Vol. 82, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

June 14,1978

Pages

BINDING TO HUMAN THYROID HYROIO HORMONES AND “REVERSE

INHIBITION

OF THYROTROPIN

Wojciech

Received

Kielczyriski

and

of Biochemistry, 01-813 WARSAW,

Department Education,

811-819

April

3anus.z

Nauman

Medical Center MARYMONCKA 99,

of Postgraduate POLAND

13,1978

SUMMARY: Triiodothyronine, e triiodothyronine and thyroxine were found to inhibit ‘““E‘!! I labelled thyrotropin binding to human thyroid plasma membranes in vitro. Both the thyrotropin binding and the effect of the above iodoaminoacids on this binding were pH, temperature and time depende t. 50% inhibition of thyrotropin binding was observed at 2x10' 9 M concentra ion of reverse triiodothyronine or thyroxine and at 1.1 x lo- 6 M concentration of triiodothyronine. The kinetic studies of thyrotropin binding revealed that the maximal capacity of receptor sites for the pituitary hormone is unaffected by the presence of thyroid hormones. On the other hand the association and dissociation constants for thyrotropin binding changed when iodoaminoa?ida were presenh ;f~ the incubation medium /Ka 8.13 x 28 Mvs 1.6 x 10 M and Kd 1.14 x 10" M vs 4.55 x 10 M respectively, depending on The double reciprocal plots showed competitive the pH/. mechanism of inhibition. The present study suggest that triiodothyronine, reverse triiodothyronine and thyroxine are able to modify the thyrotropin binding to membrane receptors. INTRODUCTION

It

:

and

thyroxine

/T4/

the

pituitary

thyrotropin

the

existence

of

thyroidal et T3

al.

/2/

inhibits as

well

binding

to

thyroid

l/ 2/

be

the

first

to

as

effect TSH

function

et

al.

/3/

endocytosis. receptors

pituitary th

Presented in part at the 8 Lyon /France/ 26 - 30 Sept. This study was supported by Polish Academy of Sciences,.

on

/i/.

have

demonstrated

of adenylate Since

is

years

hormones

suggested

activation

TSH-induced

regulating

recent

thyroid

been

/T3/ by

In

of

has

TSH-induced

of

triiodothyronine

secretion.

Shimizu,

membrane step

that

thyroid

the

direct

and the

known

/TSH/

some

cyclase

to

affect

responsivenes

Takasu,

that

well

is

the

generally

hormone

action

European 1977. 10.4.2.01.5.10

Thyroid

TSH

regarded

the

aim

of

Association Grant

from

the

0006-291X/78/0823-0811$01.00/0 811

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 82, No. 3, 1978

the

present

study

directly

influence

Although

reverse

biologically aminoacid

on

was

to

this

find

AND BIOPHYSICAL

out

whether

RESEARCH COMMUNICATIONS

thyroid

hormones

reaction.

triiodothyronine

inactive,

the

thyroid

membrane

/t-T,/ possible TSH

effect binding

is

regarded of

this

to

be

iodo-

was also

investigated. Human TSH for labelling was gifted TSH standard A was obtained Company, Chromatographically pure standards of L-T5, from MRG, iondon. L-t-T and L-T4 were prepared by Dr Henning Company /west Berlfn/. All other chemicals were purchased from commercial of highest purity grade available, hTSH was sources and wer 135 I /Amersham U.K./ by a slightly modified labelled with /4/ to a specific activity of method of Greenwood et al. Protein concentration was measured by the 75 - 120 mCi/mg. method of Lowry et al. /5/ using bovine albumin as a standard. Thyroid tissue was obtained from surgically treated patients with Graves’ disease. Crude plasma membrane fraction was prepared as previously described elsewhere /6/. Membranes were divided into smgll aliquots and used immediately or stored frozen at -20 C prior to use. Experiments were carried out in small Receptor assay. The incubation medium contained 5 fmoles yrene tubes. ,ppm I-TSH, 100 ug of membrane proteins in 25 mM Tris-HCl 0.4% bovine serum albumin, final pH 4,75 or 6,0. The amount of plasma membranes used was kept within the linear phase of binding when evaluated as a function of membrane protein concentrations. The incubation medium was enriched with either 500 fmoles of native TSH or with 100 pmoles of T or T4. In saturation experiments concentration of labe&drT2H varied from 1 - 200 fmoles per sample. In displacement studies concentration of native TSH varied from 10 fmoles to 2 pmoles and the concentration of T rT and T varied from 1 to 500 pmoles/sample. The fins: vofume of4the sample was 100 AJ~. The samples were incubated for 15 minutes at 22OC. The reaction was stopped by rapid dilution with 1 ml of icecold 25 mM Tris-HCl. The tubes were then centrifuged at 10000 x g for 10 minutes, the supernatant was discarded and the radioactivity of the resulting pellet was counted in a LKB - Wallac automatic ctro-gamma counter. Nonspecific binding of !&5 I-TSH was measured in on incubation mixture with 2 pmoles of native TSH/tube added and in tubes without membrane proteins. In all experiments nonspecific in our experimental conditions fgn~;n;gp;fb&*r 5%. Since I-TSH were bound specifically by equal amounts of membrane proteins depending on the specific activity of the label used, Car statistical reasons the final results are expressed as percentage of the maximal binding. For each experiment the results shown were the mean of those obtained in quintiplicate /most of them/ or triplicate vessels. In addition all experiments were carried out at least three times and only those findings that were consistent are presented. MATERIALS

AND METHODS

:

to us by Byk-Mallincrodt

812

Vol. 82, No. 3, 1978

Figure

1.

RESULTS

Effect

of

plasma

membranes /..../

0,4x

from

BSA,

of

studies

hormones

at to

the

medium

binding

of

labelled

to

of

T4.

that

When

100 pmoles

of

dropped

20%.

Effect

labelled

to

of

either

incubation TSH

to

human

the

pH.

binding T4

of

in

/

or

medium

were

pH 6.0.

AND BIOPHYSICAL

/-

Maximal

As

shown

caused

to

of

in

the

incubation or

time thyroid

60%.

T4,

and

The

the

813

presence

of

T

k.D.,

mM Tris-HCl

3 n=l5.

and

125 I-TSH

of at

the

to

pH 4,75, of

1,

pmoles

100 the

crude

however

effect

in

thyroid of

T3

membrane

effect

of

medium

was

binding

temperature. plasma

thyroid

pH 8,O.

a decrease to

to

Mean

Figure

TSH

rT3

the

binding

made

hTSH

25

was observed also

RESEARCH COMMUNICATIONS

/-.-.-/.

contained

pH 3,5

fraction

membrane

parallel

added

pH on

rT3 / ---/ Incubation

Effect

:

thyroid

BIOCHEMICAL

of

The membranes

rT3

was

enriched 125 I-TSH

equal

with

binding was

of studied

Vol. 82, No. 3, 1978

Figure

BIOCHEMICAL

Effect

2.

time

of

of

hTSH

in

the

to

4’C,

binding

of

incubation,

thyroid

effect

of

Effect

of

membrane

shown

2 S.D.,

= 37’C.

respectively.

was very

rapid.

T3,

2/.

22’C

of

TSH

rT3

and

different 125 I-TSH

T4

binding. T3,

Figure

rT3

TSH binding

of

these

37’C,

after

complex

m membrane

TSH

binding

membrane

of

T4

on

TSH

binding

was

was

effect

noted. on

of

different

125 I-TSH

membrane

minutes

significant

iodoaminoaclds

inhibitory

and

30

binding

3.

or

in T4

were

was found

iodoaminoacids effect

and

used

The

rT3

decrease of

on

/ and or

./

temperatures

temperatures

concentration

of

in

all

At

At all

At

dissociation

/...

n=12.

temperature

37’C,

binding /-

rT3

C = Incubation

and

the

membranes

/----/,

= 22’C,

of

inhibitory

Mean

T3

temperature

A significant 4 pmoles

plasma of

B = Incubation

concentrations is

on

= 4’C

/Figure

observed

temperature

RESEARCH COMMUNICATIONS

temperature

22’C

the

and

presence

-/. T4 /-.-. A= Incubation

at

AND BIOPHYSICAL

of

to

added

to

be

complete

reached T3

was

was

300

weaker

814

the

observed

medium. when

pmoles and

The the

per

complete

when

inhibition

concentration tube. inhibition

The

Vol. 82, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

80

0

Figure

3.

Influence rT3

of

/..../

or

thyroid

of

TSH

binding hormone

Kinetic

parameters

in

-

presence

Data

from

saturation

6,0

were

used

Burk

The

/8/

analysis

the

the

Scatchard

The

values

range for of

TSH T3

to

was

no

DISCUSSION:

and

draw

or

dissociation

I/.

lower

significant The

at In

the

by

-

results

this

interactions,

TSH

6,0 of

Ka the

815

-

values

or

while

for present

TSH

of

the

/Kd/

binding

and

sites either

were

rT3

10,

of

pH 4.75

Lineweaver

constant

plots.

and

both

the

Burk

in of

and

calculated

presence

a factor

at

when

pH 4,75

change

hormone

that

membranes

Lineweaver

of

5/.

/Ka/,

range

to n = 15.

tube.

/7/

revealed

same

2 S-D.

performed

plots

the

/----/,

T4.

4 and

of

in

per

Scatchard

capacity

T3

binding

hTSH

Mean

thyroid

-

/Figure

obtained

/Table

rT3

of

concentration

studies

constant

were

on

pmoles

12%-TSH

TB,

both

maximal

/Bmax/

500

plots

of

association of

of

type

/-.-.-/

when

exceeded of

amounts

membranes.

achieved

the

and

T4

plasma

was

thyroid

increasing

also

from

in

the

T4

the

Ka value

in

the

presence

was

observed.

study

show

that

same

Vol. 82, No. 3, 1978

Figure

4.

BIOCHEMICAL

Scatchard

/left/

plots

Table

I.

of

in

the

or

T4

Kinetic

to of

-0

data

/

of

T3

at

in

hormones

PH

/right/

thyroid

plasma

/a--a

/,

rT3

membranes /+.

. . .+/

pH 4.75.

binding

hTSH

membranes

thyroid

Lineweaver-Burk

/M/

presence

plasmi of

and

hTSH

/p-.-.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the or

to

human

absence

or

reverse

= 4.75

T

3'

pH = 6.00

Scatchard

Lineweaver-Bu*

Scatchard

.I”ewea”er-6Ork

B max.

4.5 x 16%

4.5 x lo-%

2.6 x d%

2.6xlO"M ___-

KA TSH

3.60x IO’M-’

3.61 x lO"M-'

2.40 x10%-

7.39 x 10"n"

KA Eli/T3

220x

lD*M-

2.10 x loan“

1.61 x low

1.60 x 108H-'

KA TSH/rT3

8.77x

low

8.77 x low

3.13 x 1D'M“

8.13 x 10'W'

KA TSH/l4

0.77x

ID'M-'

3.77 x IO'M-'

8.13 x 10%“

3.13 x low' __--

KD TSH

2.76 x to-'H

2.77 x IO-%

4.17 x 10%

4.19 x 10.'H

KDTSH/T3

4.55 x1o“rl

4.56 x10-%

6.23 x IO-'H

6.25 x lD.'H

KO TSH/rT3

1.14 x 10-=38n

1.14 x IO%

1.23 x lO-'M

1.23 x lO-+l

KO TSti,/T4

1.14 x 16%

1.14 x 10%

1.23 x 10%

1.23 x IO-%

816

thyroid

presence

Vol. 82, No. 3, 1978

Figure

BIOCHEMICAL

Scatchard

5.

plots

/left/ of

hTSH

membranes

in

in

vitro

both

125 I-TSH binding was

and pH,

temperature /9,10/

in our

experimental

to

membrane

to

that

was

by

that

physico-chemical

Bryson

all and

the

TS

of

at

et

of

al.

In

inhibit TSH studied

contrast

binding

to

at

pH 4,75,

change

the

membrane

binding

a value

close

exclude

the

biological

and

receptors, carried

clearly

out

other

pH 6,0

thyrotropine

To

817

pH 6,O.

membranes.

/11/.

obtained

/o--o/,

iodoaminoacids

TSH

were

plasma

competitively

maximal

occur

thyroid

at

plasma

maximal

experiments results

rT5

/right/

to /

dependent.

a low pH might properties

iodoaminoacids, pH 6,0

thyroid

conditions, to

of

O-.-.-U

and

time

observed

found

reported

/

effect

and

who

possibility

at

human

/

presence T4

inhibitory

autors

/-

hormones

to

the

Lineweaver-But-k

binding or

thyroid

binding

and the

rT 3 /+....+/

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TSH

or

in parallel

demonstrate

that

BIOCHEMICAL

Vol. 82, No. 3, 1978

the

in

were quantitative

differences

the

possibility

fT 3 and T4 was

not

TSH

types

itself

two

125 I-TSH

was

22’C

in

as

column.

The

similar

to

the

due

the

Our

outer

can be

ring

membrane

of

the

of present

factors thoroughly

binding and

than T4

in vitro

influencing

the

understood,

properties

Tg

and

is.

of

of

fully

T3,

on

thyroid

the physiological the

that the

recognized

because

outer

limited,

suggest

more

atoms in

the -

are is

two iodine

is

TSH

T4

This

Although

action even

rT3

iodoaminoacids

least

with

membranes

of TSH.

have

experiments

can at the

G-25

binding

iodination

The for

study

had

that

interrelationship.

the

was

Sephadex

plasma

shown

rT3

significant

15 minutes

mixture

binding

thyronine.

receptor

relevance

of

of TSH

of

performed.

hormone.

clearly

to both

ring

column

the

in decreased

inhibitors

the

T3,

with

were for

calibrated

preincubation

in contrast

and

of

or interaction

iodoaminoacids

from

have

effect

experiments

assay

untreated

the

experiments

the

preliminary

with

eluted

contrary,

potent

of

binding

a previously

rT 3 Or T4 resulted

T3

than qualitative

the inhibitory

their

receptor on

TSH

that to

incubated

chromatographed

On

rather

RESEARCH COMMUNICATIONS

nature.

To exclude

at

AND BIOPHYSICAL

results

not all thyroid at

are

present,

Acknowledgments,

Autors wish to express their gratitude Dr H. Steinmans from Henning Co., West Berlin for sending standards of T rT and T to W.H.O. International Lab?* for38iolog!?Gal Standards - London, for TSH standard A, and to Dr Naegele from Byk-Mallinckrodt Dietzenbach for offering us hTSH for labelling. to us

REFERENCES

Miyachi, y. and Ozawa, y. 22, 367 - 371. 2. Takasu, N., Sato, S., Tsukx, T., Yamada, T., Furihata, R. 3. Shimizu, M. /1974/ 3. Clin. and Makiuchi, Endocr. Metab. 39, 772-778. T. and Shishiba, Y. /1975/ Endocrinol. 3apon. 22, 55-60. 1.

Shishiba,

Y.,

/1975/‘Endocrinol.

Takaishi,

M.,

Japon.

818

Vol. 82, No. 3, 1978

4. 5. 6.

7. 8, 9.

10. 11.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Greenwood, F-C., Hunter, W.M. and Glover, J.S. /1963/ Biochem. J. 89, 114-123. Lowry, O.H., Rosenbrough, N.T., Farr, A.L. and Randall, R.J. /1951/ J. Biol. Chem. 193, 265-275. J. and Wasilewz, A. /1976/ in Biochemical Basis Nauman, of Thyroid Stimaulation and Thyroid Hormone Action /A. von zur MUhlen and H. Schleusener, eds./ pp. 128-132. Georg Thieme Publishers, Stuttgart. Scatchard, G. /1949/ Ann. N. Y. Acad. Sci. 51, 660-672. Lineweaver, H. and Burk, D. /1934/ J. Am. CEm. Sot. 56, 658-666. Tate, R.L., Schwartz, H.I., Holmes, J.M., Kohn, L.D. and Winand, R.J. /1975/ J. Biol. Ghem. 250, 6509-6515. Goldfine, I.D., Amir, S.M., Ingbar, S.H. and Tucker, G. /1976/ Biochim. Biophys. Acta 448, 45-56. Bryson, J.M., Joasoo, A. and Turtle, J.R. /1976/ Acta

Endocrinol.

83,

528-538.

819