Vol. 179, No. 2, 1991 September 16, 1991

BIOCHEMICAL

COMPETITIVE

Fiona

July

Klis,

E.D.L. Schmidt, W.M. Wiersinga

F5-258, of Endocrinology, Meibergdreef 9, Amsterdam,

Division

Received

INHIBITION OF T3 BINDING TO al AND 81 THYROID HORMONE RECEPTORS BY FATTY ACIDS

van der

R.M.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1011-1016

25,

H.C.

van Beeren

Academic Medical The Netherlands

and

Centre,

1991

Summary: This study was undertaken to investigate whether fatty acids inhibit the binding of T3 to the al and 81 form of the thyroid hormone receptor. Fatty acids inhibited the binding of T3 to both receptor proteins isolated of inhibition depends from a bacterial expression system. The effectiveness on the chain length and degree of saturation of the fatty acids. The inhibition of T3 binding to the al and 81 receptor by oleic acid is the Ki value was 5.4 10e6M for the c-erbh al protein competitive in nature; The findings indicate a direct and 3.3 10m6M for the c-erb 81 protein. 0 1991 Academic Pre.55, Inc. interaction of fatty acids with T3 receptor proteins.

The binding binding with

calculated

isolated

hormone

rat

effect There

of is

liver

receptor

or the

inhibition. fatty

(3,4).

variety

of sources

receptor

proteins

We investigated and 81 form vector

nuclei;

nuclear

oleic

binding

acid

(2).

proteins

laboratories

other

be desirable

isolated

the

might

were

done

thyroid

be involved

in

to investigate

isolated c-erbh

cDNA's

arise

from

two genes,

acids

could

inhibit

the

the

receptor

c-erbh

T3

inhibition

studies

than

membrane

of nuclear

of this

These

proto-oncogene have

by fatty

inhibitors

nature

of T3 to the the

proteins.

encodes cDNA's encoding

the

from

T3

a

a1 and 81

(3,5).

of the

fatty

thyroid the

c-erbA

hormone al

receptor

or 81 protein

MATERIALS Chemicals: and fatty activity

strong

competitive

therefore

that

and these

can be inhibited

are

of a nuclear

would

on the

whether

containing

for

evidence

Several

the

presence

nuclei

acids

Ki-value

It

acids

now strong

liver

fatty

demonstrated the

receptor

observed

rat

unsaturated

We have

(1).

and have

the

of T3 to isolated

Especially

acids.

obtained

binding from

of T3 to the

bacteria

in which

al a

was fused.

AND METHODS

Triiodothyronine (T3) was obtained from Hennin GmbH (Berlin, FRG) acids from Sigma Chemical Co (St Louis, USA). [ f 251]T3 (specific 2200 Ci/mmol) was purchased from New England Nuclear (Boston, USA).

1011

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

BIOCHEMICAL

179, No. 2, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Isolation of al and 61 receutor: The pEX plasmid (6) was used as a bacterial expression vector in the E.coli pop 2136 strain (7) containing the cIts857 repressor. The c-erbh coding sequence of chicken al-type (full length, encoding amino acids l-408) (3) and rat Pl-type (encoding amino acids 31-456) (5) were introduced into this vector. Both sequences contain the hormoneand DNA binding domain. C-erbA fusion proteins were isolated according to Stanley (6) and stored in solution A ( 20mM Tris-HCl, 0.25 M sucrose, 1mM EDTA, 50 mM NaCl, 5% (v/v) glycerol, pH 7.6) in liquid N2. Gelelectronhoresis: acetyl-[1251]T3 (9) and electrophoresis The gel was stained

Samples (6-7 ug total protein) incubated with Bromogel for 2 hours at 22 C were put on a 10% polyacrylamide was carried out according to the method of Laemmli (10). with Coomassie Blue and an autoradiograph was made.

T3 bindinu assay: Prior to each incubation the fusion proteins were solubilized by sonication (10 seconds, 6 microns) and the non-soluble proteins were pelleted by centrifugation (lO.OOOxg, 5 minutes). 100 1.11 c-erbh protein suspension (20-25ug protein) was incubated in solution A with [1251]T3 and 5 mM dithiothreitol in the absence or presence of llpM, 33uM, 1OOwM or 3OOuM of fatty acid for 2 hours at 22'C. Total volume was 0.5 ml (2). Incubations were stopped by chilling the samples in ice-cold water. Separation of bound and unbound hormone was performed at 4'C using a small Sephadex G25-medium column in a Pasteur pipette (8). Four 0.8 ml fractions were collected from the column, using solution A as an eluant. The radioactivity in these fractions was taken to represent the hormonal fraction bound to the c-erbh proteins. Specific binding was determined by calculating the difference between the counts bound to the c-erbh proteins in the absence and presence of an excess (10W7M) of nonradioactive T3. RESULTS J1251]T3

binding

From the approx.

to the

a,

and

gelelectrophoresis lo-15%

of

or (31 receptor. is

receptor

types

FIGURE

protein

pEX (Mw

are

in

which

as 45-50 could

Polyacrylamide-gel bacterial

3,6)

with

receptor the absence proteinase gel (right

shown not

(lane inhibitor panel).

and the

1, left

be prevented

the c-erbA

proteins al

aI receptor

observed

proteins the

(lane

1,4),

obtained vector

pEX

c-erbA

Bromoacetyl-[1251]T3 incubated with 1,2,3) or presence (lane 4,5,6) PMSF (left panel). Autoradiograph

1012

a1

from (lane in the of

the

al to

and J31

both

presence

pl of

the fused

for

in the

that

is

The Mw of the is

by incubation

of

system,

and 01 receptor

panel.

containing

we estimate

bacterial

Some degradation

electrophoresis system 2,5)

fusion by the

1lOkD)

figure kD.

expression (lane

of the

expressed

The vector calculated

1.

receptor.

pattern

total

pEX (Mw 155-160kD) receptor

Ljl

the

of

Vol.

179, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

Table

RESEARCH COMMUNICATIONS

I

Inhibition of the binding of T3 to al and 01 T3 receptor proteins by fatty acids expressed as IC50 values (the concentration of fatty acid that reduces T3 binding to 50% of controls) IC50 values in pM Fatty acid cl receptor 01 receptor Effect

of chain

length

ClO: 0 c14:o C18:O Effect

the

proteinase

proteins

was 56.4%

PMSF. Autoradiography

of

and 50.9%

to 3Opg-35ug

protein/tube).

Incubation

of the

al

fatty

acids

decreases

fatty

acids

depends

acids)

Comnetitive Fig. panel)

and acid.

progressive Lineweaver-Burk the

al

for

competitive = Kd(l+i/Ki)

type

al

being

values

protein

of

of increasing

decrease

of apparent reveals

thyroid

inhibition

for

concentration

found

for

binding

panel)

in the of oleic

a competitive receptor acid

in the

presence

than al

saturated

absence

MBC remains

al

concentration

types.

acid. protein

(left

and presence results

of

in a

unchanged.

inhibition

of T3 binding

by oleic

acid

(figure

can be deduced

from

the

to

3).

next

equation

(11):

in which Kd is the dissociation constant for T3, . . dissociation constant for T3 in the presence of inhibitor

i the

of

fatty

receptor

bv oleic

to c-erbA

by

and degree

and sl

acid

of

of T3 binding

at C14:O)

oroteins

amounts Ka whereas

hormone oleic

greatest

[1251]T3

(up

proteins.

Inhibition

more potent

are

proteins

a linear

[1251]T3

to c-erbA

(right

Addition

pEX

was 1.1% -1.4%.

of T3.

acids

plots

analysis

with

binding

of T3 binding

constant

Kd' acid),

c-erbA

and 131 receptor

and protein

binding

(being

IC50

only

The vector

ug protein/tube);

length

fatty

Scatchard

that

T3.

of T3 to a, and 13, recentor

specific

on chain Similar

and D1 type

The inhibitor

apparent

the

bind al

binding

or (31 receptor

inhibition

2 depicts

oleic

1).

(20-25 specific

bindine

(unsaturated (table

expressed

Nonspecific

the

demonstrates

receptor

panel).

to the

between

inhibit

saturation

1, right

respectively

existed

acids

to the

[1251]T3

relationship

Fatty

220 3 8

170 3.4 2

T3 (figure

binding

>300 18 220

saturation

a MW corresponding

to bind

Specific

of C18:O C18:l C18:3

inhibitor

with

failed

>300 19 170

of inhibitor

and Ki the 1013

dissociation

Kd'

the

(oleic constant

for

Vol.

1.4

179, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

h.

0.6

0.6

0.4

0.4

0.2

0.2

1.0

*\

2.0

20

30

baundT3(xlO%l) FIGURE

the

2.

inhibitor.

represent

and 3.3

binding

Scatchard plots of the panel) and Bl receptor oleic acid.

If the

-Ki

10T6M for

Kd'

is

plotted

value. the

Ki

is

c-erbA

(right

versus found

of T3 to the al receptor panel)

i,

in

the

to be 5.4

81 protein

(figure

the

absence

or

(left presence

of

intercept

of the

x-axis

10m6M for

c-erbA

a1 protein

will

4).

bound (x WQM)

W5MOA 60 -

I

/

0.5 10-5M

0.5 1O-5 M OA

OA

lo-’ M OA no OA

-1;id-#

‘/Tot

FIGURE

3.

T3 (x W9

Lineweaver-Burk of T3 to the panel).

al

M)

analysis receptor

‘I Tot T3(x

-‘/kdr

of the effect (left panel)

1014

of and

oleic acid a, receptor

on the (right

lo-‘U)

binding

Vol.

179, No. 2, 1991

BIOCHEMICAL

kd’ b~QM)

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

4c

I 165

I lo5

-Ki

M Oleic Acid

FIGURE 4. Graphical determination of the inhibitor constant on the binding of T3 to al and gl receptors.

Ki of oleic

acid

DISCUSSION In this the

study

c-erbA

both

fatty

types

are

is

found

in

the

inhibition

in that

this

studies

is

study

the

in for

the

nuclear

same order ~1 and pl

membrane

T3 receptor

are

not

involved

interaction

of

fatty

acids

A plausible

mechanism

to the

receptor

receptor the

domain

responsible Inhibitory have

also

of for

the

effects been

modulation

inhibitory

reported indicating

of

the

largely

hormonal

for

effect acids

response.

acid

studies

ICSO is

with

as the

Ki's

proteins

whole calculated

Ki values

effect is

suggest

other

than

and indicates

the

a direct

proteins. steric

a binding

would

be very

which

is

hindrance site

for

of T3 binding fatty

interesting

susceptible

acid

on the

to investigate for

fatty

acids

and

on T3 binding.

on the

angiotensin, in general

our

of nuclear

interaction

molecule

of fatty

and the

by oleic

(10W6M)

inhibiting

It

of

nuclei

The identical

though

for

of saturation of

liver

from

the T3 receptor

acids,

receptor

calculated

the

similar

and degree

al and pl receptor

presence

be postulated.

the

(13,14,15,16), presently

of this

by fatty

can also

in

rat

of T3 to is

(12).

receptor.

with

length

of magnitude

or the

binding

and degree

isolated

to the

the

of inhibition

length

of magnitude

The Ki value

the

inhibit

of chain

using

same order

of nature.

nuclei

acids

on chain

dependency

of T3 binding

competitive liver

in

fatty

The effectiveness

and depends

A similar

acids.

saturation values

that

and @l proteins.

receptor

the

The

we demonstrate

al

binding

of a ligand

to its

progesterone,estradiol the potential Its

unknown. 1015

biological

of fatty significance

receptor

and insulin acids

for remains

Vol.

179, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Wiersinga, W.M., Chopra, I.J. and Chua Teco, G.N. (1988) Metabolism 37, 996-1002. van der Klis, F.R.M., Wiersinga, W.M. and de Vijlder, J.J.M. (1989) FEBS-Letters 246, 6-12. Sap, J. Munoz, A. and Damm, K. (1986) Nature 324, 635-640. Weinberger, C., Thompson, C.C., Ong, E.S., Lebo, R., Gruol, D.J. and Evans, R.M. (1986) Nature 324, 641-646. Murray, M.B., Zilz, N.D., McCreary, N-L., MacDonald, M.J. and Towle , H.C. (1988) J. Biol. Chem. 263, 12770-12777. Stanley, K.K. and Luzio, J.P. (1984) EMBO J. 3,1429-1434. Vidal-Ingigliardi, D. and Raibaud, 0. (1985) Nucl. Acids Res. 13, 5919-5926. Hartong, R. and Wiersinga, W.M. (1985) Acta Endocrin. 108, 525-531. Mol, J.A., Dotter, R., Kaptein, E., Jansen, G., Hennemann, G. and Visser T.J. (1984) Biochem. Biophys. Res. Comm. 124,475-483. Laemmli, U.K. (1970) Nature 227, 680-685. Dixon, M. and Webb, E.G. (1979) Enzymes, pp. 334-336, Longman, London. Wiersinga, W.M. and Platvoet-ter Schiphorst, M. (1989) Int. J. Biochem., 22,269-273. Goodfriend, T.L. and Ball, D.L. (1986) J. of Cardiovasc. Pharmacol. 8, 1276-1283. Mitsuhashi, N., Takano, A. and Kato, J. (1986) Endocrinol. Japon. 33, 251-256. Vallette, G., Christeff, N., Bogard, C., Benassayag, C. and Nunez, E. (1988) J. of Biol. Chem.263, 3639-3645. Svedberg, J., Bjorntorp, P., Smith, U. and Lonnroth, P. (1990) Diabetes 39, 570-574.

1016