studies of thyroid-stimulating hormone binding to bovine thyroid plasma membranes
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Studies of Thyroid-stimulating Hormone Binding to Bovine Thyroid Plasma Membranes
Masanobu Kotani, Toshitsugu Kariya, and James B. Field
13’I-TSH prepared by the lactoperoxidate
method was used to study the binding of hormone to bovine thyroid plasma mem- branes. Specific binding was obtained using as little as 0.12 mu/ml 1311-TSH. Half-maximal binding occurred with 17.1 + 3.5 mu/ml and saturation a+ ap- proximately 40 mu/ml. Scatchard plot analysis revealed two classes of binding sites, with association constants of 1 .l f 0.06 x lO’M_‘and 1.4 x 10’1M~ for the high- and low-affinity sites, respectively. Binding of 1311-TSH was linearly related to the amount of thyroid plasma membrane protein. Other polypeptide hormones and prostaglandin E, did not inhibit specific
TSH binding. Identical results were ob- tained using two TSH preparations of dif- ferent biologic specific activity 12.5 mU/ ml unlabeled TSH decreased ’ ‘I-TSH bind- ing 50%, and 156 mu/ml caused com-
P lete inhibition. After equilibrium of 311-TSH binding was established, max-
imal displacement was achieved by 120 min using about 300 mu/ml TSH. How- ever, only about one-half of the “‘I-TSH was displaced. Although GTP potentiated the stimulation of adenylate cyclase by TSH, it inhibited binding of 1311-TSH. Bind- ing of TSH correlated very well with acti- vation of adenylate cyclase.
T HE INITIAL ACTION of thyroid-stimulating hormone (TSH) involves binding to receptors on the plasma membranetm6 and subsequent genera-
tion of increased cyclic 3’,5’-adenosine monophosphate (CAMP) in the cell.7m9 The augmented CAMP probably mediates the effects of TSH by dissociating protein kinase into its receptor and catalytic subunits.‘0-‘5 Previous reports of modification of stimulation of adenylate cyclase by TSH have not discriminated between binding of the hormone or activation of the enzyme.8~9J6J7
Although binding of TSH fo receptors on thyroid cells and plasma mem- branes has been reported, 2-6 the results have not always been consistent. Amir et al. reported that binding of ‘H-TSH did not correlate with biologic activity,* while Manley et al. obtained identical binding based on biologic potency.3 Dis-
agreement also exists as to whether there are one2.4-6 or two3 orders of binding sites. Differences of time and temperature of TSH binding have also been re-
ported.2-4.6 The present studies examined the binding of ‘3’I-TSH to thyroid plasma
membranes and correlated binding to stimulation of adenylate cyclase activity in such preparations.
From the Clinical Research Unit and the Department of Medicine, Universitv of Pirtsburgh School of Medicine. Pittsburgh, Pa.
Receivedfor publication November 12. 1974. Supported by USPHS. NIH Grant AM-06865. Reprint requesis should be addressed to James B. Field, M.D., Clinical Research Unit. 3304
Presbyterian-Universify Hospital. 230 Lothrop Street, Pittsburgh. Pa. 15261. Abbreviations used in this paper are: TSH. rh_vroid-stimulating hormone; CAMP, cvclic 3’S’-
adenosine monophosphare: BSA, bovine serum albumin; ACTH, adrenocorticotrophin: PGE,. prosraglandin E,
0 1975 bv Grune & Stratton, Inc.
Metabolism, Vol. 24, No. 8 (August), 1975 959
960
MATERIALS
Iodination of Bovine TSH
TSH was iodinated by the lactoperoxidase In . . .
KOTANI, KARIYA, AND FIELD
AND METHODS
method of Thorell and Johansson ‘* and Miyachi
et al.” Carrier free Na “‘I (l-3 mCi) was added to IO ~1 of 0.1 M potassium phosphate
buffer, pH 7.4, at 22’C. Lactoperoxidase (10 ~1, 100 rg/ml) in 0.1 M sodium acetate buffer, pH
5.6, was added, followed by 5 ~1 of bovine TSH (30 U/mg/ml). Five microliters of 0.88 mM
HzOz initiated the reaction. Hz02 (5 ~1) was added after 10 min and the reaction stopped at 20
min with 0.5 ml of 0.1 M potassium phosphate buffer. 13’I-TSH was separated from Na 13’1 by
Sephadex G-25 column chromatography (I x I5 cm). One-milliliter fractions were collected using
0.1 M potassium phosphate buffer containing 1% bovine serum albumin (BSA). 13’1-TSH eluted
as a sharp peak in fraction 5, while 13’1 was present in fractions 9-14. The specific activity of “‘I-TSH ranged from 70 to 160 pCi/pg. 13’1-TSH was further purified using Sephadex G-100
column chromatography (1 x 20 cm). One-milliliter fractions were eluted with 0.1 M potassium
phosphate buffer containing 1% BSA. They were stored at - 20” C until used.
Chromatography of 13’1-TSH (8 PCi, 5 x IO6 cpm) and unlabeled TSH (2 U, 30 U/mg) was
compared using a Sephadex G-100 column and eluted as outlined above. Radioactivity of the
l-ml fractions was determined and biologic activity assayed based on CAMP generation in dog-
thyroid slices.*‘**’ Radioactivity and biologic activity were closely associated in fractions 6-9 (Fig.
1). Fractions 7, 8, and 9 were satisfactory for binding of 13’1-TSH to plasma membranes.
Biologic activity of TSH treated with lactoperoxidase and H202 was assessed. Ten microliters of
TSH (500 pg, 1500 mu), IO ~1 of 0.1 M potassium phosphate buffer, and IO ~1 of lactoperoxidase
(IO mg/ml) in 0.1 M sodium acetate buffer were mixed. Addition of 5 rl of 88 mM H202 initiated the reaction. After IO min. 5 pl of Hz02 was again added. The reaction was terminated IO min
later by addition of 0.5 ml 0.1 M potassium phosphate buffer. An identical amount of TSH was
treated in a similar fashion except that lactoperoxidase and H202 were not added. After ap-
propriate dilutions, the reaction mixtures were assayed for biologic activity. No loss of biologic
activity resulted from treatment of TSH with lactoperoxidase and H202 (unpublished observa-
tions).
Measurement of ‘:“I-TSH Binding to Plasma Membranes
The buffer for binding contained 0.02 or 0.08 M Tris-HCI (pH 7.5) 3.5 x 10m3 M MgSOd.
I x 10m3 M EDTA, IO-* M theophylline, 2.1% BSA, 6 x 10m5 M ATP, 4 x 10m3 M cyclic AMP,
TUBE NUMBER
Fig. 1. Comparison of radioactivity and biologic activity of ‘3’I-TSH and unlabeled TSH after
column chromatography. TSH (2 U) and 13’I-TSH (8 pCi and 5 x 10’ cpm) were applied to a
Sephadex G-lOOcolumn and eluted with 0.1 M potassium phosphate buffer containing 1% BSA.
One-milliliter fractions were collected, and radioactivity and biologic activity determined.
THYROID-STIMULATING HORMONE BINDING 961
and an ATP regeneration system consisting of 2 x IO-‘M phosphoenol pyruvate and 250 pg per
ml pyruvate kinase. The final incubation volume included 100 ~1 of buffer, 15 ~1 of plasma mem-
branes (20-50 rg of protein), 20 ~1 of ‘3’I-TSH (9 x 104-20 x IO4 cpm, 90 rU-400 rU), and 25
~1 of0.08 M Tris-HCI buffer. Unlabeled TSH, when present, was added in 25 ~1 of 0.08 M Tris-
HCI buffer. Incubation was at 22°C for 120 min unless stated otherwise. ‘3’I-TSH bound to
membranes was separated from free hormone by the method of Amir et aL2 Tris-HCl-2.5% BSA
buffer (0.4 ml) was added to the incubation mixture which then was quantitatively applied to a
Millipore filter. Filters were presoaked with 10% BSA for 2 hr and washed with 1.5 ml of 0.02 or
0.08 M Tris-HCI buffer containing 2.5’4 BSA. After vacuum filtration, the filter was washed twice
with 1.5 ml of Tris-HCI-2.54/ BSA buffer. Filters were transferred to counting tubes and radio-
activity determined in a Packard Auto-gamma scintillation counter.
Nonspecific binding is defined as the 13’I-TSH which was not displaced by addition of 100~500
mU of unlabeled TSH at the onset of the incubation. Specific binding was corrected for nonspecific
binding. ‘3’I-TSH not bound to membranes but absorbed on the filter was determined using the
complete incubation mixture but without plasma membranes. Such binding of 13’I-TSH to filters
was less than 0.3% of the total radioactivity added to the incubation mixture and less than 25”,;
of the 13’ I-TSH bound by membranes. This was also subtracted in calculating specific binding.
The microcentrifugation method** gave identical results (unpublished observations).
Measurement of Dissociation of Bound 13’I-TSH
‘3’I-TSH was bound to plasma membranes as described above in a volume of 0.145 ml at 22°C
for 120 min. Unlabeled TSH was then added and the incubation continued. At the appropriate
times, free and membrane-bound hormone were separated.
Preparation of Bovine Thyroid Plasma Membranes
Bovine thyroid, rat liver, and rat kidney plasma membranes were prepared by the method of
Yamashita and Field.’ They were stored at -20°C for as long as 3 mo without loss of binding
capacity or adenylate cyclase activity. Adenylate cyclase activity was determined as previously
described.2’ Protein was measured by the method of Lowry et a1.23
Dr. J. G. Pierce, Department of Biological Chemistry. University of California School of
Medicine, Los Angeles, Calif., generously provided TSH (30 U/mg). TSH (NIH-B6, 2.5 U/mg)
and prolactin (NIH-PS-10) were supplied by the National Institutes of Arthritis, Metabolism,
and Digestive Diseases. Rat 13’1-TSH, prepared by the chloramine-T method, was donated by
Dr. P. Reed Larsen, Department of Medicine, University of Pittsburgh School of Medicine. Rat
TSH and bovine TSH had identical biologic activity (unpublished observations). Lacto-
peroxidase, grade B, was purchased from Calbiochem Corporation. Union Carbide Company
provided Na 13’1 in 0.05 N NaOH solution. Si4C ATP (27 mCi/mM) and 3H CAMP (24.1
Ci/mM) were purchased from Schwartz Bioresearch. GTP was from Sigma Chemical Company,
Dr. John Pike, Upjohn Company, kindly provided prostaglandin E’ (PGE’ ). Porcine insulin was
a gift from the Lilly Research Laboratories, and ACTH was obtained from Parke-Davis Company.
RESULTS
Binding of 13’1-TSH prepared by the lactoperoxidase method indicated that this preparation was better than chloramine-T labeled hormone (Table 1). Non-
specific binding of the former preparation varied from 6’%-21% of the total binding, compared to approximately 70% for the chloramine-T labeled hor- mone. The data in Fig. 2 demonstrate specific binding of 13’I-TSH using as little
as 0.12 mu/ml TSH and increasing in a relatively linear fashion with increasing amounts of hormone. Using 25 rg of membrane protein, binding was not sat- urated with 4.6 mU/ml of ‘3’I-TSH. Using a constant amount of 13’1-TSH (1.3 x 105cpm, 234 PU), binding was linearly related to the concentration of plasma membranes up to 0.5 mg protein per ml (Fig. 3). Approximately 500,” of bound 13’ I-TSH was displaced using 5 pg/ml TSH (12.5 mu/ml), and al-
962 KOTANI, KARIYA, AND FIELD
Table 1. Comparison of Binding to Thyroid Plasma Membranes of ‘N I-TSH Prepared by Chloramine-T and lactoperoxidase Methods
Type of Binding
(cpm boundfmg membrane protein)
Method of Preparation of 13’ I-TSH
Loctoperoxidase
Chloramine-T Before Fractionation Fraction 8
Total binding 3 1,000 45,500 50,900
Nonspecific binding 2 1,500 10,700 8,100
Specific binding 9,500 34,800 42,800
Thyroid plasma membranes (50 pg of protein) were incubated at 22°C for 2 hr in 0.16 ml containing
1 x lo6 cpm of each labeled hormone preparation. The loctoperoxidose preparation was tested before
and after purification on Sephadex G-100 as described in Materials and Methods. Nonspecific binding
was measured in the presence of 100 mU unlabeled TSH per tube. The specific radioactivities were 21
pCi/pg for the chloramine-T and 160 pCi/pg for the loctoperoxidose preparations, respectively. The
concentration of TSH used with chloromine-T was 2 U/mg while that with lactoperoxidase was 30 U/mg.
The results ore the average of closely agreeing duplicate determinations.
/
.
’ “Iv , , , , Fig. 2. Binding of ‘3’I-TSH to thyroid plasma membranes as a function of hormone
0 , 2 3 4 5 concentration. The plasma membranes (25
13’1 TSH CONCENTRATION pg of protein) were incubated with the ap- propriate amounts of 13’ I-TSH in 0.16 ml at
( mu/ml 1 22’C for 120 min.
60
I
Fig. 3. Binding of 13’ I-TSH to thyroid plasma membranes as o function of membrane concen- tration. The incubation was at 22°C for 120 min in 0.16 ml containing 234 PU 13’1-TSH (1.3 x lo5 cpm).
.li’Le 0.4 0 0.2
MEMBRANE CONCENTRATION (mg/ml)
THYROID-STIMULATING HORMONE BINDING 963
Fig. 4. Effect of unlabeled TSH and other hormones on displacement of
specific 13’ I-TSH binding to thyroid
plasma membranes. The incubation
medium contained ?ji6 pg of membran;
protein and 91 &I I-TSH (1.5 x 10
cpm). Incubation was at 22°C for 120
min. The unlabeled hormones were
added at the beginning of the incuba-
tion.
60
g 40
I
r 20
A ACTH
q Proloctin
1 Insulin
0 Prostoglandln E,
OJ 0 100 200 300
HORMONE ADDED (yg/ml)
most complete displacement occurred with 62.5 pg/ml (156 mu/ml) (Fig. 4). Similar results were obtained with both 2.5 U/mg and 30 U/mg TSH when the
data were expressed based on biologic activity (unpublished observations). The
bound TSH was not displaced by large amounts of other polypeptide hormones or by PGE,. These concentrations of PGEI were sufficient to increase adenylate
cyclase activity in thyroid plasma membranes”~25 and mimic other effects of
TSH on thyroid slices. 21*26 “‘I-TSH did not bind specifically to rat liver or kid-
ney plasma membranes (Table 2). 13’1-TSH binding based on biologic activity was identical using two TSH preparations of widely different purity (Fig. 5).
A representative Scatchard plot analysis 27 of TSH binding demonstrated two classes of binding sites (Fig. 6). The assumption was made that pure TSH con-
tains 50 U of biologic activity per mg and has a molecular weight of 28,000.
Since the unitage of pure TSH is unknown, the association constants are at best only an approximation. The average association constant, K,, of the high-
affinity and low-capacity site was 1.1 f 0.06 x lo* M-’ (five different plasma
membrane preparations). The association constant of the low-affinity and high- capacity site was 1.4 x lo7 M-‘. After 120 min incubation with 13’1-TSH, excess unlabeled TSH displaced the bound hormone (Fig. 7). Maximum dis- placement occurred by 120 min of incubation, although about 507; of the 13’1-TSH still remained bound. Displacement of previously bound 13’I-TSH was achieved with 15 mu/ml unlabeled TSH and near maximal effects with about
Table 2. Comparison of 13’ I-TSH Binding to Plasma Membranes
Prepared From Beef Thyroid, Rat Liver, and Rat Kidney
131 I-TSH Binding pU/mg Membrane Protein
Source of Membranes Experiment 1 Experiment 2
Beef thyroid 246 43
Rat liver 3 1
Rat kidney 19 0
Plasma membranes (thyroid, 25 pg. experiments 1 and 2; liver, 33 ~9, experiment 1 and 66 ~9, experi-
ment 2; kidney, 26 pg, experiment 1 and 52 pg. experiment 2) were incubated for 120 min at 22°C. The
incubation mixture contained 81 PU and 97 MU I31 I-TSH in experiments 1 and 2, respectively.
1 1
x
.
x . x NIH-TSH
/-.
x l Pierce -TSH
0 1 0 5 IO 15
TSH (mu/ml)
KOTANI, KARIYA, AND FIELD
Fig. 5. Comparison of TSH propara- tions of different purity on “’ I-TSH bind- ing to thyroid plasma membranes. The incubation medium contained 192 pU 13’I-TSH and appropriate amounts of unlabeled TSH (either NIH-B6, 2.5 U/mg or Pierce TSH, 30 U/mg). The incuba- tion was for 120 min at 22°C.
Fig. 6. Scatchard plot of the specific binding of TSH to thyroid plasma membranes. BOUND TSH (mU/ml)
Fig. 7. Time course of displace- ment of 13’ I-TSH bound to thyroid plasma membranes by addition of excess unlabeled TSH. lncobation of 27 vg of plasma membrane protein with 214 rtJ 13’ I-TSH for 120 min at 22°C in 0.145 ml was followed
. by addition of 400 mu/ml unlabeled TSH and incubation for an additional appropriate time. The specific bind-
. ing to thyroid plasma membranes 60 120 I.90 at the end of the initial 120 min
MINUTES incubation represents 100% binding.
THYROID-STIMULATING HORMONE BINDING 965
. Fig. 8. Displacement of lO0.k
‘3’I-TSH bound to thyroid plasma membranes by addition of in- creasing amounts of unlabeled 80- TSH. Incubation of 25 fig of .* plasma membrane protein with 4 \
97 FlJ 13’1-TSH for 120 min at $ \ \
22°C in 0.145 ml was followed ap 60. .\., by addition of the appropriate ~\ amount of unlabeled TSH and \LV
__ incubation for an additional 120 min. The specific binding to thy- roid plasma membranes at the end of the initial 120 min incuba- tion is taken as 100% binding.
40s
0: 0 loo 200 300 400 500 600 700
TSH ImU/mll
300 mu/ml (Fig, 8). Even with 625 mu/ml unlabeled TSH, approximately
407; of the bound 13’I-TSH could not be displaced. Binding of 1311-TSH was dependent on both temperature and time (Fig. 9).
Very little specific binding occurred at 4°C. At 22°C binding began at 1 min and increased gradually to near maximum by 120 min. Initially, binding was more
rapid at 37°C but the peak was significantly less than at 22°C. By 240 min at 37”C, binding was actually decreased. The optimal pH for binding was 7.3. At pH 6.0 binding was approximately 70% of that at pH 7.3. The Tris concentra-
tion influenced binding. It was maximal at 0.08 M Tris and reduced by 40”,/, and 6O”,b at 0.2 M and 0.3 M Tris, respectively.
Although GTP potentiates the TSH stimulation of adenylate cyclase,24 it does
not increase binding of ‘3’I-TSH to the plasma membranes (Fig. 10). With
10m3 M GTP, binding of TSH was reduced approximately 507;. Figure 11 com-
pares binding of TSH and its stimulation of adenylate cyclase activity. A very
good correlation exists between the concentrations of hormone producing both effects. The half-maximal binding of TSH occurred with 17.1 =I= 3.5 mu/ml (mean f SEM of four experiments) and half-maximal activation of adenylate
40- - 22°C
*_______* 37” c
t--4 4’C
Fig. 9. Effect of temperature and :iTe on binding of
..A.---- ,‘_..A I-TSH to thyroid
..I/ plasma membranes. .-’ Incubation medium
contained 50 fig of . plasma membrane
15 15 30 45 60 240 protein and 234 FU MINUTES ‘3’I-TSH.
966 KOTANI, KARIYA, AND FIELD
2 OLL ,o 0 to-8 10-7 10-G 10-5 10-4
’ .Jo 10-3 5
GTP (Ml Y
Fig. 10. Effects of GTP on stimulation of adenylate cyclase activity by TSH and binding of 13’1-TSH to thyroid plasma membranes. For measurement of binding of l3 I-TSH, the incubation medium contained 22 Ag of plasma membrane protein and 192 AU 13’1-TSH. Incubation was at 22°C for 120 min. Assay of adenylate cyclase activity was done using 50 Ag of plasma membrane protein in a volume of 0.11 ml. Incubation was for 10 min at 37°C. The TSH concentration was 91 mu/ml.
I 0 . . . . . . .._....- - ._.... - 1600
-G __--
_____ 0
. 5 1500- % - 1400
yg % 1200
% IOOO-
e 3
- 1000
2
600 0 500 - 5 8 -600
H 400
0 20 40 60 60 100 IO
TSH (mU/ml)
Fig. 11. Correlation of binding of TSH and activation of adanylate cyclase in thyroid plasma membranes. In the binding studies, 27 pug of plasma membrane protein was incubated with 137 plJ ‘3’I-TSH for 120 min at 22-C. Adenylate cyclase activity was measured using 50 pg plasma membmne protein incubated in 0.11 ml for 10 min at 37°C. The buffer also contained 5 x 10e5 M GTP for the adenylate cyclase assay.
THYROID-STIMULATING HORMONE BINDING 967
cyclase activity with 13.7 mu/ml (average of two experiments). Maximal ef-
fects on both parameters were obtained with about 40 mU /ml TSH.
DISCUSSION
The present study demonstrating specific binding of ‘3’I-TSH to thyroid
plasma membranes confirms and extends previous reports.2-6 Although
13’1-TSH prepared by the chloramine-T method has been used to measure TSH
binding’*’ our results indicate that the lactoperoxidase method is better (Table 1). The discrepancy between the two methods does not reflect species
differences since rat and bovine TSH had equal biologic activity when the re- sults were expressed on the basis of stated biologic potency (unpublished ob-
servations). Amir et al. reported that ‘2SI-TSH prepared using very low con- centrations of chloramine-T still retained 30x-40% of its biologic activity,
while 707; was bound by excess thyroid plasma membranes.40 In contrast, TSH treated with lactoperoxidase and Hz02 retained full biologic potency6 (un-
published observations). Over 800/o of hormone labeled by either method re-
acted with excess antibody, 3~6,40 but in view of the difference in biologic activity,
this might not be a good criterion to use. ‘H-TSH, which retained over 80% of its biologic activity,* also has been used for binding studies, but its specific radioactivity is less than iodinated preparations.4’
In our experiments specific binding represented 79%-94x of the total 13’I-TSH bound to plasma membranes, confirming similar results using isolated
thyroid cells4 and slices.’ Decreased specific binding of 13’1-TSH prepared with chloramine-T (Table 1) is consistent with the diminished biologic activity of
that preparation. However, in bovine thyroid slices, specific binding of
“‘I-TSH prepared with lactoperoxidase represented only about 33% of the total binding (unpublished observations). Half-maximal and maximal inhibi-
tion of “‘I-TSH binding to membranes by 12.5 mu/ml and 156 mu/ml TSH, respectively, is similar to what has been reported previously.3*4 This latter amount is significantly less than the 1.8 U/ml which Amir et al. reported was
necessary for complete inhibition of binding of ‘251-TSH prepared with
chloramine T.QO Binding of hormone to thyroid plasma membranes was specific for TSH
(Fig. 4), in confirmation of similar results previously published.2*4 Our observa-
tion that up to 300 pg/ml PGE’ did not inhibit binding of “‘I-TSH indicates separate receptor sites for the two substances. Moore and Wolff reached the same conclusion based on studies of ‘H PGEl binding to thyroid plasma mem-
branes.*” This amount of PGE’ exceeds that required for biologic effects on thyroid plasma membranes. 25 The absence of significant binding of ‘3’I-TSH to liver and kidney plasma membranes provides additional support for its specificity (Table 2).
In contrast to the observations of Amir et al.,* binding of 13’1-TSH was equivalent, based on biologic activity, regardless of the purity of the TSH preparation (Fig. 5). Our results confirm those of Manley et al. who also used TSH preparations varying tenfold in their biologic specific activity.3 If the biologic effects of TSH reflect initial binding to receptors on the plasma mem- brane, it is difficult to understand the discrepancy reported by Amir et al.* They
960 KOTANI, KARIYA, AND FIELD
found decreasing binding of ‘H TSH per unit biologic activity as the purity of the TSH increased.’
The present results concerning effects of time and temperature on ‘3’I-TSH binding also differ from those of Amir et al.* They originally reported that maximum binding of ‘H-TSH was immediate and inversely dependent upon temperature. More recently they found that ‘3’I-TSH binding was half-maximal by lo-15 min and maximal at 2 hr.40 They found maximum binding was similar
at 0°C 15°C and 22°C but was decreased 45% at 37°C. Lissitzky et al. found
binding of TSH at 0°C was only 25% of that present at 35”C.4 The apparent decrease of binding at 37°C compared to 22°C (Fig. 9) could reflect more rapid
degradation of the receptors at the higher temperature. Lissitzky et al. also ob-
served that binding of TSH was time dependent.4 Such rapid binding of TSH is compatible with the rapid onset of effects of the hormone on thyroid plasma membranes,’ thyroid homogenates,” and intact thyroid cells.29
Controversy exists concerning the number of types of binding sites and their
association constants. The present results indicating two different classes of
receptor sites is in accord with that of Manley et al.,3 Moore and W01ff,~i and
Amir et a1.,40 but is in contrast to several others2,4-6 which indicated only a
single class of receptors. Some of this discrepancy may reflect the different types of thyroid preparations which have been used and the failure to use high con- centrations of TSH. The association constant of the high-affinity, low-capacity
site was 1.1 f 0.06 x lo* M-‘, a value very similar to that obtained by Man-
ley et al.’ and Moore and W01ff.~’ It is two orders of magnitude less than the value of 2.6 x 10” M-l reported by Smith and Hall’ and one order of magni-
tude less than that obtained by Lissitzky et a1.,4 and Verrier et a1.6 Some of the differences might relate to the different incubation conditions, TSH prepara-
tions, and thyroid preparations which were used. Obviously the purity of the TSH preparation has a significant effect on the calculation of the association
constant. Since the specific biologic activity of pure TSH is not known, data of
binding experiments should be reported based on biologic activity. Some previous reports presented the data based on either molarity or weight of
TSH.2.4,6 Similar considerations are probably important in the reported dif- ferences for the association constant of the low-affinity, high-capacity site. Our value of 1.4 x 10’ M-l is very similar to that of 2.7 x lo7 M-’ observed by
Manley et al.,3 but is an order of magnitude less than that reported by Moore ’ and Wolff (0.7-1.0 x lo6 M- ). 41 Although the association constant for the
high-affinity, low-capacity site exceeds the physiologic concentrations of TSH,30
it is similar to that required for most of its in vitro effects.29 The explanation for this disparity is not clear.
‘311-TSH bound to thyroid plasma membranes could not be completely dis- placed by addition of a large excess of unlabeled hormone (Fig. 8). Verrier et al. reported a rapid component to the dissociation of 12’I-TSH from membranes, but even at the end of 50 min about 30% of the hormone was still bound.6 About 60% of I25 I-TSH was dissociated rapidly from cultured thyroid cells with a half-time of 3 min, while the remainder dissociated with a half-time of 30 min. Using 3H-TSH, Amir et al. observed that previously bound hormone could be
THYROID-STIMULATING HORMONE BINDING 969
completely displaced from plasma membranes by unlabeled TSH within 3 min.2
However, only 40% of “‘1-TSH bound to guinea pig thyroid slices was dis-
placed over 1 hr and 99.5”,: over 30 hr when excess unlabeled TSH was added.3
This slower dissociation with this preparation is compatible with the slower
binding which the same authors reported. The apparent retention of a signiti- cant amount of I31 I-TSH to plasma membranes in our experiments is consistent
with the demonstration of persistent binding of the hormone to beef thyroid
slices when generation of CAMP was measured. 42 This suggests that TSH is not
rapidly metabolized by thyroid tissue, in contrast to results with insulin,
glucagon and hepatic plasma membranes.3’,32 In our system the pH optimum for binding was 7.3. Moore and Wolff re-
ported maximal binding at pH 5.5,4’ while Amir et al. found it at pH 6.0.40
Nonetheless, the latter investigators did their studies at pH 7.4 where binding
was only 257; of maximum.
Potentiation by GTP of stimulation of adenylate cyclase by TSH in mem- branes (Fig. 10) confirms earlier results.24,33*34 Such potentiation of hormone
stimulation of adenylate cyclase was first observed by Rodbell et al. using
glucagon and liver plasma membranes,35 and has also been noted in other tis-
sues.36,37 The stimulating effect of GTP was associated with a reduction of 13’1-TSH binding to plasma membranes (Fig. 10). Rodbell et al.,” using gluca-
gon and liver plasma membranes, and Soloff and Swartz,3g using oxytocin and a
particulate fraction obtained from mammary gland, reported similar findings. In contrast, Moore and Wolff reported that GTP had no effect on TSH binding
to thyroid plasma membranes despite potentiation of the stimulation of adenylate cyclase.4’ Because of the inverse relationship between effects of GTP
on hormone binding and activation of adenylate cyclase, Rodbell et al. postu- lated that dissociation of hormone from its receptor sites was more important
in initiation of its action.38
The correlation between binding of 13’ I-TSH and hormone activation of adenylate cyclase in thyroid plasma membranes provides further support for the biologic validity of our system. Since the biologic actions of TSH have been
attributed to activation of the adenylate cyclase-CAMP system,2’ binding of hormone should be intimately related to stimulation of adenylate cyclase. Verrier et al. also reported good correlation between binding of TSH and acti-
vation of adenylate cyclase.6 Half-maximum activation of adenylate cyclase re- quired 1.8 nM TSH (approximately 2 mu/ml, assuming a TSH concentration
of 37 U/mg and a molecular weight of 28,000). Although Amir et al. found similar good correlation between binding of TSH and activation of adenylate
cyclase using between 1 and 8 nM TSH/ml, this relationship did not hold when more than 200 pg/ml plasma membrane protein was used in the assay.’ The concentrations of TSH which Amir et al.* used are much higher than those we required for either activation of adenylate cyclase or binding of TSH to plasma membranes. Based on a biologic activity of 6.8 U/mg, 1 nM/ml of TSH would be 196 mu/ml. Our studies were done using the same buffer system for both binding of ‘3’I-TSH and activation of adenylate cyclase so that the two activi- ties could be compared more directly. We found (unpublished observations),
970 KOTANI, KARIYA, AND FIELD
as did Verrier et a1.6 and Moore and W~lff,~l that TSH binding was signifi- cantly decreased using the adenylate cyclase buffer compared to this buffer without ATP.
ACKNOWLEDGMENT
The authors are grateful to Mary Matthews and Barbara Sheehan for excellent help in the prep-
aration of the manuscript.
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