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INTRODUCTION |
Thyroid tissue is confined to and is present in all vertebrates.
Its role is to synthesize and secrete polyiodinated thyronine molecules
that modulate gene expression in virtually every vertebrate tissue
through ligand-dependent transcription factors.
Thyroxine (T4)1 is
the primary product of thyroid secretion, a pro-hormone that must be
activated by deiodination to 3,5,3'-triiodothyronine (T3) by either type 1 or 2 iodothyronine deiodinases (D1 or D2) in order to
initiate thyroid action. To balance the activation pathway, both
T4 and T3 are irreversibly inactivated by
monodeiodination of the tyrosyl ring of the iodothyronines, a reaction
catalyzed by the type 3 iodothyronine deiodinase (D3). These three
enzymes constitute a family of selenocysteine (Sec)-containing integral membrane oxidoreductases (1).
Changes in the activity of D3 modulate both global and local tissue
thyroid status. In the global sense, D3 expression is increased by
T3 and reduced in hypothyroidism or iodine deficiency, thus
accelerating or retarding T3 inactivation to maintain
homeostasis (2-4) or to alter plasma T3 concentrations
such as occurs during tadpole metamorphosis or during fetal life
(5-7). More complex are the alterations in D3 activity in specific
tissues dictated by developmental programs that permit precisely timed
changes in their differentiation. For example, during metamorphosis in Xenopus laevis tadpoles, the eyes must shift from
a lateral to a more rostral and dorsal location to permit overlapping
visual fields. Retinal cells follow this shift with an asymmetrical
growth, a process that is thyroid hormone-dependent. To
develop asymmetrically, however, a subset of dorsal cells must grow at
a slower rate. This is achieved by an increase in D3 expression in
these cells, thus producing transient local hypothyroidism (8). Whereas D3 thus serves an essential physiological role, its inappropriate overexpression in large hemangiomas has recently been shown to cause a
unique clinical syndrome termed consumptive hypothyroidism. This occurs
in infants (and rarely in adults) when D3-catalyzed inactivation of
T3 and T4 occurs more rapidly than maximum
thyroidal production (9-11).
Understanding the cellular biology of D3 can shed light on its
function, the potential physiological cofactors, and the mechanisms regulating its degradation. The two T4-activating enzymes,
D1 and D2, are also integral membrane proteins. Whereas D1 is located in the plasma membrane (PM), D2 is an endoplasmic reticulum
(ER)-resident protein (12). Nothing is known about D3 other than it is
an integral membrane protein with a putative transmembrane domain near
the NH2 terminus (reviewed in Ref. 1). The present studies demonstrate that D3 is primarily in the PM with most of its molecule in
the extracellular space.
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MATERIALS AND METHODS |
DNA Constructs and Transfections--
The human D3 cysteine
(Cys) mutant (CysD3) was generated by replacing Sec144 with Cys using
overlap extension PCR. The CysD3 carboxyl FLAG construct (D3C-FLAG) was
engineered in a D10 vector fusing the epitope by an
EcoRI/XbaI-based strategy as described previously
for D1 and D2. These carboxyl FLAG D1 and D2 constructs (D1C-FLAG and
D2C-FLAG) encode full-length Cys mutant deiodinases fused to the FLAG
peptide at the COOH terminus (12, 13). Human embryonic kidney (HEK-293)
or mouse Neuro2A neuroblastoma cells (NB-2A) were transfected along
with TKGH to monitor transfection efficiency as described and were used
~48 h later. Transfection efficiency was estimated by measuring human
growth hormone in the media (12).
Antibodies and Chemicals--
The primary antibodies used for
immunofluorescence cytochemistry (IF) include the monoclonal antibody
anti-FLAG M2 and its biotinylated derivative (Sigma); rabbit anti-FLAG,
monoclonal anti-Na,K-ATPase
, rabbit anti-early endosome antigen-1
(EEA-1) (Affinity Bioreagents, Golden CO); affinity-purified goat
anti-BiP antibody, Research Diagnostics, Inc., (Flanders, NJ); and
monoclonal anti-clathrin heavy chain TD-1 antibodies, Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies were used at
1.25 µg/ml and included goat anti-rabbit IgG fluorescein
isothiocyanate (FITC) and goat anti-mouse IgG Texas Red-X (Molecular
Probes, Eugene, OR) or 1 µg/ml FITC-conjugated F(ab')2
fragment donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove,
PA). For Western analysis, we used the monoclonal anti-FLAG M2 or the
rabbit anti-D3-18 antibody (D3-18) (10). The D3-18 is directed against
amino acids 53-68, just downstream of the putative transmembrane
domain (residues 10-35) and is affinity-purified. Unless otherwise
specified, all other chemicals and reagents were purchased from Sigma.
Subcellular Fractionation and Western Analysis--
The cytosol
and the microsomal fractions of HEK-293 cells transiently expressing
D3C-FLAG were prepared after cells were sonicated in lysis buffer-1
(200 mM HEPES buffer, pH 7.5, containing 5 mM sodium pyrophosphate, 5 mM EGTA, 1 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
iodoacetamide, and 1 mM N-ethylmaleimide) and
ultracentrifuged at 100,000 × g for 1 h as
described (14). Proteins were resolved by SDS-PAGE and processed for
Western analysis as described (12, 15).
IF and Confocal Microscopy--
Studies were conducted in monkey
hepatocarcinoma cells (NCLP-6E) that endogenously express D3 (16) and
in transfected NB-2A or HEK-293 cells grown directly on glass slides
(12). Cells were transfected with D3C-FLAG and processed in two
different ways. (i) Paraformaldehyde-fixed cells were permeabilized
either with digitonin (25-50 µM) or acetone as
indicated, followed by incubation with primary antibodies, as
indicated, for 1 h at room temperature (RT), rinsed in
phosphate-buffered saline (PBS) containing 1% BSA (PBS/BSA), and
incubated for 30 min with the appropriate species-specific secondary
antibodies. (ii) Non-permeabilized cells were washed twice with cold
PBS and incubated for 1 h at 4 °C with mouse anti-FLAG (1:100)
and rabbit anti-D3-18 (1:80) antibodies diluted in cold DMEM containing
10% FBS. Cells were then fixed and blocked with PBS/BSA containing 1%
goat serum and incubated with secondary antibodies as above (17). In
all studies, the slides were mounted in Vectashield® mounting medium
with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA)
and examined by conventional confocal microscopy on a Bio-Rad
MCR-1024/2P system interfaced with a Zeiss Axiovert microscope.
Cell Biotinylation Assays--
For cell surface biotinylation,
HEK-293 cells transiently expressing FLAG-tagged deiodinases were
washed twice with cold PBS containing 1 mM
MgCl2 and 0.1 mM CaCl2
(PBS2+) and incubated for two 20-min periods at 4 °C
with 1.0 mg/ml sulfo-NHS-LC-biotin (sulfo-biotin; Pierce) diluted in
PBS2+. They were then quenched twice for 20 min (4 °C)
with 100 mM glycine in PBS. For intracellular
biotinylation, cells were probed for 30 min at room temperature with 1 mg/ml biocytin (Pierce) in PBS and then washed 3 times with
PBS2+ containing 2%
-mercaptoethanol. After either
method of biotinylation, cells were harvested and lysed for 30 min at
4 °C in lysis buffer-2 (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Triton X-100, 1 mM
MgCl2) containing 1,000 units of DNase (Promega, Madison,
WI), 1 mM phenylmethylsulfonyl fluoride and 1× protease
inhibitors mixture (Roche Diagnostics). The lysate was centrifuged at
12,000 × g for 10 min (4 °C) and the supernatant
incubated for 2 h with streptavidin-agarose beads (Pierce) at
4 °C. The beads were washed twice with lysis buffer-2 containing 500 mM NaCl, twice with the lysis buffer-2, and once with 50 mM Tris-HCl, pH 7.5, and finally the complexes were
processed for Western analysis with anti-FLAG M2 antibody (1:3000). In
some experiments, as indicated, cells were first treated at 37 °C
with DMEM containing 0.3 M sucrose for 15 min to inhibit
the clathrin-mediated endocytic pathway (18) or with 10 mM
methyl-
-cyclodextrin (M
CD) for 30 min (19), a compound
that inhibits clathrin- and caveolae-mediated endocytic pathways.
[35S]Met/-Cys Metabolic Labeling--
HEK-293
cells transfected with D3C-FLAG or D10 vector were incubated for 30 min
at 37 °C with methionine (Met)- and Cys-free DMEM and then labeled
with ~120 µCi/plate of [35S]Met/-Cys labeling mix
(PerkinElmer Life Sciences) for 20-180 min at 37 °C. Cells were
then washed with PBS and processed as indicated separately for each
experiment. Pulse-chases were performed with complete DMEM containing
10% FBS for 1-20 h.
In Vivo Immunoprecipitation (IP)--
HEK-293 cells transiently
expressing D3C-FLAG or D10 vector were metabolically labeled and
processed for in vivo IP as described elsewhere (20). Some
cells were treated with 10 mM M
CD during the last 30 min
of the labeling phase. The cells were then washed with cold buffer A
(PBS containing 1% BSA and 10 µg/ml goat anti-mouse IgG) for 20 min
followed by incubation on ice with the biotinylated anti-FLAG antibody
(1:300) diluted in buffer A for 30 min. Cells were then washed once
with buffer A and twice with buffer A containing 5% FBS, harvested,
and lysed with lysis buffer. The streptavidin-agarose beads were added
to the solubilized fraction, and immune complexes were pelleted by
brief centrifugation. Samples were resolved by 4-15% SDS-PAGE and
processed for autoradiography.
Studies of D3 Internalization--
HEK-293 cells transiently
expressing D3C-FLAG were cell surface-biotinylated with 2.0 mg/ml
sulfo-NHS-SS-biotin in PBS2+ for 60 min (4 °C) followed
by incubation for 5-10 min at 37 °C with DMEM containing 10% FBS
to allow internalization of biotinylated proteins (21). Similarly
treated cells were incubated with 0.3 mM primaquine in DMEM
with 5 mM HEPES, pH 7.4, to inhibit the recycling limb of
the endocytic pathway (22). The cells were then washed with ice-cold
PBS2+ and the extracellular biotinylated proteins were
freed of biotin by treatment with 50 mM reduced GSH
solution and processed for streptavidin-agarose beads pull-down and
Western analysis using FLAG M2 antibody (1:3000).
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RESULTS |
D3 Is Located in the Plasma Membrane--
NCLP-6E monkey
hepatocarcinoma cells express high level of endogenous D3 (23). These
cells were acetone-treated and processed for IF with primary antibody
D3-18, and the images show a pattern in the periphery of these cells
suggesting a plasma membrane location (Fig.
1A, a and
b). To analyze the cellular expression of this enzyme under
controlled conditions using highly specific monoclonal antibody, we
prepared a plasmid containing a recombinant human D3 protein tagged
with FLAG at its COOH terminus (D3C-FLAG). Because of the inefficient
translation of selenoproteins in general, the Sec residue at position
144 was replaced with Cys, and the protein was transiently expressed in
various cell types. This D3 retains the capacity to convert
T3 to 3,3'-diiodothyronine (T2) in a
saturable fashion with a Km of ~150 nM
(not shown). Western analysis of cytosolic and
Na2CO3 (pH 11)-washed microsomal
fractions of D3C-FLAG-expressing HEK-293 cells using anti-FLAG and a
primary antibody (D3-18) revealed an identical band of the predicted
size (33 kDa) only in the microsomal fraction of the cell lysate (Fig. 1B).

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Fig. 1.
Detection of endogenous and
transiently expressed D3 by IF and Western analyses. A,
subcellular localization of D3 using confocal immunofluorescence.
a and b, NCLP-6E cells endogenously expressing
D3. c and d, HEK-293. e and
f, NB-2A cells transiently expressing D3C-FLAG were fixed
with paraformaldehyde, permeabilized with acetone, and processed for IF
(see "Materials and Methods"; a, c, and
e, phase contrast) either with anti-D3-18 (a and
b) or anti-FLAG M2 (1:600, c-f); secondary
antibodies were anti-rabbit IgG FITC (a and b),
anti-mouse IgG Texas Red-X (c and d), and
FITC-conjugated donkey anti-mouse IgG (e and f).
All cell types are indicated at the bottom right-hand
corner; bar = 10 µm; B, Western
analysis of HEK-293 cells transiently expressing D3C-FLAG. Twenty µg
of cytosolic (100,000 × g supernatant) (lanes
a) or microsomal (lanes b) fractions of cell lysate
were resolved by 4-15% gradient SDS-PAGE and processed for Western
analysis using anti-FLAG M2 antibody (1:3000) and, after membrane
stripping, with anti-D3-18 antibody (1:1000).
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D3C-FLAG-expressing HEK-293 (Fig. 1A, c and
d) and NB-2A (Fig. 1A, e and
f) cells were processed for IF using anti-FLAG M2 antibody
and analyzed by confocal microscopy. D3 is distributed in the
periphery, confirming a PM location. The similar IF staining pattern in
the periphery to that in the NCLP-6E cells that constitutively express
wild type Sec-containing D3 (Fig. 1A, a and
b) validates the use of the FLAG-tagged Cys D3 mutant in
these studies.
To determine whether the D3 was also expressed in the ER membrane, we
exposed D3C-FLAG-expressing HEK-293 cells to 25 µM
digitonin to permeabilize the PM but not the ER (12). D3 staining with anti-FLAG M2 antibody was present only at the cell periphery (Fig. 2A, a and
b), not different from the pattern in acetone-treated cells.
To analyze further for D3 expression in the ER membrane, both HEK-293
and NB-2A cells transiently expressing D3C-FLAG were co-stained with
anti-FLAG M2 and anti-BiP, an ER-specific marker. The D3 signal was
clearly distinct from that of the ER marker (Fig. 2A,
c and d). Similar findings were obtained with
another ER-specific marker, calnexin (not shown).

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Fig. 2.
Exclusion from ER and co-localization of D3
and Na,K-ATPase in the plasma membrane. A, HEK-293
cells transiently expressing D3C-FLAG were prepared as described in
Fig. 1 and permeabilized with 25 µM digitonin
(a, phase contrast) and incubated with anti-FLAG M2, and the
secondary antibody was anti-mouse IgG Texas Red-X (b).
c, acetone-fixed cells were processed for IF with
anti-FLAG M2 and with goat anti-BiP (1:20); secondary antibodies were
anti-mouse IgG FITC and anti-goat IgG rhodamine. d is a
detail of the indicated area. B, HEK-293 cells transiently
expressing D3C-FLAG and processed for IF with rabbit anti-FLAG
(a) and mouse anti-Na,K-ATPase (1:80; b)
antibodies; secondary antibodies were anti-rabbit IgG FITC
(a) and anti-mouse IgG Texas Red-X (b); the
co-localization is shown in c and d, and the
superimposition is in e and f; d and
f, detail of the indicated selected areas in
c and d; bar = 10 µm.
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To confirm PM localization, HEK-293 cells transiently expressing
D3C-FLAG were co-stained anti-Na,K-ATPase
antibody, a typical PM
marker (Fig. 2B, a-d). The confocal analysis
indicates that a substantial fraction of transiently expressed D3
co-localizes with the Na,K-ATPase PM staining (Fig. 2B,
c-f).
The primary anti-D3 antibody (D3-18) is directed against residues
53-68, just COOH-terminal to the predicted transmembrane domain of D3
(residues 16-41). To confirm co-localization of the COOH-terminal FLAG
epitope and this peptide, non-permeabilized HEK-293 cells transiently
expressing D3C-FLAG were incubated with anti-FLAG M2 and anti-D3-18
antibodies, fixed, and then stained with secondary antibodies. Both
antibodies detected D3 on the surface of the transfected cells, and
confocal analysis confirmed their co-localization in the extracellular
space (Fig. 3A). By using the
same antibodies, acetone-permeabilized D3-expressing cells also showed
a similar co-localization (Fig. 3B).

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Fig. 3.
D3 is biotinylated in the PM and has a long
half-life. All cells are HEK-293 transiently expressing D3C-FLAG
except where indicated. A, non-permeabilized cells (see
"Materials and Methods"; a, phase contrast) were
incubated with mouse anti-FLAG M2 (1:100; b) and rabbit
anti-D3-18 (1:80; c); secondary antibodies were anti-mouse
IgG Texas Red-X (b) and anti-rabbit IgG FITC (c);
the superimposition is shown in d; bar = 10 µm. B, cells were fixed and processed for IF (see
"Materials and Methods"; a, phase contrast) with mouse
anti-FLAG M2 (1:600; b) and rabbit anti-D3-18 (1:80;
c); secondary antibodies were anti-mouse IgG Texas Red-X
(b) and anti-rabbit IgG FITC (c); the areas of
co-localization are shown in d (yellow pixels);
bar = 10 µm. C, HEK-293 cells transiently
expressing D3C-FLAG, D1C-FLAG, or D2C-FLAG were cell surface- or
intracellular-biotinylated (see "Materials and Methods"), and the
pellets were resolved by 12% SDS-PAGE and processed for Western
analysis using anti-FLAG M2 antibody (1:3000). D, cells were
[35S]Met/-Cys-labeled and chased with complete media for
0-6 h; at the indicated times cells were surface-biotinylated,
sonicated in lysis buffer-2, incubated with anti-FLAG M2 antibody
(1:100), and immunoprecipitated with protein-G/A agarose beads; half of
the IP pellet was loaded directly into 4-15% SDS-PAGE (total
IP); the other half was heated for 5 min at 100 °C in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% SDS to
release the immunocomplexes, diluted to 1 ml with 50 mM
Tris-HCl, pH 7.5, and the pull-down with streptavidin-agarose beads
loaded in the same gel; E-G, HEK-293 cells transiently
expressing D3C-FLAG (E), D1C-FLAG (F), or
D2C-FLAG (G) were [35S]Met/-Cys metabolically
labeled, chased with complete media for 0-20 h, processed for IP with anti-FLAG M2, and resolved by 10%
SDS-PAGE; bands were quantified, corrected by growth hormone levels in
the media, and plotted as mean ± S.D. of two plates.
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An independent approach was used to demonstrate the presence of D3 in
the PM of transfected HEK-293 cells. These were subjected to cell
surface biotinylation with the cell-impermeant sulfo-biotin probe (see
"Materials and Methods"). Cells were lysed with buffer containing
1% Triton X-100 and the biotinylated proteins isolated with
streptavidin-agarose beads. Western analysis with anti-FLAG M2 antibody
showed a D3 band, confirming the presence of biotinylated D3C-FLAG
(Fig. 3C). As controls we employed HEK-293 cells transiently expressing D1C-FLAG or D2C-FLAG, as they are located in PM and ER,
respectively (12). D1C-FLAG was also biotinylated by the cell-impermeant reagent, whereas D2C-FLAG was not (Fig. 3C).
However, D2 could be biotinylated with biocytin, a cell
membrane-permeable probe, indicating it was present but not accessible
to sulfo-biotin (Fig. 3C).
The chronology of D3 appearance in the PM was determined by combining
[35S]Met/-Cys metabolic labeling with cell surface
biotinylation of D3C-FLAG-expressing HEK-293 cells (see "Materials
and Methods") (24). At various times after cells were chased with
unlabeled amino acids and surface-biotinylated,
[35S]Met/-Cys-labeled D3C-FLAG was immunoprecipitated
with anti-FLAG M2 antibody and the PM D3 pool isolated on
streptavidin-agarose beads (Fig. 3D). It is notable that at
all time points the biotinylated (PM) D3 signal is at least 10-fold
less intense than that of the total lysate D3, indicating the presence
of a substantial pool of intracellular D3. However, the PM D3 signal
was most intense at 4-6 h relative to earlier or later times (Fig.
3D), indicating that previously synthesized D3 continues to
enter and exit the PM and that D3 is a relatively stable protein. The
D3 half-life was ~12 h by pulse-chase analysis (Fig. 3E)
as compared with the shorter half-lives of D1C-FLAG and D2-C-FLAG
proteins (~7 and ~2 h, respectively) (Fig. 3, F and
G).
D3 in the Plasma Membrane Is Internalized Predominantly by a
Clathrin-dependent Mechanism--
Most proteins in the PM
undergo internalization through the endocytic pathway. To determine
whether this is the case for D3, we exposed HEK-293 cells transiently
expressing D3C-FLAG to either hypertonic media (0.3 M
sucrose), a procedure that inhibits receptor-mediated endocytosis by
preventing the formation of clathrin-coated pits (18), or to 10 mM M
CD, which extracts cholesterol from the PM and
inhibits both caveolin- and clathrin-dependent endocytosis (25-28). After incubation, cells were exposed to sulfo-biotin and analyzed by Western blotting using anti-FLAG M2 antibody. Both treatments markedly and specifically increased the PM D3 while not
affecting cell permeability, as demonstrated by the absence of D2
biotinylation in similarly treated cells (Fig.
4, A and B) and
trypan blue staining (not shown).

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Fig. 4.
Internalization is through clathrin-mediated
pathway. A and B, Western blot analysis with
anti-FLAG antibody of HEK-293 transiently expressing D3C-FLAG or
D2C-FLAG treated either with 0.3 M sucrose for 15 min
(A) or 10 mM MBCD for 30 min (B),
processed for cell surface biotinylation and resolved in 4-15%
SDS-PAGE. C, HEK-293 transiently expressing D3C-FLAG were
[35S]Met/-Cys metabolically labeled, treated with 10 mM M CD during the last 30 min of labeling, and processed
for in vivo IP (see "Materials and Methods"); the
arrow indicates the D3 protein. D, cells were
fixed and processed for IF (see "Materials and Methods") with
rabbit anti-FLAG (1:600; a) and mouse anti-clathrin heavy
chain (1:100; b) antibodies; secondary antibodies were
anti-rabbit IgG FITC (a) and anti-mouse IgG Texas Red-X
(b); c, co-localization of the two proteins;
d, higher magnification of the boxed area in
c, showing details of the co-localization area;
bar = 10 µm. E, HEK-293 cells transiently
expressing D3C-FLAG were cell surface-biotinylated with the
GSH-sensitive (cleavable) reagent sulfo-NHS-SS-biotin at 4 °C ± 0.3 mM primaquine. Cells were then processed for
streptavidin-agarose beads pull-down and Western analysis with
anti-FLAG M2 antibody (1:3000) (see "Materials and Methods").
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The D3 internalization was also investigated by immuno-analysis of PM
D3 in intact [35S]Met/-Cys pulse-labeled cells with or
without M
CD (20). These were incubated with biotinylated anti-FLAG
antibody, lysed, isolated on streptavidin agarose beads, and resolved
by SDS-PAGE. Consistent with the direct biotinylation experiments,
M
CD-sensitive [35S]Met/-Cys-labeled D3C-FLAG was
clearly present in much higher quantity on the surface of
M
CD-exposed than in control cells (Fig. 4C). The D3
endocytic pathway is likely to include internalization via a
clathrin-dependent mechanism because in addition to the sensitivity to hypertonic medium (Fig. 4, A-C), confocal
analyses indicate co-localization of D3C-FLAG with clathrin (Fig.
4D, c and d).
Evidence of D3 recycling was obtained using an assay that utilizes cell
surface biotinylation with a glutathione-sensitive (cleavable)
biotinylation reagent (see "Materials and Methods") (21). After
biotinylation of D3C-FLAG-expressing cells at 4 °C, cells were
re-warmed at 37 °C to allow internalization of biotin-tagged cell
surface proteins. At the indicated times, the HS-biotin moiety was
cleaved from non-internalized cell surface D3 by exposure to 50 mM GSH. The cell lysates were subjected to streptavidin
pull-down and Western analysis.
At zero time all the biotinylated D3 is sensitive to GSH and is
therefore on the cell surface (Fig. 4E). After 5 min of
re-warming, biotinylated D3 is present inside the cells as indicated by
its resistance to GSH treatment. Longer incubation times did not result in greater biotinylated D3 accumulation inside the cells, which could
be due to recycling of the internalized, biotinylated D3 to the cell
surface. Consistent with this explanation, when cells were re-warmed in
the presence of 0.3 M primaquine, a weak base that reduces
the rate of return of endosomal proteins to the cell surface (22), the
pool of internalized (GSH-resistant) biotinylated D3 was increased
(Fig. 4E). Exposure of cells to the D3 substrate, T3, had no effect on the ratio of biotinylated PM D3 to the
total Triton X-100-soluble cell D3.
The fate of the internalized D3 was determined by specific confocal IF
analysis in cells stained with antibody to early endosomes antigen-1
(EEA-1) (Fig. 5c). The merged
images show the extensive co-localization of D3 and EEA-1 (Fig.
5d). This indicates that most D3 does not follow the
endocytic pathway to late endosomes and lysosomes but accumulates in
the early endosomes where a short-loop recycling can occur.

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Fig. 5.
D3 is in early endosomes. HEK-293
transiently expressing D3C-FLAG (a) were processed for IF
with mouse anti-FLAG M2 (b) and rabbit anti-EEA-1
(c) antibodies; secondary antibodies were anti-mouse IgG
Texas Red-X (a) and anti-rabbit IgG FITC (b);
image super-imposition is shown in d; bars = 10 µm.
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DISCUSSION |
The present studies are the first to describe the cellular biology
of D3, a critical component of the thyroid hormone inactivation pathway. Both endogenous D3 in NCLP-6E cells and D3 transiently expressed in embryonic kidney or neuroblastoma cells are found at the
cell periphery (Fig. 1). Curiously, however, only a fraction of D3 is
in the PM at any point in time, the remainder is localized just
internal to this (Figs. 2, A and B, and
5b). This is illustrated clearly in the comparison between
the staining of Na,K-ATPase
and D3 (Fig. 2B) which show
co-localization of only a portion of the D3 with the membrane marker.
D3 is external to the ER compartment, and much of it co-localizes with
clathrin or EEA-1, an early endosomal marker (Figs. 4D and
5d). Positive staining of intact cells with antibodies
directed against residues 53-68 or the COOH-terminal FLAG epitope and
biotinylation of the protein by an impermeant probe confirm the
presence of both epitopes in the extracellular space (Figs.
3C and 4, A-C). Thus, circulating iodothyronines are exposed to D3 but not to D2, which is present only in the ER, as is
re-confirmed in the present investigation (Fig. 3C) (12).
Whereas both D1 and D3 are present in the PM, previous topological
studies of D1 indicated it had a type 1 membrane protein orientation,
with the COOH terminus and the catalytic center of the protein
remaining in the cytosol as D1 is incorporated into the PM (29). Like
D1, D3 contains a single predicted highly conserved transmembrane
domain between residues 16 and 41. All five different algorithms used
predict that the COOH-terminal portion of newly synthesized D3 is in
the ER compartment, although the COOH-terminal portion of newly
synthesized D1 is in the cytosol (29). If there is only a single
transmembrane domain and the catalytic center is extracellular, it
would give ready access of plasma T4 and T3 to
this inactivating enzyme. This is consistent with the role of D3 in the
placenta, uterus, and fetal liver to block entry of maternal thyroid
hormone to the fetus (reviewed in Ref. 1) and also the capacity of the
overexpressed enzyme in infantile hemangioma cells to increase thyroid
hormone inactivation rates up to 9-fold (9).
The presence of D3 in the PM can explain another aspect of the
inactivation pathway. In some species, such as amphibians, D1 is not
expressed, and in fact, it is also not expressed in human cerebral
cortex despite its presence in human liver and kidney (30). Thus, the
balance between T4 activation by D2 and inactivation by D3
is what determines the concentration of T3 in the nuclei of
such tissues. It is not yet certain whether D2 and D3 are expressed in
the same or different cells in the brain (1). If both are in the same
cell, the differential subcellular localization would still allow for
preferential access of D2-generated T3 to the cell nucleus.
The D3 in D2-expressing cells would then act to limit the availability
of the pro-hormone T4, deiodinating it to reverse
T3, rather than inactivating T3 produced at the ER membrane.
The rapid accumulation of biotinylated D3 in the PM in the presence of
endocytosis inhibitors (Fig. 4, A-C) argues that
D3-containing PM regions are normally internalized, becoming part of an
endosomal vesicle. These vesicles seem to be predominantly
clathrin-coated as there is extensive co-localization of D3 and
clathrin (Fig. 4D) and minimal co-localization of D3 and
caveolin-1 (not shown). This suggests that during endocytosis,
internalized D3 is selected for recycling back to the cell surface.
This is supported by the data in the internalization assay in which
biotinylated D3 was retained within the cells exposed to primaquine, a
potent inhibitor of membrane transport from endosomes to the plasma
membrane (Fig. 4E) (22).
This is the first demonstration that a deiodinase undergoes
endocytosis, a phenomenon that could have important biological and
physiological consequences for D3 activity. For example, the presence
of D3 in early endosomes (Fig. 5) suggests that only a minute fraction
of internalized D3 progresses to late endosomes and to lysosomal
proteolysis, explaining the long half-life. In fact, the D3 half-life
is ~12 h, a figure that contrasts with the short half-life of D2
(~2 h), the critical ER-resident T3-producing deiodinase
(Fig. 3, E-G). This suggests that the short term adaptation of thyroid status to reduced T4 production during iodine
deficiency or hypothyroidism is due primarily to post-translational
up-regulation of D2 and not to a rapid decrease in global D3-mediated
thyroid hormone degradation, characteristic of chronic iodine
deficiency (1). On the other hand, the substantial quantity of
potentially recyclable D3 in the early endosomal pool raises the
possibility that an appropriate signal such as starvation or illness
could lead to its rapid relocation to the cell surface with a
consequent acute inactivation of circulating T4 and
T3 (1).
During the deiodination reaction the selenol in the active center of
the enzyme participates in the nucleophilic attack during which it is
oxidized, resulting in transient enzyme inactivation until it is
reduced by an as yet unidentified co-factor. The extracellular compartment is known to be an oxidizing environment, making it difficult to reduce the active center after catalysis. Because it has
been suggested that a reducing environment is present in the
prelysosomal compartment (endosomes) (31), D3 recycling could
constitute a mechanism for D3 re-activation. Our preliminary studies do
not show any acute effect of T3 on D3 partitioning within
the Triton X-100 soluble pool. However, other stimuli have not been explored.
In conclusion, the present studies show that newly synthesized D3
migrates to the plasma membrane and rapidly undergoes endocytosis to
the early endosomal pool, possibly recycling back to the plasma membrane. The signals controlling D3 partition between these two pools
are not known, although this process may facilitate regeneration of the
selenium-containing active center of the enzyme. The topological studies suggest that the catalytic portion of D3 is extracellular in
agreement with the theoretical predictions from the primary amino acid
sequence. An active center in the extracellular space would allow D3
ready access to inactivate both the active thyroid hormone
T3 and the prohormone T4 in both physiological
and pathophysiological conditions. Recent IF studies have localized D3
in the endothelial cells of human hemangiomas (10), placenta, umbilical
cord, and uterus (11). In addition D3 in the human fetus is expressed in all the epithelial surfaces in contact with amniotic fluid including
the skin, bronchial and intestinal epithelia, and amnion syncytio and
cytotrophoblast of the placentas. Its position on the PM of these cells
would facilitate its putative role to block uncontrolled access of
maternal thyroid hormone to the fetus.