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Further characterization of the low and high affinity binding components of the thyrotropin receptor
Vol. 137, No. 1, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 61-68
May 29, 1986
FURTHER CHARACTERIZATION OF THE LOW AND HIGH AFFINITY BINDING COMPONENTS OF THE THYROTROPIN RECEPTOR* Robert
HcQuadel’+,
Colin
G. Thomas’,
Jr.,
and Shihadeh
N. Nayfeh++
Department of Biochemistry and Nutrition1 and Department of Surgery2 University of North Carolina School of Medicine Chapel Hill, NC 27514
Received
March
17,
1986
Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the highand low-affinity TSH binding components exhibited two similar 1251-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves’ disease inhibited 12’I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the highand low-affinity components share simlar subunit composition and antigenic determinants. 0 1986 Academic Press, Inc.
Equilibrium receptor
saturation
results
in a nonlinear
Kinetic
and thermodynamic
complex
behavior
binding
sites;
other
low affinity
have
shown
affinity
(6)
analysis
is
best
(concave
analyses explained
one exhibiting and high that
component
of the
high capacity
the highby a number
(Ha)
interaction
upward)
Scatchard
of TSH binding by the affinity for
have
existence
TSH (4,s).
including
with
plot
of at least
Further from gel
the
its
(l-4).
shown that
and low capacity,
can be separated
of methods,
of TSH
this two
and the studies low-
(La)
filtration
*Presented Antonio, +Present
in part at the 65th Annual Meeting of The Encodrine Society, San Texas, 1983. This work was supported by NIH Grant AM 23080. address: Schering-Plough Corporation, 60 Orange Street, Bloomfield, New Jersey ++To whom all correspondence should be addressed. Abbreviations : DSS, disuccinimidyl suberate; PMSF, phenylmethylsulfonyl fluoride; STI; soybean trypsin inhibitor; DFP; diisopropyl fluorophosphate; PPM, partially-purified membranes; PM, purified membranes; SH, solubilixed membranes; SDS-PAGE, sodium dodecyl sulfate - polacylarylamide gel electrophoresis; DTT, dithiothreitol; Ha and La, highand low-affinity binding components, respectively. 0006-291X/86 61
All
Copyright !F 1986 rrghts of reprodwtion
$1.50
by Ac,ademrc Press. Inc. in at?y form rcwrv~*d.
Vol. 137, No. 1, 1986
chromatography continuous
on Sepharose sucrose
to be the
gradient
responsible
the
AND BIOPHYSICAL
6B, lectin
density
receptor
neither
(7-9))
BIOCHEMICAL
for
chemical
identity
affinity
RESEARCH COMMUNICATIONS
chromatography,
centrifugation. activation nor
and dis-
Although of adenylate
Ha appears cyclase
the physiological
role
of La
has been established. The present structure
of the high-
to elucidate
their
of Graves’
sera.
and La is
inhibited
properties, similar
investigation
and low-affinity
Our results
subunits
size
with
show that
by Graves’
(a)
IgG and (b)
(1,4-6), with
to identify
components
mode of interaction
including protein
was initiated
the
Mr values
MATERIALS
of TSH receptor
anti-TSH binding although
receptor
differing each
and
antibodies
of 1251-TSH
two components of 80,000
the subunit
to Ha in
contain
several two
and 68,000.
AND METHODS
Purified bovine TSH (40 International Units/ mg), a gift from Dr. John Pierce (UCLA), was iodinated with 1251 (Amersham IM-30) to a specific radioactivity of 50-90 pCi/pg by a modification of the lactoperoxidase method, as previously described (1). Disuccinimidyl suberate (DSS) was purchased from the Pierce Chemical Co. (Rockford, IL). Porcine thyroids were obtained from the Pel-Freeze Co. (Rogers, AR). DEAE Affi-Gel Blue was purchased from Bio-Rad (Richmond, CA). All other chemicals were of the highest quality commercially available. Preparation of Purified and Solubilized Membranes Partially purified membranes (PPM) were prepared as previously described (l), except for the presence of the protease inhibitors, 2.0 mM diisopropyl fluorophosphate (DFP), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mg/ml soybean trypsin inhibitor (STI)), and stored at -9ooc. PPM were subsequently purified on discontinuous sucrose density The purified membrane pellets gradients, as previously described (10). (PM) were resuspended in 10 mM sodium phosphate buffer, pli 7.4, containing 0.5 mM DFP, 1 mM PMSF, and 0.1 mg/ml ST1 (Buffer A) at an approximate concentration of 1 mg/ml. Protein concentrations were determined by the Lowry method of (11). Membranes were solubilized in 1% Triton X-100 in Buffer A (6). Solubilized membranes (SM) were stored at -90% at an approximate protein concentration of l-2 mg/ml. Protein concentrations were determined by the Markwell modification of the Lowry method (12). Binding
and cross-linking of 1251-TSH to purified membranes Bindinn of rzSI-TSH to thvroid PM was carried out in Buffer A in the presence or-absence of excess unlabeled TSH, as described previously (1). Radioiodinated TSH was covalently linked to PM by a modification of the disuccinimidyl suberate method of Pilch and Czech (13), as described previously (14). Labeled membranes were then solubilized in 1% Triton X-100 in Buffer A, and the 100,000 xg supernatant containing solubilized membranes was fractionated by centrifugation on discontinuous sucrose gradThe gradients were fractionated from ients as described previously (6). the bottoms of the tubes and bound 1251-TSH was assayed by precipitation with polyethylene glycol and filtration (6,15). Aliquots from the sucrose gradient peaks of radioactivity corresponding to the low and high affinity 62
Vol.
137,
No. 1, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
binding components were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (16). Statistical analysis was made using Student’s t test or analysis of variante . Statistical significance was established at P < 0.05. RESULTS AND DISCUSSION Cross-linking followed in the
by analysis appearance
The lighter is
La,
peak
(peak
I
followed
sucrose
represents
Ha while
La was relatively
the procedure
labeling
component.
membranes
density
gradients
bound
1251-TSH
the heavier
by chromatography
by Scatchard
autoradiography,
thyroid
of specifically
demonstrated
of the
of this
to purified
of two peaks
columns
labeling
1251-TSH
on discontinuous
as previously
Sepharose the
of
analysis
To minimize
DSS resulted
(Fig.1).
peak
(Peak
on Concanavalin (6).
low and could
was therefore
with
However, not
modified
II) A-
since
be visualized to maximize
the dissociation
of labeled
by the TSH
20.0
2 ; ~ 15.0 -z 4 3x $E 2 8 c: 10.0 ::
5.0
i TOP
4
k
Fraction
Number
J
8--T Bottom
Figure 1: Discontinuous sucrose density gradient ultracentrifugation of SM covalently cross-linked to 1251-TSH. Thyroid PM (1 mg) were incubated with ‘251-TSH (0.6 nM) in the presence or absence of 1.25 IlJ of native TSH for 1 h at room temperature. Following removal of unbound TSH by washing and centrifugation, '251-TSH was covalently linked to the membrane by incubation with 0.3 mM DSS as described in the text. The labeled membranes were washed and then solubilized with 1% Triton X-100. The labeled SM were layered on top of discontinuous gradients of 5,10,20 and 50 percent (v/v) sucrose and were centrifuged at 22,000 x g for 2 h at 4OC. The gradients were fractionated from the bottom of the tube, and each aliquot was assayed for bound ‘251-TSH by PEG precipitation and filtration. Bound radioactivity was corrected for nonspecific and filter binding. Results were expressed as the average of duplicate determinations. 63
Vol.
137,
No.
1, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
M, x 10d 18 ‘: 0 -x
16
1
8068-
3
TOP I
5
7
9 Fraction
II
13
15
Figure 2: Discontinuous sucrose density gradient ultracentrifugation of thyroid PM following labeling with radioiodinated TSH. Thyroid PM (1 mg) were labeled with 2.4 nM ‘251-TSH and cross-linked as described in Fig. 1. However, the membranes were not washed to remove unbound TSH prior to cross-linking. Following solubilization with Triton X-100, the membranes were analyzed as described in Fig. 1. Figure 3: Electrophoretic mobility of the 1251-TSH labeled high- and lowaffinity binding components. Aliquots of the binding peaks corresponding to the covalently labeled high- and low-affinity binding components in Fig. 2 were boiled in 2% SDS containing 50 mM DTT. The labeled membranes were then subjected to SDS-PAGE on 7.5% acrylamide gels. Following electrophoresis, the gels were dried and analyzed by autoradiography. High- and low-affinity binding peaks cross-linked to ‘2sI-TSH in the presence (lane A and C) or absence (lanes B and D) of excess unlabeled TSH, respectively.
from La, prior
unbound
TSK was not
to cross-linking
increased
the
phoresis
with
specific
removed DSS.
labeling
and autoradiography
by washing
of the
As can be seen in Fig. of La in comparison
been
TSH receptor identified
identified
(14).
to represent
procedure
of Ha.
Electro-
labeled bands with
the main
binding
Mr
These two bands have subunits
of the
The band near the bottom of the gels has also been
as unbound TSH (14).
The bands on the top of the gels could
not be resolved in 5% acrylamide and therefore aggregates.
The labeling
for
membranes
when the
2, this
to that
of 80,000 and 68,000 (Fig. 3, Lanes B and D).
previously
membranes
of the peaks corresponding to Ha
of aliquots
and La resulted in the appearance of two similarly values
labeled
were
were assumedto be protein
of the 80,000 and 68,000 bands was specific, incubated
in the presence
64
of excess
native
BIOCHEMICAL
Vol. 137, No. 1, 1986
hormone , the bands ionally
a third
of the
low
were
not
labeled
affinity
DTT (50 mM), suggesting Similar
units. of intact linking
with
however
it
whose
is
the 200,000
tion
has previously
not
with efficiently
upon boiling
been
reported
(Mr = 197,000)
some of which
denatured
It Graves’ appear
is well
established
disease
possess
to be directed
Some of these binding
antibodies
of 1251-TSH
80,000
and 68,000
utilized
against
Protease-free
IgG fractions
Graves ’ disease
were
and tested
for
membranes,
as described
findings
(20,14),
inhibited while were the
their
ability
the
the binding normal
then
solubilized
high
shown
(14,20)
sera
and sera
ability
from
in a dose-dependent shown).
with Blue,
to thyroid with
previous
disease
patients
fashion
(P < O.OOl),
These
the binding
binding
components.
65
was
of La and Ha.
of patients
to block
and low affinity
as to the
bindings
some Graves’
20).
the
binding
In agreement
not
see ref.
on DEAE Affi-Gel
12%-TSH
(14).
from
to inhibit
as well
by chromatography
(data
oligomer,
review
properties
to inhibit
their
(for
been
This
that
some of which
the physicochemical
effect
observa-
suffering
to inhibit
IgG had little
Alter-
who showed
ability
IgG fractions
DSS.
in SDS.
membranes
previously
a component
Similar
(10)
antibodies,
1251-TSH
for
et al.
autoimmune
of
examined
with
of patients
of normal
prepared
may represent
sera
(14).
analyze
it
a noncovalently-linked
have previously
Mr bands
unknown;
of the TSH holoreceptor
the TSH receptor
to thyroid
to further
is
the
several
still
in SDS (16,18).
by boiling that
is
and cross-
some of the TSH holoreceptor
by Islam
the TSH holoreceptor is not
band
sub-
in SDS extracts
‘251-TSH
cross-linked
band may represent
dissociated
with
DSS or
of
of disulfide-linked observed
labeling of this
seen on gels
in the presence
composed
some molecules
crosslinked
that
was not
following
Occas-
A and C).
was also
been occasionally
represent
were
that
was not
RESEARCH COMMUNICATIONS
Lanes
was observed
The identity
may either
of the receptor natively,
it
3,
Mr of 200,000,
band
has also
DSS (14).
subunits
This
membranes
(Fig.
with
that
peptide
thyroid
observed
band,
peak.
AND BIOPHYSICAL
JgG fractions of
12’I-TSH
Solubilized
to
Vol. 137, No. 1, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0
i
4
6 Fraction
Top
8
12 Bottom
10
Number
Figure 4: Effects of IgG on the labeling of the high and low affinity TSH binding components. Thyroid SM (0.5 mg) were preincubated with buffer IgG ( 1. 5 mg) (m), normal (O-*-*-*-*0) or Graves’ ( l II,,,,,,,,,II,,,,,II,IIII~I~) followed by incubation for an additional hour with ‘251-TSH (0.6 MI) in the presence or absence of excess unlabeled hormone,(1.25 Ill). The labeled membranes were then analyzed on discontinuous sucrose gradients as described in Fig. 1.
thyroid
membranes
IgG for
1 h at room temperature.
I*%-TSH
were
and analyzed
preincubated
with
buffer
The membranes
on discontinuous
density
IgG from
normal
4, preincubation
with
decrease
labeling
of both
peaks
patients
resulted
in marked
Although
not
Graves’ both
peaks
suggest with
disease
(P < 0.001).
that
La and Ha are
Graves’
IgG through
conclusion
is
supported
by the
antibodies
are
highly
homogeneous
determinant Previous for
adenylate
on the
receptor
evidence cyclase
(P > 0.05).
that
arising
labeled
sera
of the
Graves’
As can a slight
IgG from labeling
these
and possibly
against
with
caused
However,
determinants.
suggestion
or Graves'
gradients.
conclusive.
related
antigenic
then
inhibition
entirely
structurally similar
were
sucrose
be seen in Fig. in the
(control),normal
data
interact This
anti-TSH
a single
of
latter receptor
antigenic
(20).
indicates activation
(7-9)
that
Ha is
by TSH, whereas 66
the receptor La does not
required appear
to
Vol.
137,
No.
BIOCHEMICAL
1, 1986
play
any significant
size
than
role
Ha (6),
receptor
it
induced
in this
by hormone insulin
that
Concanavalin
A and La does not,
of Ha (23).
It
is
apparatus
(21,22).
units
to mannose it
artifact
of the membrane
strated
is unlikely
on intact
unpublished
that that
rat
(6)
differ
the
that
affinity
multiple
forms
of TSH with
present
Ha binds
of the previously to
precursor
processed
it
bind
added
by the
Golgi
concanavalin
A.
is
formed
as an
has recently
been
demon-
(Gennick,
of receptors
as
catecholamines,
glucagon
their
wheat
recently
complexes
exist
Thomas and Nayfeh,
not
cells
(see
with
the separation the
wheat
hepatic
to determine in the
using
monoclonal
tumors
lacking
Acknowledgements.
the
responsiveness antibodies
low
affinity We wish
purified
bovine
TSH and Drs.
critical
review
of the
receptor
of target raised
against
binding
(7,8).
to thank Sharon
manuscript.
Dr.
It
John
component
G. Pierce,
discussion). (24)
have
in their
inter-
will
be of
low-affinity
to hormone
this
and
binding
differ
of the
cells
of
angiotensin,
nucleotides.
of involvement
interaction
additional
that
of
interaction
and low-affinity
and guanine
degree
the
Mason and Tager
of high-
glucagon
germ lectin
24 for
columns,
for the
dopamine, ref.
size,
The participation
includes
insulin,
previous forms
including
to be unique
but
our
in two distinct
composition.
germ lectin-Sepharose
for
and extend
properties,
membranes,
target
reported
components
confirm
appears
such hormones with
results
and carbohydrate
plasma
component
since
component
cells
physicochemical
thyroid
interest
to that
that
since
thyroid
TSH-receptor
in several
binding
action
similar
in
co-translationally
affinity
preparations,
greater
results).
findings
Using
low
La is
a metabolic
are subsequently
the
COMMUNICATIONS
an aggregate
La contains
glycoproteins
that
FRTL-5
In conclusion,
that
La represents
Alternatively,
containing
However,
Since
La may represent
conceivable
oligosaccharide
RESEARCH
a phenomenon
action,
for
BIOPHYSICAL
interaction.
is possible
demonstrated
core
AND
action, as well
as
UCLA for
the
Gennick
and John E. Wilson
for
We also
thank
and
67
Cindy
Hickey
their
Vol. 137, No. 1, 1986
Sylvia
Gault
BIOCHEMICAL
for preparation
AND BIOPHYSICAL
of the manuscript.
RESEARCH COMMUNICATIONS
Supported by NIH grant
#AM 23080. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N. (1980) J. Biol. Chem. 255, 4001-4010. Gr, S.M., Goldfine, I.D., and Ingbar, S.H. (1976) J. Biol. Chem. 25l, 4693-4699 Powell-Jones, C.H.J. Thomas, C.G., and Nayfeh, S.N. (1979) Proc. Natl. Acad. Sci. USA76, 705-709. Pekonen, F. and Weintraub, B.C. (1979) Endocrinology 104 352-359. Saltiel, A.R, Thomas, C.G., and Nayfeh, S.N. (1982) MorCell. Endocrinol. 28, 299-311. Drummond,R.W., McQuade, R., Grunwald, R., Thomas, C.G., and Nayfeh, S.N. (1982) Proc. Natl. Acad. Sci. USA 79, 2202-2207. Saltiel, A.R., Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N. (1980) Biochem. Biophys. Res. Commun. 95, 395-403 Saltiel, A.R. Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N., (1981) Cancer Res. 41 2360-2365 Lefort, G.P.,Amr, S., Carayon, P., and Nisula, B., (1984) Endocrinology -9 114 1005-1011. Saltiel, A.R.,
Powell-Jones, C.H.J., Thomas, C.G. and Nayfeh, S.N., (1981) Endocrinology 109, 1578-1588. Lowry, O.H., Rosebrough ,N.J., Farr, A.L., and Randall, R.J., (1951) J. Biol. Chem. 193, 265-275. Markell, M.A., Haas, S.M., Bieber, L.L., and Tolbert, N.E., (1978) Anal. Biochem. 87, 206-210. Pilch, P.F., and Czech, M.P., (1979) J. Biol. Chem. 254 , 3375-3381. McQuade, R., Thomas, C.G., and Nayfeh, S.N., (1985) Arch. Biochem. Biophys. (In Press) Cuatrecasas, P., (1972) Proc. Nat. Acad. Sci. USA69, 318-322. Laemmli, U.K., (1970) Nature 227, 680-685. Robinson, N.C., and Tanford, C., (1975) Biochemistry 14. 369-374. Pierce, J.G., and Parsons, T.F. (1981) Ann. Rev. Biochem. 50 465-495. Islam, M.N., Briones-Urbina, R., and Farid, N.R. (1983) Endocrinology -113 436-438. Farid, N.R., Briones-Urbina, R., and Bear, J.C., (1983) Molec. Aspects Med. 6, 355-457. Schecter, J., Cheng, K., Jacobs, S., and Cuatrecasas, P., (1979) Proc. Natl. Acad. Sci. USA 76, 2720-2725. Heffetz, D., and Zick, Y., (1986) J. Biol. Chem. 261, 889-894. Ronnet G.V., Enuston, V.P., Kohauski, R.A., Simpson, T.L., and Lane, M.D., (1984) J. Biol. Chem. 259, 4566-4575. Mason, J.C., and Tager, H.S., (1984) Proc. Natl. Acad. Sci. U.S.A. U.S.A. 8& 6835-6839.
68
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 61-68
May 29, 1986
FURTHER CHARACTERIZATION OF THE LOW AND HIGH AFFINITY BINDING COMPONENTS OF THE THYROTROPIN RECEPTOR* Robert
HcQuadel’+,
Colin
G. Thomas’,
Jr.,
and Shihadeh
N. Nayfeh++
Department of Biochemistry and Nutrition1 and Department of Surgery2 University of North Carolina School of Medicine Chapel Hill, NC 27514
Received
March
17,
1986
Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the highand low-affinity TSH binding components exhibited two similar 1251-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves’ disease inhibited 12’I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the highand low-affinity components share simlar subunit composition and antigenic determinants. 0 1986 Academic Press, Inc.
Equilibrium receptor
saturation
results
in a nonlinear
Kinetic
and thermodynamic
complex
behavior
binding
sites;
other
low affinity
have
shown
affinity
(6)
analysis
is
best
(concave
analyses explained
one exhibiting and high that
component
of the
high capacity
the highby a number
(Ha)
interaction
upward)
Scatchard
of TSH binding by the affinity for
have
existence
TSH (4,s).
including
with
plot
of at least
Further from gel
the
its
(l-4).
shown that
and low capacity,
can be separated
of methods,
of TSH
this two
and the studies low-
(La)
filtration
*Presented Antonio, +Present
in part at the 65th Annual Meeting of The Encodrine Society, San Texas, 1983. This work was supported by NIH Grant AM 23080. address: Schering-Plough Corporation, 60 Orange Street, Bloomfield, New Jersey ++To whom all correspondence should be addressed. Abbreviations : DSS, disuccinimidyl suberate; PMSF, phenylmethylsulfonyl fluoride; STI; soybean trypsin inhibitor; DFP; diisopropyl fluorophosphate; PPM, partially-purified membranes; PM, purified membranes; SH, solubilixed membranes; SDS-PAGE, sodium dodecyl sulfate - polacylarylamide gel electrophoresis; DTT, dithiothreitol; Ha and La, highand low-affinity binding components, respectively. 0006-291X/86 61
All
Copyright !F 1986 rrghts of reprodwtion
$1.50
by Ac,ademrc Press. Inc. in at?y form rcwrv~*d.
Vol. 137, No. 1, 1986
chromatography continuous
on Sepharose sucrose
to be the
gradient
responsible
the
AND BIOPHYSICAL
6B, lectin
density
receptor
neither
(7-9))
BIOCHEMICAL
for
chemical
identity
affinity
RESEARCH COMMUNICATIONS
chromatography,
centrifugation. activation nor
and dis-
Although of adenylate
Ha appears cyclase
the physiological
role
of La
has been established. The present structure
of the high-
to elucidate
their
of Graves’
sera.
and La is
inhibited
properties, similar
investigation
and low-affinity
Our results
subunits
size
with
show that
by Graves’
(a)
IgG and (b)
(1,4-6), with
to identify
components
mode of interaction
including protein
was initiated
the
Mr values
MATERIALS
of TSH receptor
anti-TSH binding although
receptor
differing each
and
antibodies
of 1251-TSH
two components of 80,000
the subunit
to Ha in
contain
several two
and 68,000.
AND METHODS
Purified bovine TSH (40 International Units/ mg), a gift from Dr. John Pierce (UCLA), was iodinated with 1251 (Amersham IM-30) to a specific radioactivity of 50-90 pCi/pg by a modification of the lactoperoxidase method, as previously described (1). Disuccinimidyl suberate (DSS) was purchased from the Pierce Chemical Co. (Rockford, IL). Porcine thyroids were obtained from the Pel-Freeze Co. (Rogers, AR). DEAE Affi-Gel Blue was purchased from Bio-Rad (Richmond, CA). All other chemicals were of the highest quality commercially available. Preparation of Purified and Solubilized Membranes Partially purified membranes (PPM) were prepared as previously described (l), except for the presence of the protease inhibitors, 2.0 mM diisopropyl fluorophosphate (DFP), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mg/ml soybean trypsin inhibitor (STI)), and stored at -9ooc. PPM were subsequently purified on discontinuous sucrose density The purified membrane pellets gradients, as previously described (10). (PM) were resuspended in 10 mM sodium phosphate buffer, pli 7.4, containing 0.5 mM DFP, 1 mM PMSF, and 0.1 mg/ml ST1 (Buffer A) at an approximate concentration of 1 mg/ml. Protein concentrations were determined by the Lowry method of (11). Membranes were solubilized in 1% Triton X-100 in Buffer A (6). Solubilized membranes (SM) were stored at -90% at an approximate protein concentration of l-2 mg/ml. Protein concentrations were determined by the Markwell modification of the Lowry method (12). Binding
and cross-linking of 1251-TSH to purified membranes Bindinn of rzSI-TSH to thvroid PM was carried out in Buffer A in the presence or-absence of excess unlabeled TSH, as described previously (1). Radioiodinated TSH was covalently linked to PM by a modification of the disuccinimidyl suberate method of Pilch and Czech (13), as described previously (14). Labeled membranes were then solubilized in 1% Triton X-100 in Buffer A, and the 100,000 xg supernatant containing solubilized membranes was fractionated by centrifugation on discontinuous sucrose gradThe gradients were fractionated from ients as described previously (6). the bottoms of the tubes and bound 1251-TSH was assayed by precipitation with polyethylene glycol and filtration (6,15). Aliquots from the sucrose gradient peaks of radioactivity corresponding to the low and high affinity 62
Vol.
137,
No. 1, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
binding components were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (16). Statistical analysis was made using Student’s t test or analysis of variante . Statistical significance was established at P < 0.05. RESULTS AND DISCUSSION Cross-linking followed in the
by analysis appearance
The lighter is
La,
peak
(peak
I
followed
sucrose
represents
Ha while
La was relatively
the procedure
labeling
component.
membranes
density
gradients
bound
1251-TSH
the heavier
by chromatography
by Scatchard
autoradiography,
thyroid
of specifically
demonstrated
of the
of this
to purified
of two peaks
columns
labeling
1251-TSH
on discontinuous
as previously
Sepharose the
of
analysis
To minimize
DSS resulted
(Fig.1).
peak
(Peak
on Concanavalin (6).
low and could
was therefore
with
However, not
modified
II) A-
since
be visualized to maximize
the dissociation
of labeled
by the TSH
20.0
2 ; ~ 15.0 -z 4 3x $E 2 8 c: 10.0 ::
5.0
i TOP
4
k
Fraction
Number
J
8--T Bottom
Figure 1: Discontinuous sucrose density gradient ultracentrifugation of SM covalently cross-linked to 1251-TSH. Thyroid PM (1 mg) were incubated with ‘251-TSH (0.6 nM) in the presence or absence of 1.25 IlJ of native TSH for 1 h at room temperature. Following removal of unbound TSH by washing and centrifugation, '251-TSH was covalently linked to the membrane by incubation with 0.3 mM DSS as described in the text. The labeled membranes were washed and then solubilized with 1% Triton X-100. The labeled SM were layered on top of discontinuous gradients of 5,10,20 and 50 percent (v/v) sucrose and were centrifuged at 22,000 x g for 2 h at 4OC. The gradients were fractionated from the bottom of the tube, and each aliquot was assayed for bound ‘251-TSH by PEG precipitation and filtration. Bound radioactivity was corrected for nonspecific and filter binding. Results were expressed as the average of duplicate determinations. 63
Vol.
137,
No.
1, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
M, x 10d 18 ‘: 0 -x
16
1
8068-
3
TOP I
5
7
9 Fraction
II
13
15
Figure 2: Discontinuous sucrose density gradient ultracentrifugation of thyroid PM following labeling with radioiodinated TSH. Thyroid PM (1 mg) were labeled with 2.4 nM ‘251-TSH and cross-linked as described in Fig. 1. However, the membranes were not washed to remove unbound TSH prior to cross-linking. Following solubilization with Triton X-100, the membranes were analyzed as described in Fig. 1. Figure 3: Electrophoretic mobility of the 1251-TSH labeled high- and lowaffinity binding components. Aliquots of the binding peaks corresponding to the covalently labeled high- and low-affinity binding components in Fig. 2 were boiled in 2% SDS containing 50 mM DTT. The labeled membranes were then subjected to SDS-PAGE on 7.5% acrylamide gels. Following electrophoresis, the gels were dried and analyzed by autoradiography. High- and low-affinity binding peaks cross-linked to ‘2sI-TSH in the presence (lane A and C) or absence (lanes B and D) of excess unlabeled TSH, respectively.
from La, prior
unbound
TSK was not
to cross-linking
increased
the
phoresis
with
specific
removed DSS.
labeling
and autoradiography
by washing
of the
As can be seen in Fig. of La in comparison
been
TSH receptor identified
identified
(14).
to represent
procedure
of Ha.
Electro-
labeled bands with
the main
binding
Mr
These two bands have subunits
of the
The band near the bottom of the gels has also been
as unbound TSH (14).
The bands on the top of the gels could
not be resolved in 5% acrylamide and therefore aggregates.
The labeling
for
membranes
when the
2, this
to that
of 80,000 and 68,000 (Fig. 3, Lanes B and D).
previously
membranes
of the peaks corresponding to Ha
of aliquots
and La resulted in the appearance of two similarly values
labeled
were
were assumedto be protein
of the 80,000 and 68,000 bands was specific, incubated
in the presence
64
of excess
native
BIOCHEMICAL
Vol. 137, No. 1, 1986
hormone , the bands ionally
a third
of the
low
were
not
labeled
affinity
DTT (50 mM), suggesting Similar
units. of intact linking
with
however
it
whose
is
the 200,000
tion
has previously
not
with efficiently
upon boiling
been
reported
(Mr = 197,000)
some of which
denatured
It Graves’ appear
is well
established
disease
possess
to be directed
Some of these binding
antibodies
of 1251-TSH
80,000
and 68,000
utilized
against
Protease-free
IgG fractions
Graves ’ disease
were
and tested
for
membranes,
as described
findings
(20,14),
inhibited while were the
their
ability
the
the binding normal
then
solubilized
high
shown
(14,20)
sera
and sera
ability
from
in a dose-dependent shown).
with Blue,
to thyroid with
previous
disease
patients
fashion
(P < O.OOl),
These
the binding
binding
components.
65
was
of La and Ha.
of patients
to block
and low affinity
as to the
bindings
some Graves’
20).
the
binding
In agreement
not
see ref.
on DEAE Affi-Gel
12%-TSH
(14).
from
to inhibit
as well
by chromatography
(data
oligomer,
review
properties
to inhibit
their
(for
been
This
that
some of which
the physicochemical
effect
observa-
suffering
to inhibit
IgG had little
Alter-
who showed
ability
IgG fractions
DSS.
in SDS.
membranes
previously
a component
Similar
(10)
antibodies,
1251-TSH
for
et al.
autoimmune
of
examined
with
of patients
of normal
prepared
may represent
sera
(14).
analyze
it
a noncovalently-linked
have previously
Mr bands
unknown;
of the TSH holoreceptor
the TSH receptor
to thyroid
to further
is
the
several
still
in SDS (16,18).
by boiling that
is
and cross-
some of the TSH holoreceptor
by Islam
the TSH holoreceptor is not
band
sub-
in SDS extracts
‘251-TSH
cross-linked
band may represent
dissociated
with
DSS or
of
of disulfide-linked observed
labeling of this
seen on gels
in the presence
composed
some molecules
crosslinked
that
was not
following
Occas-
A and C).
was also
been occasionally
represent
were
that
was not
RESEARCH COMMUNICATIONS
Lanes
was observed
The identity
may either
of the receptor natively,
it
3,
Mr of 200,000,
band
has also
DSS (14).
subunits
This
membranes
(Fig.
with
that
peptide
thyroid
observed
band,
peak.
AND BIOPHYSICAL
JgG fractions of
12’I-TSH
Solubilized
to
Vol. 137, No. 1, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0
i
4
6 Fraction
Top
8
12 Bottom
10
Number
Figure 4: Effects of IgG on the labeling of the high and low affinity TSH binding components. Thyroid SM (0.5 mg) were preincubated with buffer IgG ( 1. 5 mg) (m), normal (O-*-*-*-*0) or Graves’ ( l II,,,,,,,,,II,,,,,II,IIII~I~) followed by incubation for an additional hour with ‘251-TSH (0.6 MI) in the presence or absence of excess unlabeled hormone,(1.25 Ill). The labeled membranes were then analyzed on discontinuous sucrose gradients as described in Fig. 1.
thyroid
membranes
IgG for
1 h at room temperature.
I*%-TSH
were
and analyzed
preincubated
with
buffer
The membranes
on discontinuous
density
IgG from
normal
4, preincubation
with
decrease
labeling
of both
peaks
patients
resulted
in marked
Although
not
Graves’ both
peaks
suggest with
disease
(P < 0.001).
that
La and Ha are
Graves’
IgG through
conclusion
is
supported
by the
antibodies
are
highly
homogeneous
determinant Previous for
adenylate
on the
receptor
evidence cyclase
(P > 0.05).
that
arising
labeled
sera
of the
Graves’
As can a slight
IgG from labeling
these
and possibly
against
with
caused
However,
determinants.
suggestion
or Graves'
gradients.
conclusive.
related
antigenic
then
inhibition
entirely
structurally similar
were
sucrose
be seen in Fig. in the
(control),normal
data
interact This
anti-TSH
a single
of
latter receptor
antigenic
(20).
indicates activation
(7-9)
that
Ha is
by TSH, whereas 66
the receptor La does not
required appear
to
Vol.
137,
No.
BIOCHEMICAL
1, 1986
play
any significant
size
than
role
Ha (6),
receptor
it
induced
in this
by hormone insulin
that
Concanavalin
A and La does not,
of Ha (23).
It
is
apparatus
(21,22).
units
to mannose it
artifact
of the membrane
strated
is unlikely
on intact
unpublished
that that
rat
(6)
differ
the
that
affinity
multiple
forms
of TSH with
present
Ha binds
of the previously to
precursor
processed
it
bind
added
by the
Golgi
concanavalin
A.
is
formed
as an
has recently
been
demon-
(Gennick,
of receptors
as
catecholamines,
glucagon
their
wheat
recently
complexes
exist
Thomas and Nayfeh,
not
cells
(see
with
the separation the
wheat
hepatic
to determine in the
using
monoclonal
tumors
lacking
Acknowledgements.
the
responsiveness antibodies
low
affinity We wish
purified
bovine
TSH and Drs.
critical
review
of the
receptor
of target raised
against
binding
(7,8).
to thank Sharon
manuscript.
Dr.
It
John
component
G. Pierce,
discussion). (24)
have
in their
inter-
will
be of
low-affinity
to hormone
this
and
binding
differ
of the
cells
of
angiotensin,
nucleotides.
of involvement
interaction
additional
that
of
interaction
and low-affinity
and guanine
degree
the
Mason and Tager
of high-
glucagon
germ lectin
24 for
columns,
for the
dopamine, ref.
size,
The participation
includes
insulin,
previous forms
including
to be unique
but
our
in two distinct
composition.
germ lectin-Sepharose
for
and extend
properties,
membranes,
target
reported
components
confirm
appears
such hormones with
results
and carbohydrate
plasma
component
since
component
cells
physicochemical
thyroid
interest
to that
that
since
thyroid
TSH-receptor
in several
binding
action
similar
in
co-translationally
affinity
preparations,
greater
results).
findings
Using
low
La is
a metabolic
are subsequently
the
COMMUNICATIONS
an aggregate
La contains
glycoproteins
that
FRTL-5
In conclusion,
that
La represents
Alternatively,
containing
However,
Since
La may represent
conceivable
oligosaccharide
RESEARCH
a phenomenon
action,
for
BIOPHYSICAL
interaction.
is possible
demonstrated
core
AND
action, as well
as
UCLA for
the
Gennick
and John E. Wilson
for
We also
thank
and
67
Cindy
Hickey
their
Vol. 137, No. 1, 1986
Sylvia
Gault
BIOCHEMICAL
for preparation
AND BIOPHYSICAL
of the manuscript.
RESEARCH COMMUNICATIONS
Supported by NIH grant
#AM 23080. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N. (1980) J. Biol. Chem. 255, 4001-4010. Gr, S.M., Goldfine, I.D., and Ingbar, S.H. (1976) J. Biol. Chem. 25l, 4693-4699 Powell-Jones, C.H.J. Thomas, C.G., and Nayfeh, S.N. (1979) Proc. Natl. Acad. Sci. USA76, 705-709. Pekonen, F. and Weintraub, B.C. (1979) Endocrinology 104 352-359. Saltiel, A.R, Thomas, C.G., and Nayfeh, S.N. (1982) MorCell. Endocrinol. 28, 299-311. Drummond,R.W., McQuade, R., Grunwald, R., Thomas, C.G., and Nayfeh, S.N. (1982) Proc. Natl. Acad. Sci. USA 79, 2202-2207. Saltiel, A.R., Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N. (1980) Biochem. Biophys. Res. Commun. 95, 395-403 Saltiel, A.R. Powell-Jones, C.H.J., Thomas, C.G., and Nayfeh, S.N., (1981) Cancer Res. 41 2360-2365 Lefort, G.P.,Amr, S., Carayon, P., and Nisula, B., (1984) Endocrinology -9 114 1005-1011. Saltiel, A.R.,
Powell-Jones, C.H.J., Thomas, C.G. and Nayfeh, S.N., (1981) Endocrinology 109, 1578-1588. Lowry, O.H., Rosebrough ,N.J., Farr, A.L., and Randall, R.J., (1951) J. Biol. Chem. 193, 265-275. Markell, M.A., Haas, S.M., Bieber, L.L., and Tolbert, N.E., (1978) Anal. Biochem. 87, 206-210. Pilch, P.F., and Czech, M.P., (1979) J. Biol. Chem. 254 , 3375-3381. McQuade, R., Thomas, C.G., and Nayfeh, S.N., (1985) Arch. Biochem. Biophys. (In Press) Cuatrecasas, P., (1972) Proc. Nat. Acad. Sci. USA69, 318-322. Laemmli, U.K., (1970) Nature 227, 680-685. Robinson, N.C., and Tanford, C., (1975) Biochemistry 14. 369-374. Pierce, J.G., and Parsons, T.F. (1981) Ann. Rev. Biochem. 50 465-495. Islam, M.N., Briones-Urbina, R., and Farid, N.R. (1983) Endocrinology -113 436-438. Farid, N.R., Briones-Urbina, R., and Bear, J.C., (1983) Molec. Aspects Med. 6, 355-457. Schecter, J., Cheng, K., Jacobs, S., and Cuatrecasas, P., (1979) Proc. Natl. Acad. Sci. USA 76, 2720-2725. Heffetz, D., and Zick, Y., (1986) J. Biol. Chem. 261, 889-894. Ronnet G.V., Enuston, V.P., Kohauski, R.A., Simpson, T.L., and Lane, M.D., (1984) J. Biol. Chem. 259, 4566-4575. Mason, J.C., and Tager, H.S., (1984) Proc. Natl. Acad. Sci. U.S.A. U.S.A. 8& 6835-6839.
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