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.

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