Download Characterization of a AT-Bromoacetyl-L-Thyroxine Affinity

0013-7227/91/1294-20ll$03.00/0
Endocrinology
Copyright © 1991 by The Endocrine Society
Vol. 129, No. 4
Printed in U.S.A.
Characterization of a AT-Bromoacetyl-L-Thyroxine
Affinity-Labeled 55-Kilodalton Protein as Protein
Disulfide Isomerase in Cultured Glial Cells
MARJORIE SAFRAN AND JACK L. LEONARD
Molecular Endocrinology Laboratory, Departments of Medicine, Nuclear Medicine, and Molecular and
Cellular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
characterized glial-p55 and compared it with purified rat hepatic
PDI. Glial-p55 appears to be identical to PDI by peptide fragmentation analysis, using multiple methods. The hydrodynamic
properties of glial-p55, determined by molecular sieve chromatography and sucrose density centrifugation, showed that this
protein has a native molecular mass of 115 kDa, which is in
close agreement with that reported for PDI. Like PDI, glial-p55
is an acidic protein with a pi of 4.8 or less. Thus, the 55-kDa
thyroid hormone-binding protein in glial cells appears to be PDI.
(Endocrinology 129: 2011-2016,1991)
ABSTRACT. In glial cells, thyroid hormone regulates the
polymerization state of the actin cytoskeleton by a mechanism
that does not require protein synthesis or the nuclear T3 receptor. Using the affinity label iV-bromoacetyl-L-T4, we identified
a thyroid hormone-binding protein of 55 kDa (glial-p55) in
cultured glial cells that is unique from the type II iodothyronine
5'-deiodinase and which exhibits a T4-dependent shift from a
membrane-associated pool to the F-actin cytoskeleton. In a
number of other cell types, a 55-kDa thyroid hormone-binding
protein has been identified and shown to be a subunit of the
enzyme protein disulfide isomerase (PDI). In this study we have
I
N THE brain, thyroid hormone regulates the levels of
type II iodothyronine 5'-deiodinase (5'D-II) by modulating the turnover of the enzyme polypeptide (1). T4
increases the degradation of this short-lived membranebound protein in cultured glial cells by dynamically regulating the polymerization of the actin cytoskeleton
through an energy-dependent mechanism that does not
require protein synthesis or the nuclear T 3 receptor (2).
This extranuclear action of thyroid hormone differs from
the traditional pathway of thyroid hormone action, which
requires access to a chromatin-bound receptor with subsequent modulation of gene expression and protein synthesis (3). Recently, several thyroid hormone-binding
proteins in addition to the nuclear T 3 receptor have been
identified in the cell (4) and form a group of candidate
proteins that may mediate extranuclear effects of thyroid
hormone (5, 6). These specific thyroid hormone-binding
proteins have been described in mitochondria, cytoplasm,
and plasma membranes of many tissues (4).
Affinity labeling with bromoacetyl derivatives of T 4
and T 3 has proven to be a valuable method for identifying
Received May 20,1991.
Address all correspondence and requests for reprints to: Dr. Marjorie
Safran, Division of Endocrinology and Metabolism, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01655.
* This work was supported by Grants DK-38772 and DK-32520 from
the NIDDK, NIH (Bethesda, MD).
many of these thyroid hormone-binding proteins (7-11).
In glial cells, iV-bromoacetyl-L-T4 (BrAcT4) is found
predominately in three proteins of 55,000, 29,000, and
18,000 mol wt (9). The 29-kDa protein has been identified
as the substrate-binding subunit of 5'D-II (9, 12), while
the identities of the 55- and 18-kDa proteins remain to
be established. In other organs and cell lines, a 55-kDa
affinity-labeled protein has been identified and proposed
as a plasma membrane thyroid hormone transport protein (13). Subsequent analysis has shown this protein to
be a subunit of the enzyme protein disulfide isomerase
(PDI) (14, 15), which is localized to the endoplasmic
reticulum and nuclear envelope (16). However, in glial
cells, the affinity-labeled 55-kDa protein exhibits a T4dependent shift from a Triton X-100-soluble (membranes/cytosol) to a Triton X-100-insoluble pool (cytoskeleton), presumably due to an association with F-actin
(17). Thus, the observation that the BrAcT4-labeled 55kDa protein (glial-p55) becomes associated with the actin
cytoskeleton in the presence of thyroid hormone suggests
that glial-p55 in glial cells may play a role in ^-dependent actin polymerization.
In this study we have characterized the 55-kDa thyroid
hormone-binding protein present in cultured astrocytes
and compared it with purified liver PDI. Glial-p55 appears to be identical to PDI by peptide fragmentation
analysis. The hydrodynamic properties of glial-p55, de-
2011
2012
BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS
termined by sucrose density centrifugation and molecular sieve chromatography, showed that the taurodeoxycholate-solubilized glial-p55 has a sedimentation coefficient of 3.94S, a Stokes radius of 4.14 nm, and a native
molecular mass of 115 kDa, in good agreement with the
mol wt reported for PDI (18). Glial-p55 has a pi of 4.8
by isoelectric focusing, which is also similar to the pi
reported for PDI (19). Thus, the 55-kDa thyroid hormone-binding protein in glial cells appears to be PDI.
Since previous work in our laboratory has demonstrated
that affinity-labeled glial-p55 becomes actin associated
in a T4-dependent manner (17), PDI may play a role in
the thyroid hormone-dependent internalization of shortlived membrane proteins.
Materials and Methods
Materials
Dulbecco's Modified Eagle's Medium, antibiotics, Hanks'
Salt Solution, glucose, and trypsin were obtained from Gibco
(Grand Island, NY); supplemented bovine calf serum from Hyclone, Inc. (Logan, UT); (Bu)2cAMP, Staphlococcal aureus V8protease (V8-protease), and hydrocortisone from Sigma (St.
Louis, MO); cyanogen bromide (CNBr) from Fluka (Buchs,
Switzerland); acrylamide and iV,JV'-methylene-bis-acrylamide
from U.S. Biochemical Corp. (Cleveland, OH); ammonium persulfate and AT,iV,A^iV'-tetramethylethylenediamine (TEMED)
from Bio-Rad Laboratories (Richmond, CA); Ampholines from
LKB (Rockville, MD); and dithiothreitol (DTT) from Calbiochem (La Jolla, CA). BrAc-[125I]T4 was synthesized as described previously (7). Its purity was greater than 90%, as
determined by reverse phase HPLC analysis (8).
Culture conditions and affinity labeling of astrocytes
Neonatal rats were decapitated, and glial cells were prepared
as previously described (20). This study was approved by the
Animal Research Committee and complies with the institutional assurance certification of the University of Massachusetts Medical Center. Cells were seeded at 4 X 105 cells/cm2 in
75-cm2 flasks and used during passages 2-6. The cells were
cultured in Dulbecco's Modified Eagle's Medium containing
10% supplemented bovine calf serum, 50 U/ml penicillin, and
90 U/ml streptomycin in a humidified atmosphere of 5% CO2
and 95% air at 37 C. Cells were used at confluence in 25- or
75-cm2 flasks.
Glial cells were changed to serum-free medium 24 h before
affinity labeling. Sixteen hours before affinity labeling, 1 mM
(Bu)2cAMP and 100 nM hydrocortisone were added to induce
5'D-II activity. At the time of labeling, medium was removed,
and the cells were incubated with 2.7 nM BrAc-[125I]T4 (5 X 107
cpm/75-cm2 flask) in 3 ml serum-free Hanks' solution for 20
min at 37 C. After washing twice with 5 ml ice-cold 20 mM
PBS (pH 7.4), the cells were harvested by scraping and then
collected by centrifugation at 500 X g for 10 min. Cell pellets
were sonicated in 10 mM HEPES (pH 7.0) containing 10 mM
DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride
(lysis buffer) and kept frozen at -20 C until use. Samples used
Endo • 1991
Vol 129 »No 4
for sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis were treated by the addition of 0.25 vol
5X SDS-PAGE sample buffer [250 mM Tris-HCl (pH 6.8), 50%
(vol/vol) glycerol, 5% (wt/vol) SDS, 5% (vol/vol) mercaptoethanol, and 0.01% (wt/vol) bromphenol blue] and denatured by
heating at 70 C for 5 min.
Purification of PDI
PDI was isolated from rat liver using an adaptation of the
procedure described by Lambert and Freedman (18). Briefly,
liver microsomes were prepared as previously described (21),
solubilized in 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA and 1% Triton X-100 (vol/vol), heated to 54
C for 5 min, cooled on ice, and centrifuged for 7.2 x 106 g-min.
The clarified supernatant was fractionated by NH4SO4 and the
55-85% saturated NH4SO4 fraction was dissolved in 25 mM
sodium citrate buffer (pH 5.3) and diafiltered using a Centricon30 microconcentrator (Amicon, Danvers, MA) with three
changes of the same buffer. The dialyzed sample was applied
to a 1 x 5-cm Sephadex C-50 column at 4 C and eluted with
the 25 mM sodium citrate buffer (pH 5.3). The unretarded
fraction was collected, and the buffer was changed to 20 mM
sodium phosphate (pH 6.3) by diafiltration using three changes
of buffer. This sample was applied to a 1 x 5-cm DEAESephacel column equilibrated in 20 mM sodium phosphate (pH
6.3). Proteins were eluted at a flow rate of 20 ml/h with a 0- to
700-mM NaCl gradient in equilibration buffer and monitored
at OD28o- Two-milliliter fractions were collected, and the identity of the eluted proteins was determined by SDS-PAGE.
SDS-PAGE analysis
Heat-denatured samples (70 C for 5 min) were electrophoresed on a 12.5% SDS-PAGE gel according to the method of
Laemmli (22). Gels were fixed in 25% isopropyl alcohol (vol/
vol) and 10% acetic acid (vol/vol), dried, and exposed to XAR5 film (Eastman Kodak, Rochester, NY) with intensifying
screens for 3-7 seven days at -70 C. Radioautograms were used
to identify affinity-labeled proteins. When indicated, the affinity-labeled proteins were cut from the dried gels and cleaved
directly in the gel slices, as described previously (12).
Affinity labeling of purified PDI
Purified PDI (10 fig) was affinity labeled with 130 pM BrAc[125I]T4 (5 X 105 cpm) in a total volume of 100 /A containing
100 mM potassium phosphate buffer (pH 7), 1 mM EDTA, and
10 mM DTT by incubating at 37 C for 20 min. Labeling was
stopped by the addition of 0.25 vol 5x SDS-PAGE sample
buffer, and the affinity-labeled PDI was stored at -20 C.
N-Terminal sequencing was used to confirm the identity of
the purified PDI. Affinity-labeled PDI was heat denatured,
electrophoresed on SDS-PAGE, and transferred to Imobilon-P
(Millipore, Bedford, MA), using the method of Towbin et al.
(23). Membranes were stained with 0.1% Coomassie blue (wt/
vol) in 50% methanol (vol/vol) for 5 min, followed by destaining
for 10 min in 50% methanol (vol/vol) and 10% acetic acid (vol/
vol) to identify the 55-kDa protein. N-Terminal sequencing
was determined by automated Edman degradation in the Protein Sequencing Core Facility at the University of Massachusetts Medical School.
BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS
Molecular sieve chromatography and sucrose density
centrifugation
Procedures have been previously described in detail (12). In
brief, taurodeoxycholate-solubilized BrAc-[125I]T4-labeled glial p55 was analyzed by molecular sieve chromatography, using an
LKB TSK 3000 column (Rockville, MD), and by isopynic centrifugation on 5-20% linear sucrose gradients in H2O and 2H2O.
Fractions were analyzed by SDS-PAGE, and the resulting
radioautographs were analyzed by scanning densitometry.
1.0
800
0.8 --
eod
3 0.6 --
3 .
CM
1
>
0.4-
200
0.2 --
0.0 -h
Isoelectric focusing and two-dimensional gel analysis
Isoelectric focusing of affinity-labeled glial-p55 was performed using the Mini-Protean II (Bio-Rad Laboratories, Richmond, CA). Samples were mixed with an equal volume of
sample buffer (9.5 M urea, 1% Nonidet P-40, 5% j8-mercaptoethanol, and 0.4% pH 3.5-10 and 1.6% pH 5-8 Ampholines)
and applied to gels containing 4% polyacrylamide, 9 M urea,
and 0.4% pH 3.5-10 and 1.6% pH 5-8 Ampholines. Gels were
focused for 3 h using 10 mM H3PO4 as the anode buffer and 20
mM NaOH as the cathode buffer. Focused gels were then
separated on a 12.5% SDS-PAGE gel, which was analyzed by
radioautography. The pH gradient was determined separately
on concurrently focused gels by allowing 0.5-cm fragments to
equilibrate for 10 min in water and then measuring the pH.
2013
40
60
80
Fraction
FIG. 1. OD280 absorbance of fractions obtained by gradient elution (0700 mM NaCl) of DEAE-Sephacel during purification of PDI from rat
liver microsomes. Fractions 37-41, 57-63, and 80-94 were pooled to
make peaks 1, 2, and 3, respectively.
B
Mr
x10"3
55
—
Other procedures
Protein was determined by the method of Bradford (24).
Results
Affinity-labeled glial cells contain three proteins, of
55,000, 29,000, and 18,000 mol wt, that contained greater
than 90% of the affinity label (9). The 29-kDa protein
has been identified as the substrate-binding subunit of
the type II iodothyronine 5'D-II (9,12). To confirm that
the 55-kDa protein was not a dimer of the 5'D-II substrate-binding subunit, the two proteins were compared
by peptide fragmentation. Peptide fingerprinting of the
glial 29-kDa protein and glial-p55 using CNBr fragmentation and V8-protease digests confirmed that the two
proteins are unique (data not shown). These data are
consistent with earlier work demonstrating that the affinity-labeled 55-kDa protein in rat liver and kidney
microsomes differs from the 27-kDa substrate-binding
subunit of 5'D-I (7).
Since the BrAcTVlabeled 55-kDa protein in other cell
types had previously been identified as PDI (14, 15), we
compared glial-p55 to purified PDI. The procedure of
Lambert and Freedman (18) was used to isolate PDI
from rat liver microsomes. As shown in Fig. 1, 3 peaks
of proteins were eluted from the DEAE-Sephacel column
when monitored by absorbance at OD28o- When analyzed
by SDS-PAGE, protein was identified by Coomassie
staining of the gels only in peaks 1 and 2. Peak 1
contained a protein of 65 kDa and was labeled very poorly
with BrAcT4 (Fig. 2). Greater than 99% of peak 2 was
36
Peak
1
1
FIG. 2. Protein staining and affinity labeling of proteins eluting off
the DEAE-Sephacel column during purification of PDI from rat liver
microsomes. Proteins were affinity labeled and separated by SDSPAGE, as described in Materials and Methods. A, Coomassie staining
of peaks 1 and 2. B, The radioautograph of BrAc-[125I]T4-labeled peaks
1 and 2 in the same gel as A. Mr, Mol wt.
comprised of a 55-kDa protein that incorporated 0.1 pmol
BrAcT4/mg protein (Fig. 2). N-Terminal sequencing of
the peak 2 protein showed that the first 13 amino acids
were identical to those of the N-terminus of PDI. This
PDI was used without further purification.
Glial-p55 was compared to PDI by peptide fingerprinting of the isolated proteins. Partial digests of glial-p55
and PDI using V8-protease showed 11 labeled fragments
of 5,700-39,000 mol wt (Fig. 3). While the intensity of
the fragments differed between the two preparations,
fragment mol wt were identical. Cyanogen bromide digests of glial-p55 and affinity-labeled PDI revealed identical fragmentation patterns, with fragments of 45,000,
40,000, and 35,000 mol wt (Fig. 4). Similarly, partial
digests of the two proteins using 2 ng trypsin also yielded
identical fragmentation patterns (data not shown).
Detergent-solubilized membranes from BrAc-[125I]T4-
BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS
2014
Glial-p55
Mr
x10"3
0
0.04
PDI
0.2
0
0.04
Endo • 1991
Vol 129 • No 4
1.0
0.2
1
0.9
ug V8
0.8
pSS-II
-
<3)O
0.7
i
0.6
2
3
St okes Radius
55 —
4
^
\
5
o
29
30
40
50
Fraction Number
FIG. 5. Molecular sieve chromatography of glial-p55. Taurodeoxycholate-solubilized BrAcT4 affinity-labeled glial membranes were chromatographed on a LKB TSK 3000 column. Glial-p55 was identified by
the distribution of the affinity-labeled 55-kDa protein. Data shown are
representative of three experiments. The Stokes radius of the protein
was determined using the following protein standards: 1) cytochromec, 2) a-chymotrypsin, 3) ovalbumin, 4) BSA, and 5) alcohol dehydrogenase. The data reported in the inset represent the mean of four
experiments.
12.4 —
FlG. 3. Peptide fragmentation patterns obtained with Staphfococcal
V8-protease (V8) digests of glial-p55 and PDI. Gel slices containing
affinity-labeled glial-p55 and PDI were loaded along with the indicated
quantity of V8-protease onto a 15% SDS-PAGE gel with a 4% stacking
gel. Proteolysis was allowed to occur within the stacking gel with the
current turned off for 30 min. Electrophoresis was then allowed to
continue as usual. The peptide map shown is representative of three
experiments. Mr, Mol wt.
Mr
GlialPDI
p55
XKT3
55
36
FlG. 4. Peptide fragmentation patterns obtained with cyanogen bromide (CNBr) digests of glial-p55 and PDI. Gel slices containing affinity-labeled glial-p55 and PDI were treated with 0.8 M cyanogen bromide
for 2 h at room temperature. Treated gel slices were then loaded onto
a 15% SDS-PAGE gel with a 4% stacking gel, and electrophoresis was
performed as usual. The peptide map shown is representative of three
experiments. Mr, Mol wt.
labeled glial cells were applied to a TSK 3000 column,
and the distribution of the 55-kDa protein was determined by SDS-PAGE. As shown in Fig. 5, the majority
(73%) of glial-p55 eluted in fractions 31-32 (p55-I). The
Stokes radius of this peak, determined graphically from
the Kd1/3 was 4.14 ± 0.1 nm (Fig. 5, inset). A second
smaller peak (27%) present in fractions 35-37 (p55-II)
migrated with an estimated mol wt of ~54,000 and a
Stokes radius of 3.07 ± 0.09 nm, and most likely represents a protein monomer.
The hydrodynamic properties of glial-p55 were determined from the fractional radial migration of detergentsolubilized proteins in the presence of H2O and 2H2O.
Solubilized glial membranes were centrifuged on 5-20%
linear sucrose gradients prepared in both H2O and 2H2O.
The migration of glial-p55 was determined from the
distribution of the affinity-labeled protein and is shown
in Fig. 6. Glial-p55 migrated with a r/r 0 = 1.392 ± 0.034
in H2O and r/r 0 = 1.302 in 2H2O. Occasionally, a second
peak of affinity-labeled p55 was present which sedimented faster than glial-p55, most likely secondary to
protein aggregates. The S2o,w of glial-p55, as determined
by interpolation from the sedimentation in H2O, was
3.94 ± 0.40S (mean ± SE; n = 6) and was similar in 2H2O
(4.05S). The calculated partial specific volume of glialp55 was 0.74 ± 0.07 cm3/g, which is consistent with it
being a globular protein. The mol wt of p55-I, calculated
from the Stokes radius of the major peak, partial specific
volume, and sedimentation coefficient, was determined
to be 114,800 ± 39,000.
The pi of BrAcT4-labeled glial-p55 was 4.8 ± 0.2 (mean
± SE; n = 7), and the protein focused as a single spot,
with no microheterogeneity (data not shown).
Discussion
The affinity labels BrAcT4 and BrAcT3 have identified
a widely distributed 55-kDa thyroid hormone-binding
BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS
I-LO
Ho0
CD
2.0
FlG. 6. Sucrose density centrifugation of glial-p55. Taurodeoxycholate-solubilized BrAc-[125I]T4 affinity-labeled glial membranes were
sedimented on 5-20% linear sucrose gradients in the presence of H2O
and 2H2O. The migration of glial-p55 was determined from radioautographs of the sucrose gradient fractions after analysis by SDS-PAGE.
The results shown are representative of individual gradients repeated
six (H2O) or four (2H2O) times. The protein standards used were
carbonic anhydrase, BSA, and alcohol dehydrogenase.
protein (7-11). Cheng and co-workers (10, 11, 14) and
Yamauchi etal. (15) purified a 55-kDa protein from GH3
(a rat pituitary tumor cell line), A431 (a human epithelioid carcinoma), and 3T3-4 (Swiss mouse fibroblast cell
line) cells and isolated a cDNA encoding this protein
that showed 85% homology at the nucleotide level to the
endoplasmic reticulum luminal protein, PDI (14). In glial
cells, a 55-kDa protein contains ~25% of the total BrAcT4 incorporated into cell proteins (9). Since p55 has
been identified as PDI in other cells, we sought to determine whether glial-p55 was also PDI.
The current study indicates that the 55-kDa thyroid
hormone-binding protein in glial cells is similar if not
identical to PDI. Partial peptide fragmentation of PDI
isolated from rat liver and glial-p55, using multiple methods yielded identical peptide fingerprints. The native
undenatured mol wt of detergent-solubilized glial-p55 is
115,000, in close agreement with that of PDI determined
by gel filtration on Sephadex G-200 (18). These data
indicate that both glial-p55 and PDI are homodimers. In
addition, both PDI and affinity-labeled glial-p55 are
acidic proteins with pi values of 4.8 or less, and both
proteins show little or no microheterogeneity. Up to 27%
of glial-p55 was present as a monomer.
PDI has been shown to act as a molecular chaparone
catalyzing native disulfide bond formation and is abundant in tissues that secrete proteins (16). In addition,
PDI has also been demonstrated to be a component of
other multimeric enzymes and proteins, such as prolyl4-hydroxylase (25) and the microsomal triglyceride
transfer protein complex (26). By both subcellular fractionation and ultrastructure analyses, PDI has been
shown to be an extrinsic membrane protein localized to
the lumen of the endoplasmic reticulum (16) and the
2015
pore complex of the nuclear envelope (27). However, ~ 5 10% of PDI has been found on the external surface of
the plasma membranes of rat exocrine pancreatic cells,
suggesting that the protein may be deposited on the cell
surface during secretion (28).
The presence of the small amount of PDI on the cell
surface raises the possibility that the molecule is missorted and carried to this site by secretory vesicles. The
rescue of this misdirected protein may explain the observation of Cheng and co-workers that PDI functioned as
a thyroid hormone transporter. They followed the migration of endocytic vesicles containing a rhodamine-T3PDI conjugate from the cell surface to the perinuclear
space (29). Since endocytic vesicles are initially targeted
to the perinuclear space for sorting (30), the movement
of T 3 bound to PDI in these vesicles would mimic nuclear
translocation of the hormone. Other cell surface receptors, such as the apolipoprotein receptor, may also nonspecifically carry thyroid hormone to the perinuclear
space, since T 3 bound to apolipoprotein is internalized
into cells by endocytosis (31). However, the thyroid
hormone still has to cross the vesicle bilipid layer and
the nuclear envelope to attain access to its chromatinbound receptor. The ability of cell surface receptors to
passively carry thyroid hormone to the perinuclear space
implies that PDI may not be a specific thyroid hormone
transport protein.
The identification of two peaks of glial-p55 by molecular sieve chromatography suggests that PDI may exist
in two different forms in the glial cell. Since the majority
of PDI is confined to the lumen of the endoplasmic
reticulum, it is unlikely to be accessible to the actin
cytoskeleton. Monomeric glial-p55 comprises more than
25% of the total glial PDI. The presence of two forms of
PDI raises the possibility that one of these may be
localized in a different subcellular site, possibly the cytosol, where it could act to modulate actin polymerization. More work is needed to evaluate this possibility.
The ability of PDI to associate with the actin cytoskeleton in a hormone-dependent manner in cells derived
from the brain has not been observed previously. The
addition of T 4 to thyroid hormone-deficient glial cells
causes the percentage of polymerized actin to increase
from 65% to 90% of the total cellular actin (17). The
observation that this treatment also resulted in the rapid
movement of glial-p55 from a membrane-associated pool
to the F-actin cytoskeleton (17) implied that glial-p55
may participate in T4-dependent regulation of actin polymerization. The current demonstration that glial-p55
is PDI may represent a new role for this multifunctional
polypeptide in cells of the central nervous system.
Acknowledgments
We thank Ms. Susan Dubord for technical assistance, and
Dr. Jacqueline Anderson for performing the protein sequencing.
2016
BrAcT4-LABELED 55-kDa PROTEIN IN GLIAL CELLS
References
1. Leonard JL, Siegrist-Kaiser CA, Zuckerman CJ 1990 Regulation
of type II iodothyronine 5'-deiodinase by thyroid hormone: inhibition of actin polymerization blocks enzyme inactivation in
cAMP-stimulated glial cells. J Biol Chem 265:940-946
2. Siegrist-Kaiser CA, Juge-Aubry C, Tranter MP, Ekenbarger DM,
Leonard JL 1990 Thyroxine-dependent modulation of actin polymerization in cultured astrocytes: a novel, extranuclear action of
thyroid hormone. J Biol Chem 265:5296-5302
3. Shambaugh III GE 1986 Thyroid hormone action: biologic and
cellular effects. In: Ingbar SH, Braverman LE (eds) Werner's The
Thyroid. Lippincott, Philadelphia, pp 201-218
4. Sterling K 1986 Thyroid hormone action at the cellular level. In:
Ingbar SH, Braverman LE (eds) Werner's The Thyroid. Lippincott, Philadelphia, pp 219-233
5. Davis PJ, Davis FB, Bias SD 1982 Studies on the mechanism of
thyroid hormone stimulation in vitro of human red cell Ca2+ATPase activity. Life Sci 30:675-682
6. Segal J, Ingbar SH 1985 In vivo stimulation of sugar uptake in rat
thymocytes. An extranuclear action of 3,5,3'-triiodothyronine. J
Clin Invest 76:1575-1580
7. Koehrle J, Rasmussen UB, Rokos H, Leonard JL, Hesch RD 1990
Selective affinity labeling of a 27-kDa integral membrane protein
in rat liver and kidney with iV-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine. J Biol Chem 265:6146-6154
8. Koehrle J, Rasmussen UB, Ekenbarger DM, Alex S, Rokos H,
Hesch RD, Leonard JL 1990 Affinity labeling of rat liver and
kidney type I 5'-deiodinase: identification of the 27-kDa substrate
binding subunit. J Biol Chem 265:6155-6163
9. Farwell AP, Leonard JL 1989 Identification of a 27-kDa protein
with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells. J Biol Chem 264:2056120567
10. Horiuchi R, Johnson ML, Willingham MC, Pastan I, Cheng S 1982
Affinity labeling of the plasma membrane 3,3'5-triiodo-L-thyronine receptor in GH3 cells. Proc Natl Acad Sci USA 79:5527-5531
11. Cheng S 1983 Structural similarities in the plasma membrane
3,3'5'-triiodo-L-thyronine receptors from human, rat and mouse
cultured cells. Analysis by affinity labeling. Endocrinology
113:1155-1157
12. Safran M, Leonard JL 1991 Comparison of the physicochemical
properties of type I and type II iodothyronine 5'deiodinase. J Biol
Chem 266:3233-3238
13. Cheng S, Hasumura S, Willingham MC, Pastan 11986 Purification
and characterization of a membrane-associated 3,3',5-triiodo-Lthyronine binding protein from a human carcinoma cell line. Proc
Natl Acad Sci USA 83:947-951
14. Cheng S, Gong Q, Parkinson C, Robinson EA, Appella E, Merlino
GT, Pastan I 1987 The nucleotide sequence of a human cellular
thyroid hormone binding protein present in endoplasmic reticulum.
J Biol Chem 262:11221-11227
15. Yamauchi K, Yamamoto T, Hayashi H, Koya S, Takikawa H,
Toyoshima K, Horiuchi R 1987 Sequence of membrane-associated
Endo • 1991
Vol 129 • No 4
thyroid hormone binding protein from bovine liver: its identity
with protein disulphide isomerase. Biochem Biophys Res Commun
146:1485-1492
16. Freedman RB 1984 Native disulphide bond formation in protein
biosynthesis: evidence for the role of protein disulphide isomerase.
Trends Biochem Sci 9:438-441
17. Farwell AP, Lynch RM, Okulicz WC, Comi AM, Leonard JL 1990
The actin cytoskeleton mediates the hormonally regulated translocation of type II iodothyronine 5'-deiodinase in astrocytes. J Biol
Chem 265:18546-18553
18. Lambert N, Freedman RB 1983 Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a
rapid high-yielding procedure. Biochem J 213:225-234
19. Mills ENC, Lambert N, Freedman RB 1983 Identification of protein disulphide-isomerase as a major acidic polypeptide in rat liver
microsomal membranes. Biochem J 213:245-248
20. McCarthy KD, de Vellis J 1978 Alpha-adrenergic receptor modulation of beta-adrenergic, adenosine and prostaglandin E! levels
increased adenosine 3':5'-cyclic monophosphate levels in primary
cultures of glia. J Cyclic Nucleotide Res 4:15-26
21. Leonard JL, Rosenberg IN 1978 Subcellular distribution of thyroxine 5'-deiodinase in the rat kidney: a plasma membrane location.
Endocrinology 103:274-280
22. Laemmli UK 1970 Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685
23. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:43504354
24. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248-254
25. Tasanen K, Parkkonen T, Chow LT, Kivirikko KI, Pihlajaniemi
T 1988 Characterization of the human gene for a polypeptide that
acts both as the /3 subunit of prolyl 4-hydroxylase and as protein
disulfide isomerase. J Biol Chem 263:16218-16224
26. Wetterau JR, Combs KA, Spinner SN, Joiner BJ 1990 Protein
disulfide isomerase in a component of the microsomal triglyceride
transfer protein complex. J Biol Chem 265:9800-9807
27. Akagi S, Yamamoto A, Yoshimori T, Masaki R, Ogawa R, Tashiro
Y 1988 Distribution of protein disulfide isomerase in rat hepatocytes. J Histochem Cytochem 36:1533-1542
28. Akagi S, Yamamoto A, Yoshimori T, Masaki R, Ogawa R, Tashiro
Y 1988 Localization of protein disulfide isomerase on plasma
membranes of rat exocrine pancreatic cells. J Histochem Cytochem
36:1069-1074
29. Cheng S, Maxfield FR, Robbins J, Willingham MC 1980 Receptormediated uptake of 3,3',5-triiodo-L-thyronine by cultured fibroblasts. Proc Natl Acad Sci USA 77:3425-3429
30. Kelly RB 1990 Microtubules, membrane traffic, and cell organization. Cell 61:5-7
31. Benvenga S 1989 The 27-kilodalton thyroxine (T4)-binding protein
is human apolipoprotein A-I: identification of a 68-kilodalton high
density lipoprotein that binds T4. Endocrinology 124:1265-1269