Vol. 73, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAI. RESEARCH COMMUNICATIONS

THE STRUCTURE AND FUNCTION OF GLYCOPROTEIN HORMONE RECEPTORS: GANGLIOSIDE George

Lee,l

INTERACTIONS

Salvatore

WITH HUMAN CHORIONIC GONADOTROPIN

M. Aloj,l'

2 Roscoe

0. Brady,3

and Leonard

D. KohnI

' Section on Biochemistry of Cell Regulation, Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. 2 Centro

di Endocrinologia

ed Oncologia

Sperimentale

C.N.R.,

Naples,

Italy

3 Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health Received

September

17,

1976

SUMMARY: Gangliosides inhibit the binding of 125 I-labeled human chorionic gonadotropin to rat testes membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with the ganglioside can be detected by fluorescence specinhibition and fluorescence studies, human chotroscopy . In both the binding rionic gonadotropin recognizes an oligosaccharide sequence on the ganglioside molecule distinct from the sequence recognized by thyrotropin. In a previous labeled

thyrotropin

membranes.

This

and location efficacy

report binding

In this interaction

acid

having

The inhibition

and gangliosides, regard,

in

the

than

fluorescence

gangliosides

could

receptors

on bovine

was critically

residues

from studies

a distinct

within

following

resulted

rather

resulted

that

by gangliosides

sialic

of inhibition

we showed

to the thyrotropin

inhibition

of the

G * M3 ' GDla'

(l),

from the

the

interaction

showed

that

conformational

*

altered

the ganglioside

order:

inhibit

q-

thyroid

plasma

by the number structure,

the

G Dlb > GTl > Gm > GM2 =

interaction

of thyrotropin

of membrane

(TSH)*

and gangliosides.

the ganglioside-thyrotropin change

in

the TSH molecule

Abbreviations: TSH, thyrotropin; hCG, human chorionic gonadotropin: LH, GM~, E-acetylneurluteinizing hormone; FSH, follicle-stimulating hormone; GM~, ~-acetylgalactosaminyl-[~-acetylneuraminylgalactosylglucosylceramide; aminyll-galactosylglucosylceramide; GMl, galactosyl-IJ-acetylgalactosaminyl[z-acetylneuraminyll-galactosylglucosylceramide; GDla, E-acetylneuraminylgalactosyl-~-acetylgalactosaminyl-[~-acetylneuraminyl]-galactosylglucosylceramide; GDlb' galactosyl-~-acetylgalactosaminyl-[~-acetyl-neuraminyl-~-acetylneuraminyll-galactosylglucosylceramide; GTl, ~-acetylneuraminylgalactosyl~-acetylgalactosaminyl-[~-acetylneuraminyl-~-acetylneuraminyl]-galactosylglucosylceramide; DNS, dimethylaminonaphtalenesulphonate.

Copyright AN rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

370

Vol. 73, No. 2, 1976

and,

more important,

mation" the

BIOCHEMICAL

that

to an "inhibitory

gangliosides

lioside-like

than

this

quantities

Second,

TSH receptor

was correlated

with

(2) ;

this

tumor

mally

responsive In the

by which

toxin

only

of the

other

teinizing finding toxin

suggested in their

that

ganglioside

or ganglioside-like

validity

result

by TSH or cholera of this

(1,

from

toxin

suggestion

of the TSH re-

First,

gangliosides

thyroid

membranes

which

content

could of its

unresponsive

2),

it

to the cell

B subunit

in

tissue not

(1).

bind

TSH

membranes

to TSH but

was noted

that

had features

in

toxin

of TSH (1,

nor-

their

(1,

4).

insofar

but

with

extends

B subunits lu-

(hCG)*, (3).

This

to TSH and cholera

that

distinct

In the present

sequence the

(FSH)*

their

target

of carbohydrate

structures

as it

common with

gonadotropin

be similar

recognition

receptor

also

hormone

interaction

the mechanism

had a peptide

3) but

human chorionic

of receptor

might

of

or gang-

in extraneural

tumor

activity

hCG, LH, and FSH might

specificity

nized

found

and follicle-stimulating

mechanism

a ganglioside

in bovine

of cholera

hormones:

(LH)*,

the ability

component

ganglioside

studies

the B protein

glycoprotein

hormone

the

message

the

confor-

and fluoride.

of the above its

that

findings.

previously

cyclase

to prostoglandins

in common with

two

of a thyroid in

a "noninhibitory paralleled

or functional with

deficiency

a deficiency

and that

exactly

we suggested

had been

has an adenylate

course

from

shown to be present

than

TSH transmitted

cholera

(l),

suggestion

GM3 were

much higher the

studies

RESEARCH COMMUNICATIONS

TSH binding.

was a structural

We supported

more complex

which

125 I-labeled

of these

structure

ceptor.

was a progression

conformation"

to inhibit

As a result

not

there

AND BIOPHYSICAL

report

organ

sequences from

those

we support

to the structure

on recogthe

and function

of the hCG receptor. MATERIALS

AND METHODS

Rat testes membranes as well as 1251-hCG were prepared as described by Bellisario and Bahl (5). The hCG was obtained from Dr. Robert E. Canfield, Columbia University College of Physicians and Surgeons, New York, through the Center for Population Research, NICHD, NIH. TSH was a bovine preparation pre1251-hCG binding to plasma membranes was assayed by pared as described (6-9). a filtration technique already described (1, 8, 9). In addition to the agents tested for their ability to influence binding, binding assays contained in a

371

Vol. 73, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

120-1-11 volume, 0.025 M Tris-acetate, pH 6.0, 0.6% bovine serum albumin, approximately 150,000 cpm (2 x lo-' M) 1251-labeled hCG, and 200 pg of membrane protein. The amount of plasma membranes used was within the linear range of binding when evaluated as a function of membrane protein concentration; the incubation was for 2 hours at 23". To insure that the binding and the inhibition of binding measured in these assays were specific, control incubations containing 1.5 x 10e5 M unlabeled hCG or no membranes were included in each individual experiment. Gangliosides GM2 and GDla were obtained as previously described (10). Gangliosides GMJ, GDlb, and GT~ were isolated from commercial preparations (Supelco, Inc., Bellefonte, Pennsylvania) by preparative thin-layer chromatogEach ganglioside used in these experiments was at least 99% pure raphy (11). after rechromatography (11). Gangliosides were quantitated by their sialic acid content using a micromodification of the resorcinol method of Svennerholm (12) Dimethylaminonaphtalenesulphonate (DNS)* conjugates of hCG were prepared as Unreacted dye was separated previously described for bovine growth hormone (13). by filtration through a Sephadex G-25 (superfine) column (0.5 cm x 15 cm) eluted The conjugate solution was finally dialyzed with 0.02 M glycine-NaOH, pH 8.6. The number of bound DNS molecules overnight against 0.02 M Tris-acetate, pH 8.2. Fluorescence measurements were carried out with a Turper mole of hCG was 1.1. ner model 210 spectrofluorometer. Samples for fluorescence studies contained 50 ul of the hCG-DNS conjugate and 1.45 ml of 0.02 M Tris-acetate, pH 7.5; absorption was within 0.1 absorbance unit at the excitation wavelength (340 nm). RESULTS Gangliosides the efficacy

inhibit

of the

inhibition

125 I-labeled being

hCG binding related

GANGLIOSIDES

FIG.

1.

Binding was for

ADDED

to rat

testicular

to the oligosaccharide

membranes, structure

of

lnmolesl

GangZioside inhibition of I25 I-hCG binding to rat testes membranes. 2 hours at 23O in 0.02 M T&?-acetate, ph' 6.0.

372

Vol. 73, No. 2, 1976

the ganglioside GT1'

(Fig.

one of the most

ganglioside

was the

membranes cell

BIOCHEMICAL

(1)

least

(14,

were

determined

i.e.,

0.02

M Tris-acetate,

ments

did

not

studies

having

a higher

inhibitor

using for

salt

TSH binding

The assay

alter

the specificity which

content

that, This

to thyroid toxin

were

having

a higher

0.02 added)

under

both

to those

to thyroid

plasma

membranes,

used

in these

pH:

using

0.05

conditions

assay

conditions

M Tris-HCl,

binding

using

salt

content

pH and a higher

pH 7.2, hCG caused

assay

conditions

conditions

M Tris-acetate,

0.05

?l Tris-Cl,

containing

pH 7.2,

1 mM CaCl

55

35

18

GDlb

50

542

27.5

9

%a

13.5

0

hCG binding

Fig. were

of 125I-hCG unlabeled

was evaluated however,

b

1. identical

binding

save

to rat

bovine

at this

differences

2

of binding

G Dla

a Data taken from b Assay conditions

experi-

by comnarison

unlabeled

pH 6.0 a

than

to fat

similar

performed

% inhibition

concentrations

same plasma

binding

of hCG binding

of 125 I-hCG

inhibition

a 50% inhibition

after

conditions

conditions

and a higher

Thus,

Gangzioside both

binding

pH 6.0.

of hCG binding

Ganglioside

trations,

study

was GDla'

of cholera

Assay

(30 nmol

this

of TSH binding

inhibitor

to be optimal

1 mM CaC12 (5).

TABLE I.

in

of hCG binding

performed

significantly

to previous

notable

inhibitors

effective

RESEARCH COMMUNICATIONS

15).

previously

containing

was especially

effective

experiments

both

It

and was an ineffective

membranes These

1).

AND BIOPHYSICAL

TSH.

higher

for

testes !&en

the

change

membranes ganglioside

pH and at these

in ganglioside

373

noted

specificity

in buffer.

at loo-fold

lower of 1251-

inhibition higher were

salt

concen-

amplified

Vol. 73, No. 2, 1976

(Table

I).

BIOCHEMICAL

Thus,

although

in these

conditions,

the

relative

to GI,lb,

TABLE II.

G2,

Effect

the

ability

inhibitory

Experiment

of G,,la becomes

of peincubation

lowered

more pronounced

of membraneand ganglioside on inhibition

components

% Inhibition

.3 4

Ganglioside before

5

is

to rat testes membranesa

None....................... Membranes + 125 I-hCG............... Ganglioside + 125 I-hCG..............

2

to inhibit

and Gm.

Preincubation

1

RESEARCH COMMUNICATIONS

of gangliosides effect

125 I-hCG binding

of

AND BIOPHYSICAL

+ membranes assay

Ganglioside before

86 84

without

centrifugation

. . . . . . . , . . . . . . . . . .

+ membranes assay

84

followed

84

by centrifugation

. . . . . . . . . . . . . . . . . .

11

a In the control experiment where no preincubation was performed (experiment l), all components (membranes, gangliosides, and 1251-hCG) were added within 10 seconds, mixed, and incubated for a total of 2 hours prior to filtration. In experiments 2', 3, and 4, the noted components were preincubated in assay buffer for 15 minutes before the missing component, ganglioside, membranes, and 1251-hCG, respectively, were added; the binding assay then proceeded for 2 hours before filtration. In experiment 5, after the ganglioside and membranes were preincubated for 15 minutes, the mixture was centrifuged at 12,000 x g for 15 minutes to sediment the membranes. The membranes were then resuspended in buffer, and the missing component, 1251-hCG was added. The ganglioside preparation used in these experiments was a mixed preparation (from bovine brain) containing 47% GDla, 25% GTl, 16% Gblb, and 70 nmol were added. In addition to serving as a control for exper12% Gm; iment 5, experiment 2 shows that gangliosides can "chase" bound hCG off the membrane. Preincubation and binding was at 23'.

As in the plasma

case of the

membranes,

was the

result

ganglioside

inhibition

of the

interaction

of the

of an interaction

from

the

cubation

following when with

inhibition

the

than

hCG binding

ganglioside

the

with

gangliosides 125 I-hCG

of hCG binding ganglioside Evidence

the membranes.

experiments.

First, were

(Table

of TSH binding

there

preincubated II).

Second,

374

with for

to thyroid

to testes the hormone this

inhibition

the membranes

preincubation

rather

conclusion

was no significant with

membranes

came of

prior to in125 I-hCG with of

Vol. 73, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

2

4

6

RESEARCH COMMUNICATIONS

6

[Gangliidesl

10

MM

FIG. 2. Effect of gangliosides on the fluorescence of DNS-hCGin 0.02 T&-acetate, pH 7.5, at 21°. The final concentration of WG-DNS is 2 ukl; excitation tdas at 340 nm.

gangliosides it

resulted

was subjected

in a change

to gel

filtration

of 125 I-hCG

and gangliosides

hCG prevented

the altered

studies

demonstrated

associated linked

with

a large

gangliosides

(l),

oligosaccharide efficacy organ

with

elution

(Fig.

2).

the magnitude

which

the

pattern

As in of the

in

of the

125 with

emission

I-hCG. hCG.

intensity

the case of the fluorescence

of the

gangliosides

ganglioside

inhibits

contrast,

of a l,OOO-fold

interact

of the

of the 125 I-hCG when

pattern

chromatography;

gangliosides increase

structure

elution

in the presence

that

to the hormone

in the

binding

preincubation excess

Last, This

of unlabeled fluorescence

interaction

is

of DNS covalently interaction

changes

tested

(Fig.

of TSH with

depends

on the

2) and reflects

of 125 I-hCG

to the

the target

membranes. DISCUSSION The present

which

report

has a ganglioside

suggests

that

hCG, like

or ganglioside-like

TSH, interacts

structure

375

Jl

and that,

with like

a receptor TSH and

Vol. 73, No. 2, 1976

cholera

toxin,

its

receptor

ride

moiety

moiety

BIOCHEMICAL

hCG undergoes structure, of the

testicular

G M2

7

hCG receptor

th an the

membranes

(GDlb

in

ability inhibitors

minal

sialic

oligosaccharide

acid

of TSH binding, galactose

Since are

data

of gangliosides coprotein

bition

organs,

are

ganglioside change

to target

evaluating salt

the or ion

possibility

to thyroid

have

the most group

mem-

membranes. a ter-

effective

linked

or ganglioside-like

or ganglioside-like

differences

integral

concerning

at

[i]

with

thyroid,

to the

receptor organ

message

by the data

and hormonal

further

extent

change of the

I)

specificity

that

the

and

that

interaction amplify

of inhi-

conformational

propagation:

in Table

gly-

In addition,

the

of the hormone might

and other

initiated.

relationship

with

and biosynthesis

testes,

the conformational

site

structures

content

correlating

and hormone

at the

the

have been

the

(suggested

to target

of the

further

determining

concentrations

in this

testes

is

of TSH and hCG interactions

organs

specificity

ganglioside

suggested

studies

target

[ii]

organ

plasma regard

to rat

a disialyl

ganglioside

ganglioside

induces;

in this

GT1 and GDla,

whereas

to

> GDla > GDlb >

to thyroid

TSH binding

have

membranes

aimed

by a particular

(GTl

Most dramatic

of the specificity

sensitive

experiments

of hCG binding

specificity

chain,

the oligosaccha-

the oligosaccharide

inhibition

of hCG binding,

that

in the plasma

hormone

current

suggest

target

from

with

(1).

determinants

respective

that

of hCG binding

GTl and GDlb,

residue

these

important

their

in

on the

distinct

to inhibit

the most effective

interacting

of TSH binding

as an inhibitor

to its

upon

suggests

ganglioside

inhibition

Thus,

internal

is

a different

ganglioside

of GDla

contrast

inhibitors

report

> GT1 > GMl > GM2 > GDla).

the effectiveness branes

the

the

exhibits

RESEARCH COMMUNICATIONS

in conformation

structure

since

membranes

Gm)

a change

In addition,

on the TSH receptor

rat

AND BIOPHYSICAL

[iii]

alterations with

its

the specificity which

are

report.

ACKNOWLEDGMENT Dr. Fellowship

Health Service Research George Lee is a recipient of a U. S. Public (1 F 22 ZY 01983-01) from the National Eye Institute, National

376

BIOCHEMICAL

Vol. 73, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Institutes of Health, Bethesda, Maryland. We are indebted to Dr. Peter H. Fishman (National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health), not only for providing purified ganglioside standards required for the identification and characterization of the different gangliosides used in this report, but also for his helpful advice throughout the project.

REFERENCES 1.

2. 3.

Mullin, B. R., Fishman, P. H., Lee, G., Aloj, S. M., Ledley, F. D., Winand, R. .I., Kohn, L. D., and Brady, R. 0. (1976) Proc. Nat. Acad. Sci. !FA 2, 842-846. Meldolesi, M. F., Aloj, S. M., Fishman, P. H., Brady, R. O., and Kohn, L. D. (1976) Proc. Nat. Acad. Sci. USA73, in press. Ledley, F. D., Mullin, B. R., Lee, G., Aloj, S. M., Fishman, P. H., Hunt, L. T., Dayhoff, M. O., and Kohn, L. D. (1976) Biochem. Biophys. ?es.

Comniun.2, 4. 5. 6. 7. 8. 9. 10.

852-859.

Mullin, Brady, Bellisario, Winand, Kohn, L. Amir, S.

B. R., Aloj, S, M., Fishman, P. H., Lee, G., Kohn, L. D., and R. 0. (1976) Proc. Nat. Acad. Sci. USA73, 1679-1683. R., and Bahl, 0. P. (1975) J. Biol. C'hem.250, 3837-3844. R. J., and Kohn, L. D. (1970) J. Biol. hem. 245, 967-975. D., and Winand, R. J. (1971) J. BioZ. Chem.246, 6570-6575. M., Carraway, T. F., Jr., Kohn, L. D., and Winand, R. .J. (1972) J. BioZ. Chem. 248, 4092-4100. Tate, R. L., Schwartz, H. I., Holmes, .I. M., Kohn, L. D., and Winand, R. .I. (1975) J. BioZ. Chem.250, 6509-6515. Fishman, P. H., McFarland, V. W., Mora, P. T., and Brady, R. 0. (1972)

Biochem. Biophys. Res. Commun.48, 48-57. 11.

12. 13.

Fishman, P. H., Brady, R. O., Bradley, R. M., Aaronson, S. A., and Todaro, G. J. (1974) Proc. Nat. Acad. 5%. USA71, 298-301. Svennerholm, L. (1957) Biochim. Biophys. Acta 14, 604-615. Burger, H. G., Edelhoch, H., and Condliffe, P. C. (1966) J. Biol. !%em.

241, 449-457. 14. 15.

Cuatrecasas, P. (1973) Biochemistry 12, 3547-3558. Craig, S. W., and Cuatrecasas, P. (1975) Proc. mat. Acad. Sci. 3844-3848.

377

US.42,