In our previous study (19), we demonstrated
that substitution of Lys5 by Ser in the short cytoplasmic N
terminus of 11-HSD1 inverted its topology in the ER mem-
brane. Surprisingly, no difference was found for the oxoreduc-
tion of 11-dehydrocorticosterone between the mutant enzyme
with cytoplasmic orientation and wild-type 11-HSD1 upon
expression in HEK-293 cells.
The N-terminal transmembrane span of 11-HSD1 is highly
similar to that of the 50-kDa esterase/arylacetamide deacety-
lase (E3) (23), two proteins that are otherwise unrelated (Fig.
1A). E3, which is highly expressed in liver and adrenal glands
and to a lesser extent in small intestine, stomach, kidney, and
pancreas (25), acts as an N-deacetylase catalyzing hydrolytic
reactions and plays a potential role in the prevention of aryl-
amine-induced carcinogenesis (26). E3 might be involved, like
the putative triacylglycerol hydrolase E1, in the assembly of
hepatic very low density lipoprotein in the ER lumen (25). A
significantly reduced expression of E3 was observed in insulin-
deficient diabetes, which is characterized by a severe decrease
in the secretion of hepatic very low density lipoprotein triacyl-
glycerol. Unlike Lys5 in 11-HSD1, substitution of the analo-
gous Lys4 in E3 had no effect on topology, suggesting the
existence of a second determinant for the orientation of these
enzymes in the ER membrane (19, 23).
Here we tested the hypothesis whether the negatively
charged residues on the luminal side of the transmembrane
span might act as a second determinant for the orientation of
these two enzymes in the ER membrane, and we studied the
effect of charged amino acid residues on 11-HSD1 activity. In
addition, we investigated the role of the luminal orientation of
11-HSD1 on the oxidation of cortisol and the oxoreduction of
cortisone and 7KC.
EXPERIMENTAL PROCEDURES
MaterialsCell culture reagents were purchased from Invitrogen;
[1,2,6,7-3H]cortisone and [1,2,6-3H]7KC were from American Radiola-
beled Chemicals, St. Louis, MO; [1,2,6,7-3H]cortisol was from Amer-
sham Biosciences; 7KC and 7-hydroxycholesterol were from Steraloids
(Wilton, NH); and fluorescence-labeled antibodies were from Molecular
Probes. Proteinase K was from Roche Diagnostics, and all other chem-
icals were from Fluka AG, Buchs, Switzerland, and were of the highest
grade available.
Construction of PlasmidsThe plasmid for expression of FLAG
epitope-tagged human 11-HSD1 was constructed as described (19).
Mutant 11-HSD1 constructs were generated by site-directed mutagen-
esis according to the QuickChange mutagenesis kit (Stratagene, La
Jolla, CA). The rabbit E3-enhanced green fluorescent protein (EGFP)
chimera, containing the N-terminal membrane anchor sequence of E3
with a C-terminal EGFP, was constructed as described (23). For immu-
nofluorescence experiments, a construct containing the N-terminal 34
amino acids of rabbit E3 followed by a FLAG epitope tag was generated
by three-piece ligation of an EcoRI-BamHI fragment containing the
N-terminal 34 amino acids of E3, a BamHI-XbaI fragment encoding the
FLAG epitope (MDYKDDDD), and an EcoRI-XbaI pcDN3 expression
vector fragment. Mutant E3-FLAG and-EGFP constructs were made by
site-directed mutagenesis. All constructs were verified by sequencing.
Cell Culture and Transient TransfectionHEK-293 cells were grown
in Dulbeccos modified Eagles medium supplemented with 10% fetal
calf serum, 4.5 g/liter glucose, 50 units/ml penicillin, 50 mg/ml strepto-
mycin, and 2 mM glutamine. For microscopy experiments, cells were
seeded at 10% confluence onto glass coverslips placed in 6-well plates.
After growth for 24 h at 37 C under 5% CO2, cells were transfected by
Ca2-phosphate precipitation with 2 g/well of the corresponding ex-
pression plasmid and 1 g/well EGFP control plasmid. After incubation
for 8 h, the medium was replaced to remove the Ca2-phosphate pre-
cipitate. For isolation of microsomes and activity analysis, cells were
seeded to 50% confluence in 10-cm dishes and transfected, and medium
was replaced or cells were washed three times with steroid-free Dul-
beccos modified Eagles medium (doubly charcoal-treated).
Selective Permeabilization and Immunofluorescence AnalysisIm-
munofluorescence analysis was performed 48 h post-transfection as
described previously (24). Briefly, paraformaldehyde (4%) fixed cells
coexpressing the corresponding FLAG-tagged construct and EGFP con-
trol were washed four times with the buffer NAPS (150 mM sodium
phosphate, pH 7.4, 120 mM sucrose). For complete permeabilization of
membranes, cells were blocked in NAPS buffer containing 1% milk
powder and either 0.5% Triton X-100, for 11-HSD1 constructs, or 1%
saponin, for E3 constructs. The fluorescence signal obtained for E3
constructs was much stronger when saponin was used instead of Triton
X-100, an observation made previously with N-terminally FLAG-tagged
11-HSD1. The reason is unknown, but it seems that Triton X-100
interferes with antibody-epitope interactions in close proximity to the
membrane. For semipermeabilization of the plasma membrane, 25 M
digitonin was used (19). FLAG-tagged constructs were detected using
anti-FLAG antibody M2 as primary antibody and ALEXA-594 goat
anti-mouse secondary antibody. EGFP served as a transfection effi-
ciency control. Following incubation with antibodies in NAPS contain-
ing 0.1% milk, samples were washed four times with NAPS and treated
with Slow Fade Antifade kit (Molecular Probes). Samples were
analyzed on a Carl Zeiss confocal microscope LSM410 (Carl Zeiss,
Goettingen, Germany).
Isolation of Microsomes from HEK-293 CellsHEK-293 cells were
transfected with 8 g of the corresponding expression plasmid per
10-cm dish. Cells from four dishes were collected 48 h post-transfection,
washed with phosphate-buffered saline, and centrifuged at 150 g for
3 min, and the pellet was resuspended in 1.2 ml of buffer containing 10
mM Tris-HCl, pH 7.5, 0.5 M MgCl2, and protease inhibitor (Complete,
Roche Diagnostics). Cells were sonicated; 1.2 ml of isotonic buffer (1 M
sucrose, 10 mM Tris-HCl, pH 7.5, 2.5 M NaCl, 1 mM dithiothreitol) was
added, and lysates were centrifuged at 1,000 g for 10 min at 4 C,
followed by centrifugation at 11,000 g for 10 min at 4 C and
100,000 g for 1 h at 4 C. The pellet was resuspended in 300 l of
buffer containing 10 mM Tris-HCl, pH 7.5, 0.5 M sucrose, 1.25 M NaCl,
and 0.5 mM dithiothreitol. The protein concentration was adjusted to 1
mg/ml, and microsomal preparations were shock-frozen in liquid nitro-
gen and stored at 70 C until analysis.
Protease Protection Assay and ImmunoblottingMicrosomes (20 g
of proteins) were incubated in a total volume of 25 l with 0.25 g/l
proteinase K (Roche Diagnostics) for 30 min on ice in the presence or
absence of 0.5% Triton X-100. Proteinase K was inactivated by adding
2.5 l of 10 mM phenylmethylsulfonyl fluoride in isopropyl alcohol for 5
min, followed by solubilization of proteins with SDS sample buffer and
boiling for 5 min. Proteins were subjected to SDS-PAGE and Western
blot analysis using anti-FLAG antibody M2 or anti-EGFP antibody as
primary antibodies and secondary horseradish peroxidase-conjugated
antibody (Roche Diagnostics). Antibody binding was visualized using
the enhanced chemiluminescence Western detection system (Pierce).
Assay for 11-HSD11-HSD1-dependent oxidation and oxoreduc-
tion were measured as described previously (19, 27). Briefly, the rate of
conversion of cortisol to cortisone or the reverse reaction was deter-
mined in a final volume of 20 l in a TG1 buffer (20 mM Tris-HCl, pH
7.4, 1 mM EGTA, 1 mM EDTA, 1 mM MgCl2, 100 mM NaCl, 20% glycerol)
supplemented with 400 M NADP or NADPH, 30 nCi of 3H-labeled
substrate, and unlabeled substrate at different concentrations ranging
from 12.5 nM to 2 M. Cell lysates were prepared by washing transfected
cells with phosphate-buffered saline, centrifugation for 3 min at 150
g, removal of the supernatant, and quick-freezing the cell pellet in a dry
ice ethanol bath. Cell pellets were resuspended in buffer TG1, soni-
cated, and used immediately for activity assays. The reactions were
started by mixing 10 l of cell extract corresponding to 25 g of total
proteins with 10 l of reaction mixture and incubated for 10 20 min at
37 C. When measuring activities in intact cells, steroid-free medium
was used, and cofactor was omitted. To assess the effect of cyclosporin
A, cells were preincubated for 15 min with 50 M cyclosporin A prior to
addition of cortisone. The reactions were stopped by adding an excess of
unlabeled steroids in methanol, followed by separation of steroids by
TLC. Enzyme kinetics were analyzed by nonlinear regression using
Data Analysis Toolbox (MDL Information Systems Inc.) assuming first-
order rate kinetics. Hill coefficients for measurements in lysates were
ranging between 0.94 and 1.17. Data represent mean S.D. of at least
four independent transfections.
 Topology of 11-HSD1 and Esterase E3 31133

FIG. 1. A, schematic representation of the N-terminal membrane anchoring region of 11-HSD1 and E3. The positively charged residues on the
cytoplasmic side and the negatively charged residues on the luminal side that are important for determining the orientation of 11-HSD1 and E3
are indicated in boldface italic. B, wild-type and mutant constructs of rabbit E3; C, human 11-HSD1. The name of the mutant protein is given
on the left. Mutated residues are in boldface italic, and the orientation of the corresponding construct in the ER membrane, as determined by
immunohistochemistry and protease protection assay, is indicated on the right.

Oxoreduction of 7KC was determined as described (8). Briefly,
freshly prepared lysates were suspended in buffer TG1 and 400 M
NADP, 400 nM 7KC, and 30 nCi of 3H-labeled 7KC added, followed by
incubation at 37 C for 15 or 30 min. Intact cells were incubated in
steroid-free medium in the absence of cofactor for 30 or 60 min. The
reactions were stopped by adding an excess of unlabeled oxycholesterols
in methanol, followed by separation by TLC and scintillation counting.
RESULTS
Sequence Comparison of the N Termini of 11-HSD1 and
E3An alignment of the sequences of human 11-HSD1 and
rabbit E3 revealed that both proteins share a similar N-termi-
nal region (Fig. 1A) with a short and positively charged N-
terminal cytoplasmic part, a single transmembrane span fol-
lowed by negatively charged residues immediately downstream
of the membrane helix, and a large C-terminal luminal domain.
In previous studies, we have shown that substitution of Lys5 by
Ser led to inverted orientation of 11-HSD1 in the ER mem-
brane (19), whereas substitution of the analogous Lys4 by Ile in
E3 had no effect on topology (23), indicating the existence of a
second determinant for the topology of these two proteins.
The Cytoplasmic Lys4 and the Luminal Asp25 Determine the
Topology of E3To test the hypothesis that the negatively
charged residues immediately downstream of the membrane
span represent this second topology determining motif, we
generated a fusion protein encoding the N-terminal 34 amino
acids of E3 followed by a FLAG epitope for facilitated immu-
nodetection, and we subjected this construct to site-specific
mutagenesis (Fig. 1B). Cells coexpressing the corresponding
FLAG-tagged construct and EGFP control protein were either
completely permeabilized with 1% saponin or the plasma mem-
brane was selectively permeabilized with 25 M digitonin, al-
lowing restricted access of the anti-FLAG antibody to the cy-
tosolic compartment. Cells were then incubated with anti-
FLAG antibody and red fluorescent secondary antibody and
analyzed by fluorescence microscopy. Cells expressing EGFP
were analyzed for the presence of red fluorescence signal from
the corresponding E3 construct, whereby 200 300 cells were
counted in a typical experiment. By using saponin, over 95% of
cells expressing wild-type E3 stained positive but less than 5%
when digitonin was used, in line with the luminal orientation of
E3 (Fig. 2). Mutant K4I showed wild-type orientation, confirm-
ing previous findings (23). To investigate the role of negatively
charged residues at the luminal side of the transmembrane
helix, we replaced Asp25, Glu28, and Glu29 to Lys in either
wild-type E3 or mutant K4I (Fig. 1B). Substitution of the neg-
atively charged residues by Lys led to inverted insertion into
the ER membrane in mutant K4I/D25K/E28K/E29K but not in
mutant D25K/E28K/E29K (Fig. 2). Additional mutagenesis re-
vealed that substitution of Asp25/Glu28/Glu29 to Asn25/Gln28/
Gln29 and a single substitution of Asp25 by Lys in mutant K4I
both were sufficient to invert the orientation in the ER mem-
brane. All of the mutant proteins analyzed showed exclusive
localization to the ER membrane, indicating that they are not
important for ER retention. These results demonstrate that
both Lys4 and Asp25 are essential for determining the topology
of E3. The effects on topology were confirmed by subjecting
microsomal vesicles expressing wild-type or mutant constructs
31134 Topology of 11-HSD1 and Esterase E3

FIG. 2. Orientation in the ER membrane of constructs containing wild-type or mutant membrane anchoring regions of rabbit E3.
Wild-type or mutant proteins indicated at the top of each lane were coexpressed with EGFP in HEK-293 cells. Plasma membranes of cells were
selectively permeabilized with 25 M digitonin (1st two rows) or membranes completely permeabilized with 1% saponin (3rd and 4th rows), followed
by detection of the C-terminally attached FLAG epitope by mouse monoclonal anti-FLAG antibody M2 and secondary red fluorescent ALEXA-594
anti-mouse antibody using confocal microscopy. Representative experiments of cells expressing key mutant constructs are shown (results
summarized in Fig. 1).

to proteinase K digestion in the presence or absence of deter-
gent (data not shown).
The Luminal Residues Glu25 and Glu26 Are Important De-
terminants for the Topology of 11-HSD1In analogy to Asp25
of E3, 11-HSD1 contains two glutamate residues (Glu25 and
Glu26) immediately downstream of the membrane span. To
investigate whether the luminal di-glutamate motif plays a
role in determining the topology of 11-HSD1, we generated a
series of constructs with a C-terminal FLAG epitope (Fig. 1C)
and analyzed them in fully permeabilized (0.5% Triton X-100)
or semipermeabilized (25 M digitonin) cells by immunofluo-
rescence detection. Substitution of both Glu25 and Glu26 to Lys
led to an inverted orientation of 11-HSD1 in the ER mem-
brane (Fig. 3). Substitution of Glu25 by Lys and Glu26 by Gln
(mutant E25K/E26Q) did not change the orientation of the
mutant enzyme, indicating that the change from positive to
negative charge at both positions is necessary to invert the
topology of 11-HSD1.
We have shown previously that mutant K5S but not K6S
showed inverted topology. To assess a potential role of Lys6 in
determining topology, we mutated Glu25 and Glu26 in mutant
K6S. Independent of the residue at position 6, the orientation of
the enzyme was only inverted when both Glu25 and Glu26 were
changed to Lys (Fig. 1C and Fig. 3). These results indicate that
Lys6 is not involved in determining the topology of 11-HSD1.
In addition, all mutations analyzed in the present study did not
alter the restricted expression of 11-HSD1 in the ER mem-
brane (not shown).
To confirm the effect of these mutations on 11-HSD1 topol-
ogy, microsomal vesicles expressing FLAG-tagged wild-type or
mutant 11-HSD1 constructs were subjected to proteinase K
digestion in the presence or absence of detergents, followed by
separation on SDS-PAGE and detection with anti-FLAG anti-
body on Western blots. As seen in Fig. 4, in intact vesicles
mutants E25K/E26K and K6S/E25K/E26K were completely di-
gested by proteinase K, whereas wild-type 11-HSD1 and all
other mutants were protected from proteinase K-dependent
digestion. Upon addition of Triton X-100, all constructs were
digested. These results are consistent with the findings from
fluorescence microscopic analyses of semipermeabilized cells,
demonstrating an essential role of Glu25 and Glu26 in deter-
mining the orientation of 11-HSD1 in the ER membrane.
The Effect of Charged Residues in the N-terminal Region of
11-HSD1 on Enzymatic Activity in LysatesTo assess the
effect of charged residues in the N-terminal region of 11-
HSD1 on enzymatic activity, the reduction of cortisone to cor-
tisol was measured in lysates of cells expressing wild-type or
mutant 11-HSD1. None of the mutations had a significant
effect on apparent Km values (Table I); however, replacement of
both Glu25 and Glu26 by Gln or Lys led to a significant decrease
in activity (p 0.01), with a tendency for a loss of Vmax when
substituting the negatively charged Glu residues first by polar
Gln and then by positively charged Lys. The fact that mutant
K6S/E25K/E26Q with luminal orientation and mutant K6S/
E25K/E26K with cytoplasmic orientation both had similarly
reduced activities suggests that the di-glutamate motif at po-
sition 25/26 stabilizes enzymatic activity independent of the
orientation of 11-HSD1 in the ER membrane. In addition,
mutant K5S/K6S showed kinetic values comparable with that
of wild-type 11-HSD1, indicating that the orientation in the
ER membrane itself does not affect enzymatic activity. Similar
effects of these mutations were observed for the oxidation of
corticosterone (data not shown).
We also investigated the effect of the replacement of Lys35
and Lys36 by Ser immediately upstream of the conserved co-
factor binding site on intracellular localization and enzymatic
activity. Although expression level and intracellular distribu-
tion were not altered, this mutation led to a complete loss of
function, demonstrating that Lys35 and Lys36 are essential for
enzymatic activity of 11-HSD1.
Effect of the Orientation of 11-HSD1 in the ER Membrane
on the Interconversion of Glucocorticoids in Intact CellsTo
 Topology of 11-HSD1 and Esterase E3 31135

FIG. 3. Orientation of wild-type (wt) or mutant human 11-HSD1 in the ER membrane. HEK-293 cells were cotransfected with
C-terminally FLAG-tagged wild-type or mutant 11-HSD1 (indicated at the top of each lane) and EGFP. Plasma membranes of cells were
selectively permeabilized with 25 M digitonin (1st two rows) or membranes completely permeabilized with 0.5% Triton X-100 (3rd and 4th rows),
followed by immunofluorescence detection. Representative samples from cells expressing key mutant constructs are shown (results are summa-
rized in Fig. 1).

FIG. 4. Proteinase K sensitivity of wild-type and mutant 11-HSD1 in ER microsomes. HEK-293 cells were transfected with FLAG
epitope-tagged wild-type 11-HSD1 (A), mutant E25K/E26K (B), K6S/E25K/E26Q (C), E25K/E26Q (D), or K6S/E25K/E26K (E), and microsomal
vesicles were isolated as described under Experimental Procedures. Microsomal preparations were incubated for 30 min at 4 C in the presence
() or absence () of 0.25 g/l proteinase K and 0.5% Triton X-100. Proteins were separated on 12.5% SDS-PAGE and transferred to
nitrocellulose, followed by detection with mouse monoclonal anti-FLAG antibody M2 and secondary anti-mouse horseradish peroxidase antibody.

elucidate the potential role of the luminal orientation of 11-
HSD1 on its activity, we compared the interconversion of
cortisone and cortisol in lysates and intact HEK-293 cells ex-
pressing either luminally oriented wild-type 11-HSD1 or
cytoplasmically oriented mutant K5S/K6S. Measurements in
lysates yielded similar kinetic parameters for the oxidation of
cortisol and the oxoreduction of cortisone with both enzymes
(Tables II and III). In intact cells, the oxoreduction of cortisone
was not different between wild-type 11-HSD1 and mutant
K5S/K6S; however, mutant K5S/K6S showed 50% lower Vmax
(p 0.01) than wild-type for the oxidation of cortisol without a
change in Km. Approximately 50% lower Vmax but similar Km
was also observed for the oxidation of corticosterone by mutant
K5S/K6S (not shown).
Next, we investigated whether the inhibition of P-glycopro-
tein, which has been shown to catalyze the efflux of various
steroids from the cytoplasm (28, 29), by cyclosporin A (30) may
differentially affect the kinetic parameters of wild-type 11-
HSD1 and mutant K5S/K6S. As shown in Tables II and III,
cyclosporin A led to a 60% increase in catalytic efficiency of the
cytoplasmic mutant K5S/K6S (p 0.05 compared with un-
treated cells). In contrast, the catalytic efficiency of wild-type
11-HSD1 increased only by 18% (not significant). The effect
on K5S/K6S tended to be more pronounced than that on wild-
type enzyme, although the difference did not reach statistical
significance. These results suggest that the inhibition of the
activity of endogenous P-glycoprotein in HEK-293 cells leads to
an increase in cytoplasmic cortisone concentration, favoring
the activity on the cytoplasmic side.
The Luminal Orientation of 11-HSD1 Is Required for the
Oxoreduction of 7KC in Intact CellsRecently, we have shown
(8) that 11-HSD1 plays an essential role in the metabolism of
7KC in intact cells. Here we compared the ability of luminally
oriented wild-type enzyme and cytoplasmic mutant K5S/K6S to
catalyze the oxoreduction of 7KC in lysates and intact HEK-
293 cells. Both enzymes readily converted 7KC to 7-hydroxy-
cholesterol in lysates, but only wild-type 11-HSD1 catalyzed
the oxoreduction of 7KC in intact cells (Fig. 5). Mutant K5S/
K6S showed background activity comparable with that of un-
transfected cells. These results demonstrate that the luminal
orientation of 11-HSD1 is essential for the oxoreduction of
7KC.
DISCUSSION
To gain insight into the molecular mechanisms by which
11-HSD1 exerts its physiological functions, we investigated
the impact of the N-terminal membrane anchoring region on
11-HSD1 function. The two type II anchored ER membrane
proteins 11-HSD1 and E3 share a highly similar N-terminal
region consisting of a conserved Lys in the short cytoplasmic
31136 Topology of 11-HSD1 and Esterase E3
TABLE I remaining on the cytoplasmic side of the membrane (31, 32).
Reduction of cortisone by wild-type and mutant 11-HSD1 in lysates The positive-inside rule correctly predicts both the topology of
Reduction of cortisone by lysates of HEK-293 cells transfected with
wild-type E3, with N-terminal charge 1 and C-terminal
various 11-HSD1 constructs was determined as described under Ex-
perimental Procedures. Activities are expressed in terms of apparent charge 3 (1/3), when considering six residues downstream
Km and apparent Vmax. Data were obtained from 4 to 6 independent of the membrane span, and 11-HSD1 (2/2). However, the
experiments and are presented as mean S.D. present analysis of several mutant proteins revealed that spe-
Construct Kma Vmaxa,b cific amino acid residues rather than net charge distribution
determine the topology of these two enzymes.
nM nmol h1 mg1
We have shown previously that E3 mutant K4I (0/3) still
11-HSD1 (wild type) 336 39 1.88 0.23
E25Q 307 35 1.55 0.15 adopts wild-type topology (Ncyt/Clum) (23). Therefore, we now
E25K 346 20 1.66 0.27 analyzed the role of luminal, negatively charged amino acid
E26K 303 37 1.23 0.33c residues. Substitution by Lys of the three negatively charged
K6S/E25K 314 21 1.63 0.30
residues that are closest to the membrane span (mutant D25K/
E25Q/E26Q 308 29 1.07 0.13d
K6S/E25Q/E26Q 302 22 0.80 0.17d E27K/E28K (1/3)) did not alter its orientation in the ER
E25K/E26Q 300 28 0.84 0.15d membrane despite the excess of positive charges introduced at
K6S/E25K/E26Q 314 34 0.57 0.17d the luminal side (Fig. 2). Thus, the topology of this mutant
E25K/E26K 299 22 0.37 0.08d
clearly does not follow the positive-inside rule. However, sub-
K6S/E25K/E26K 344 49 0.59 0.13d
K5S/K6S/E25K/E26K 340 36 0.39 0.02d stitution of Asp25 by Lys (mutant K4I/D25K (0/1)) or of Asp25
K5S/K6S 295 25 1.75 0.14 by Asn and Glu25/Glu26 by Gln (mutant K4I/D25N/E27Q/E28Q
K35S/K36S NDe NDe (0/0)) led to an inverted orientation in the ER membrane. This
a
Apparent Km (nM) and apparent Vmax (nmol h1 mg1 of total indicates that Lys4 is the major determinant of E3 topology,
protein) values were calculated by nonlinear regression using Data whereby the introduction of several positively charged residues
Analysis Toolbox (MDL Information Systems Inc.) assuming first-order on the C-terminal end of the membrane span cannot overcome
rate kinetics (Hill coefficients ranging between 0.94 and 1.17).
b
For calculation of Vmax, the amount of 11-HSD1 protein per mg of the dominant N-terminal signal. In mutants lacking the N-
total proteins was determined, and values were normalized to the terminal signal, Asp25, the negatively charged residue closest
expression of FLAG-tagged wild-type 11-HSD1 by semiquantitative to the membrane span, dictates the topology.
densitometric analysis of Western blots That the topology of 11-HSD1 is determined by the appro-
c
p 0.05 compared with wild type.
d
p 0.01 compared with wild type. priate charge at a specific position rather than by the net
e
Activity was not detectable despite normal expression levels. charge distribution is demonstrated by the fact that mutant
K5S (1/2) adopts Nlum/Ccyt topology, whereas K6S shows
TABLE II
wild-type Ncyt/Clum orientation (19). The importance of Lys5 is
A comparison of the interconversion of cortisone and cortisol between
wild-type 11-HSD1 and mutant K5S/K6S also reflected by its conservation in all known species, whereas
HEK-293 cells were transfected with cDNA for wild-type 11-HSD1 Lys6 is replaced by Thr in squirrel monkey and by Asn in
or mutant K5S/K6S and cultured in steroid-free medium 16 h prior to mouse. Substitution of both Glu25 and Glu26 by Lys (Fig. 1C)
the determination of activities in lysates or intact cells as described led to an inverted Nlum/Ccyt orientation (Figs. 3 and 4), indicat-
under Experimental Procedures. Activities (mean S.D.) are ex-
ing that in 11-HSD1 the introduction of two positive charges
pressed in terms of apparent Km and apparent Vmax values and were
obtained from at least four independent experiments. at the C-terminal side of the membrane span is dominant over
the N-terminal Lys5 signal. This stands in contrast to E3,
Oxidation of cortisol
where the effect of Lys4 is not affected by the substitution of
Cell lysates Intact cells
Construct three negative by positive charges at the C-terminal side of the
Km Vmax Km Vmax membrane span. In mutant K6S, like in wild-type 11-HSD1,
nM nmol h1 mg1 nM nmol h1 mg1 both Glu25 and Glu26 had to be replaced by Lys to invert its
Wild type 344 32 1.4 0.2 826 51 0.64 0.17 topology, further demonstrating that Lys6 is not important for
K5S/K6S 360 35 1.5 0.5 755 47 0.31 0.10a topology.
Oxoreduction of cortisone In addition to determining the luminal orientation in the ER
Cell lysates Intact cells
membrane, the N-terminal regions of 11-HSD1 and E3 are
Construct sufficient to mediate retention in the ER membrane. We show
Km Vmax Km Vmax
that removal by mutagenesis of the cytoplasmic Lys signal, the
nM nmol h1 mg1 nM nmol h1 mg1 luminal negatively charged residues immediately downstream
Wild type 336 39 1.9 0.2 803 47 0.72 0.18 of the membrane helix or of Lys35 and Lys36 in 11-HSD1, does
K5S/K6S 295 25 1.8 0.2 799 72 0.67 0.15
not affect retention in the ER membrane. Similarly, all of the
a
p 0.01. constructed mutants of the fusion between the N-terminal 34
residues of E3 and green fluorescent protein showed restricted
sequence, a membrane spanning helix with a cluster of Tyr localization to the ER membrane, suggesting that intrinsic
residues, and a segment of negatively charged amino acid res- properties of the transmembrane sequence may be responsible.
idues C-terminal to the membrane span (Fig. 1A). Otherwise, Furthermore, the truncated splice variant 11-HSD1B, lacking
the two proteins are unrelated. Previous analyses revealed that the transmembrane span, was found to be attached to the ER
substitution of the conserved Lys5 by Ser inverted the orienta- membrane, suggesting that the hydrophobic domain located at
tion of 11-HSD1, but the substitution of the analogous Lys4 by amino acid residues 136 158 also sufficiently mediates reten-
Ile had no effect on the orientation of E3, indicating the exist- tion to the ER membrane (21).
ence of a second topological determinant (19, 23). In the present Previous studies provided evidence that the N-terminal re-
study, we identified Glu25/Glu26 in 11-HSD1 and Asp25 in E3 gion of 11-HSD1 has an important stabilizing effect on enzy-
as the second topological determinant. matic activity (19 22); however, the residues involved were not
According to the positive-inside rule, net charges on the identified. A fusion protein containing the transmembrane an-
polypeptide sequences flanking the transmembrane span de- chor of 11-HSD2 followed by residues 40 292 of 11-HSD1 as
termine the orientation, with the more positive of the two well as the truncated 11-HSD1 protein alone (residues 40
 Topology of 11-HSD1 and Esterase E3
TABLE III
Effect of cyclosporin A on oxoreduction of cortisone by wild-type 11-HSD1 and mutant K5S/K6S in intact cells
HEK-293 cells transiently expressing wild-type 11-HSD1 or mutant K5S/K6S were cultured in steroid-free medium for 16 h, preincubated with
50 m cyclosporin A, followed by determination of activities as described under Experimental Procedures. Activities (mean S.D.) are expressed
in terms of apparent Km, Vmax, and kcat (Vmax/Km) values and were obtained from four independent experiments.
Effect of cyclosporin A on oxoreduction of cortisone in intact cells
Absence of cyclosporin A
Construct Km Vmax Vmax/Km
nM nmol h1 mg1 h1 mg1 103
Wild type 803 47 0.72 0.18 0.90
K5S/K6S 799 72 0.67 0.15 0.84
a
p 0.05.
FIG. 5. The luminal orientation of 11-HSD1 is required for the
reduction of 7KC. HEK-293 cells were transfected either with a
control plasmid, wild-type 11-HSD1, or mutant K5S/K6S and cultured
in steroid-free medium. The reduction of 7KC to 7-hydroxycholesterol
was determined by incubation of lysates in the presence of NADPH and
400 nM 7KC for 15 (A) or 30 min (B) at 37 C. Intact cells were incubated
in steroid-free medium containing 400 nM 7KC without cofactor for 30
(C) or 60 min (D). 7-Oxycholesterol metabolites were analyzed by sep-
aration on TLC and scintillation counting as described under Experi-
mental Procedures. White bars, 7KC; black bars, 7-hydroxycholes-
terol. Results are expressed as a percentage of initially supplied 7KC
(mean S.D. from four independent experiments).
292, starting with Met-Thr-Gly) were catalytically inactive
(19). This may be explained by the present finding that Lys35
and Lys36 are essential for enzymatic activity. The positive
charge at positions 35/36 is highly conserved throughout dif-
ferent species with only cattle and sheep having Arg instead of
Lys36. Recently, Blum et al. (21) reported that the splice vari-
ant 11-HSD1B, lacking the first 30 amino acids but contain-
ing Lys35 and Lys36, could be partially reactivated upon solu-
bilization from the microsomal fraction. Substitution of Val149
by Arg in this truncated splice variant rendered the protein
more soluble; however, this protein was like 11-HSD1B and
31137
Presence of 50 m cyclosporin A
Km Vmax Vmax/Km
nM nmol h1 mg1 h1 mg1 103
815 74 0.87 0.18 1.07
673 98 0.91 0.27 1.35a
not stable, indicating that the more N-terminal residues are
required for the stability, e.g. for full activity of the enzyme. In
line with this assumption, Walker et al. (20) purified a trun-
cated enzyme lacking the first 23 amino acids, which retained
its activity. The present finding that mutagenesis of residues
Glu25 and Glu26 led to a significantly reduced Vmax, independ-
ent of the orientation of 11-HSD1 in the ER membrane and
not affecting expression level, suggests that these residues
stabilize a conformationally active enzyme.
In a previous study, we demonstrated that the N-terminal
region determines the topology of 11-HSD1 to the ER mem-
brane (19), but no difference in oxoreduction of 11-dehydrocor-
ticosterone was found between luminally oriented wild-type
11-HSD1 and a cytoplasmic mutant enzyme; thus the physi-
ological consequences of the luminal orientation remained un-
clear. Here we addressed the question of the impact of luminal
orientation on enzymatic function in more detail and compared
the function of the luminally oriented wild-type 11-HSD1
with the cytoplasmic mutant K5S/K6S in intact HEK-293 cells
not expressing endogenous 11-HSD activity. In line with the
previous study, no difference was found for the oxoreduction of
cortisone between wild-type and mutant enzyme. Similar re-
sults were also obtained when Chinese hamster ovary cells
were used, a cell line not expressing endogenous 11-HSD
activity (not shown). This is rather surprising because the
cytoplasm is considered to have a more reducing environment
than the ER lumen, and cofactor concentrations may be differ-
ent between the two compartments. This finding also implies
that the substrate has similar access to both compartments.
However, regeneration systems exist for cofactors in the cyto-
plasm and in the ER lumen, and 11-HSD1 accepts both cofac-
tors with a slight preference for NADP(H), and neither glyco-
sylation nor disulfide bridges are required for the catalytic
activity of the enzyme (19, 33, 34).
It was shown previously (28, 29) that P-glycoprotein acts as
an energy-dependent efflux pump that exports a wide range of
substrates including corticosteroids. P-glycoprotein is endog-
enously expressed in HEK-293 cells, although at a relatively
low level, and can be efficiently inhibited by cyclosporin A (30).2
The more pronounced increase in catalytic efficiency of the
oxoreduction of cortisone observed for the cytoplasmic mutant
K5S/K6S compared with wild-type 11-HSD1 indicates that
the inhibition of P-glycoprotein by cyclosporin A predominantly
enhanced cortisone concentrations in the cytoplasm. The tend-
ency of an increase of catalytic efficiency of wild-type 11-
HSD1 upon cyclosporin treatment indicates that the substrate
is exchanged between the cytoplasm and the ER lumen.
In contrast to the oxoreduction of cortisone, a 50% decrease
in the oxidation of cortisol was observed for mutant K5S/K6S in
the cytoplasm compared with wild-type 11-HSD1, thus dem-
onstrating that the luminal orientation is essential for appro-
2
David Clarke, University of Toronto, personal communication.
31138 Topology of 11-HSD1 and Esterase E3
priate function of 11-HSD1. The oxidative activity of 11-
HSD1 has been suggested to protect mature Leydig cells from
the inhibitory effect of glucocorticoids on testosterone produc-
tion (35). In addition, dehydrogenase activity of 11-HSD1 is
predominant in adipose stromal cells, where it may represent
an autocrine protective mechanism that facilitates preadipo-
cyte proliferation, but upon differentiation to adipocytes 11-
HSD1 switches to oxoreductase activity (36). Recent studies
(37) provided evidence that this switch from oxidation to oxo-
reduction is dependent on the activity of hexose-6-phosphate
dehydrogenase, which may act as a regulator of the redox
potential in the ER. Thus, the luminal orientation may not only
be essential for efficient dehydrogenase activity as demon-
strated in the present work but also for adequate functional
interaction with hexose-6-phosphate dehydrogenase.
Moreover, we demonstrate that the luminal orientation of
11-HSD1 is required for oxoreduction of 7KC, because no
conversion of 7KC to 7-hydroxycholesterol was detected for
mutant K5S/K6S with cytoplasmic orientation. The molecular
mechanism underlying the required luminal orientation of 11-
HSD1 for oxoreduction of 7KC is unclear but may be dependent
on other enzymes localized in the ER lumen. 7-KC has been
suggested to play a role in atherosclerosis, for example, by
inhibiting free cholesterol efflux from macrophages (38) and
disturbing cholesterol trafficking between the ER membrane
and the plasma membrane (39). In macrophages, the subcellu-
lar distribution of 7KC mirrors that of cholesterol, which is
localized in the plasma membrane and intracellular mem-
branes but also cycles constitutively between the plasma mem-
brane and subcellular organelles (reviewed in Refs. 10 and 40).
The cholesterol content of mammalian cells is controlled by the
sterol regulatory element-binding protein pathway. This path-
way is regulated by sterol regulatory element-binding protein
cleavage-activating protein (SCAP) (41). Upon sterol accumu-
lation, SCAP no longer moves from the ER to the Golgi, thus
restricting the cleavage of sterol regulatory element-binding
proteins and preventing the transcription of the cholesterol-
synthesizing enzymes (42, 43). Recently, Brown et al. (44)
showed that among other sterols, 7-hydroxycholesterol but
not 7KC reduces the activity of SCAP in the ER membrane
presumably by causing cholesterol to translocate from the
plasma membrane to the ER, where it interacts with the sterol
sensing domain of SCAP and produces conformational change
of the protein. In line with this hypothesis, conversion of 7KC
to 7-hydroxycholesterol in the ER lumen by 11-HSD1 may
have a beneficial effect by protecting cells from lipid accumu-
lation and may play a role in macrophage pathophysiology in
atherosclerosis (45). The molecular mechanism of the oxoreduc-
tion of 7KC by 11-HSD1 require further studies; nevertheless,
this novel role of 11-HSD1 should be kept in mind for the
design of specific inhibitor molecules for therapeutic purposes.
In conclusion, we identified the residues determining the
orientation of 11-HSD1 and E3 toward the ER lumen. Anal-
ysis of the charged residues in the N-terminal region of 11-
HSD1 revealed that the di-lysine motif at position 35/36 and
the di-glutamate motif at position 25/26 are essential for enzy-
matic activity. Most important, we demonstrate that the lumi-
nal orientation of 11-HSD1 is essential for efficient oxidation
of cortisol and for the oxoreduction of 7KC. In future studies,
the characterization of the expression and activities of E3 and
11-HSD1 constructs with cytosolic orientations of their cata-
lytic moiety should contribute further to the understanding of
the physiological function of these proteins.
AcknowledgmentsWe thank Mario Ziegler, Heidi Jamin, and
Regula Suter for excellent technical support and Dr. Zoltan Balazs,
Roberto Schweizer, and Dr. Bernhard Dick for helpful discussion and
advice.
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