Pituitary binding sites for [3H]-labelled luteinizing hormone releasing factor (LRF)

BIOCHEMICAL

Vol. 50; No. 3,1973

PITUITARY

BINDING SITES FOR [3H]-LABELLED

LUTEINIZING

HORMONE RELEASING FACTOR (LRF).

G. Grant, The Salk

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

W. Vale

Institute,

December

and .I. Rivier

La Jolla,

18,

California

92037

1972

pituitary cells in culture possess two SUMMARY : Normal rat anterior One is a high affinity (2 X lO"M), low capacity binding sites for [3H]LRF. site which corresponds to the half-maximal biological potency of LRF. The second site has low affinity (2 X 10%) and an enormously higher capacity to bind LRF. This second site shows only partial specificity in that inof [3~l~~~. The active LRF-analogues, although not TRF, compete for binding high affinity site is competible by LRF, LRF-agonists and LRF-antagonists even in the presence of an excess of an inactive LRF-analogue which saturates the low affinity binding site. The interaction with

respect

various

of LRF with

to the biological

synthetic

pituitary

cells

of luteinizing

(FSH)

pituitary

potencies

LRF analogues

gonadotropic

release

its

(1, is

hormone

has been

and intrinsic

2).

The effect

an integrated

activities

of

of an LRF peptide

process

(LH) and follicle

discussed

resulting

in

stimulating

on the

hormone

(3).

It at the

has been level

mechanism

established

that

of the pituitary

of stored

thyroid

has been postulated

(7)

thyrotropin

plasma stimulating

phylogenetically

related

characteristics

of binding

of labelled

cells

reported.

In vitro

apparent

affinity

Although to equal

may be similar

constant only that

receptors,

no direct

of ca.

0.5

Copyright @ 19 73 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

771

the

release

(4-6). that

experiments

of the potency

X lo-'M

have been

acts

Although

the

to be expected

it

thyrotropic to possess

examining

the

of gonadotropic of LRF gives

an

LRF (8).

have been made which

of LRF, several

(TRF)

LRF to the receptors assay

a few analogues

(TSH)

indicates

enough

factor

to initiate

hormone

and some evidence

cells

have been

releasing

membrane

and gonadotropic

close

receptor

synthesized

possess which

potencies competitively

Vol.50;No.3,

1973

BIOCHEMICAL

AND BIOPHYSICAL

antagonize the action of LRF (2) and have either intrinsic

activity.

intrinsic

lower or no detectible

It has been deduced from the biological

that most of the LRF agonistic full

RESEARCH COMMUNICATIONS

activity

analogues have lower affinity

for the LRF receptor,

activity

constants but

while at saturating

concentra-

tions the LRF antagonist5 are unable, or at least only partially activate

a response.

At the level

biological

activity

able, to

of the receptor this would suggest that

all the analogues examined act on the samephysiological This study was carried

data

out to substantiate

data and.to characterize

LRF receptor.

our deduction5 about the

the specificity

of r3H]LRF

binding to the LRP receptor at the membranelevel. MATERIALSANDMETHODS Peptide synthesis The tritiated

luteinizing

hormone releasing factor

[3H-Prog]LRF was The

synthesized by solid-phase methodology on a benzhydrylamine resin. general synthetic

approach and purification

described by Rivier et al. analogues. specific

After

activity

methods used here have been

(9) and Monahan and Rivier

purification

a homogeneousdecapeptide (final

35.6 C/mmole) was obtained;

amino acid ratio

(10) for LRF and radioactive

it possessed the correct

for LRF. Peptides were stored frozen in dilute

acetic

acid. Biological

potencies of LRF, labelled LRF and LRF analogues were

determined in vivo and in vitro. activity

based on the quantitation

Anterior

pituitary

Pituitary (8).

After

addition

of

[3H-Prog]LRP had maximal biological of radioactivity

and amino acid content.

cell5

cells were dispersed and prepared as reported by Vale et al. several day5 in culture 10m3M

the cell5 were removed from the dish by

EDTA in Hepes buffer with glucose (5 minutes) followed by

gentle scraping of the dish with a rubber policeman. [3H]LRF binding The experiments were carried out essentially 772

as those reported for

BIOCHEMICAL

Vol. 50, No. 3,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Binding curve of [3H-Prog]LRF to dispersed cultured normal rat Non-competible binding was controlled by addition of pituitary cells. excess (1 mM) LRF to the incubation mixture.

[3H]TRF binding (4). filter

Two saline washes of the incubation mixture on the

were used to reduce the non-specific

established by the addition of a final

binding.

Background was

concentration

of 10% LRF. Filtra-

tion of whole cell preparations was carried out using only WhatmanGF/c filters

as aromatic, hydrophobic peptides adsorb to cellulose nitrate

filters

used previously

in the assay of TRF binding sites

tions were carried out at 0.2 ml final

(4, 5).

volume in Hepes buffer

Incuba-

(8).

RESULTSANDDISCUSSION Analogous to the binding kinetics their

cellular

receptors

(Q.,

[3H]LRF binds to two distinct has an affinity

TRF (ll), sites

of a number of peptide hormones to oxytocin

(Figure la).

close to the determined biological

(12) and insulin

One [%I]LRF binding site activity

ca. 2 X lo-'M LRF; the other has a much lower affinity but a higher capacity

to bind [$]LRF.

The role,

(13)),

constant of

(ca. 2 X 10'8M LRF)

if any, of a low affinity

Vol. 50, No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I I

I

1 IO COMPETITOR

I 100

CONCENTRATION

(ycg)

Competition curves of LRF and LRF analogues for the binding of to the low affinity, high capacity, non-specific binding site. Percent biological potent of each analogue is iven in parentheses A. LRF (100) B. [OMe-Tyr3 ILRF (35) C. des-Arg B-LRF (< 10e4) D. des-
site in the activation to its high capacity,

of the secretion rate of LH and FSHis unknown; due its

presence on the cells renders the examination of

the second, high affinity,

apparently

physiological,

receptor technically

very difficult.

In preliminary

the low affinity

site it was possible to show that although this site has

the ability

to recognize [3H]LRF with somedegree of preference,

not show the same specificity (8).

experiments examining the specificity

characteristics

as the physiological

Although TRF does not compete with r3H]LRF for binding,

of

it does receptor

high activity

analogues (such as [OMe-Tyr5]LRF (35% potency of LRF) compete for r3H]LRF binding with a lower than expected affinity LRF derivatives

and someessentially

inactive

such as des-Arg8-LRF (<.OOOl%potency of LRF) compete to an

equal degree (1) for this high capacity site suggest that the dispersed pituitary

(Figure 2).

cells possess a site

These observations (affinity

constant

2 X lo-8M LRF) for binding LRF related peptides, but that this site is not 774

Vol. 50; No. 3, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3H-LRF

(MX

IO-‘)

Figure 3. Binding curve of [31GProg]LRF to the high affinity site in the absence (A. A-A) and presence (B. o----o) of the des-Arg*-LRP analogue (1.5 mM).

the physiological

LRF binding

affinity

and lack

of specificity.

reported

for

neoplastic but

the

low affinity

pituitary

to a degree

addition

titration analogues. absence

which

site

affinity

of excess

Figure

site

of

which

3 shows

contrast

altered

by strenuous

affinity, curve

demonstrating

775

close

that

to that

by saturating

the

binding

t3H]LRF

and

(11).

was accomplished

examining

of

analysis

qualitatively

[%I]LRP

normal specificities

constant

The saturation

low LRF the observations

in both

slightly

and then

its

with

binding

was examined

a binding

of 1.5 mM des-Arg*-LRF,

[3I]TRF

from both

has an affinity

site.

high

in

demonstrable

des-Arg8-LRF

residual

is

indicated

experiments

low

of the

as concluded This

was only

binding

in secretion

the non-specific, the

cells

which

The [3HlLRF obtained

site

LRF binding inactive

competitive

by various in

by

the presence

the kinetic

LRF and

parameters

of

Vol. 50, No. $1973

Table

1.

BIOCHEMICAL

Competition

pituitary

for

cells

in

AN0

BIOPHYSICAL RESEARCH COMMUNICATIONS

the binding

of

the presence

[3H]LRF

to normal

rat

of 1.5 mM des-Arg*-LRF.

Treatmentb

cpm [3H]LRFa

None (21)

bound

+ S.E.M.

1007

f

28

30 ng LRF (6)

820

f

39

100 ng LRF (6)

799

+

50

300 ng LRF (6)

807

f

18

1 ug LRF (5)

651

f

38

100 ng LRF (3)

611

k

51

758

f

29

688

k

34

662

f

36

855

i

80

756

f

53

619

zk

21

797

f

100

680

-+

53

10 pg des-His'-LRF

(6)

100 ng des-His'-LRF

(6)

1 mg des-His2-LRF

(6)

10 ug [Gly21LRF

(3)

100 ng [Gly2]LRF

(3)

1 mg [Gly2]LRF

(3)

100 ng [OMe-Tyr5]LRF

(3)

1 ug [OMe-Tyr5]LRF aFinal b(

concentration ) = number

the binding presence binding

of the

tion order

of 1 X lo-*M

of replicates

to this

high

inactive

of a number

two antagonistic binding

(3)

above background curves

indicate

of magnitude

per

affinity

treatment.

r3H]LRF binding Table

LRF analogue.

of high

peptides.

[3~l~~~.

and low Although

activity

competition as the affinity

for

r3H]LRF constants

‘7’76

are

1 shows data LRF agonist

the marginal

makes quantitation

site

level

virtually binding ascertained

unaffected

by the

on the

competitive

analogues

and also

of competible impossible,

at levels

concentraof the

biologically.

same

BIOCHEMICAL

Vol. 50, No. 3,1973

These

data

substantiate

antagonistic

LRF peptides

common binding

sites

The dispersed experiments detected having

sites

analogues The second

site

having

anterior

of ca.

to the known

between

for

used in these

(3H]LRF.

1 X 18'M

of these

to the

constant

activities

(2 X 10%)

and inactive

LRF receptor

LRF and a specificity

activities

binding

One of the

of the physiological

relevance

active

and

of LH and FSH via

cells

sites

biological

affinity

the agonistic

rate

pituitary

the parameters

constant

both

secretion

binding

has no apparent

to distinguish

for analogues.

of the

and lacking

LRF derivatives,

LRF

releasing

an ability

but

able

to

them from TRF. The binding

only

rat

that

fashion.

two distinct

a lower

the

competitive

possesses

similar

discern

in

possess

(2)

conclusions

manipulate

normal

an affinity

factor

our

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

LRF agonists

equivalent

i3H]LRF

but

to their

to the high

also

known

the

affinity

two reported

inhibitory

site

is

competible

LRF antagonists

by not

at concentrations

activities.

ACKNOWLEDGEMENTS We would A. Nussey, (Contract

like

to acknowledge

the technical

expertise

C. Otto

and J. White.

This

is

No. AID/csd

2785),

the Ford

research Foundation

supported

of W. Hewitt, by AID

and the Rockefeller

Foundation. REFERENCES 1. 2. 3. 4. 5. 6. 7.

Vale, W., G. Grant and R. Guillemin, in kin 1973, L. Martini and W. Ganong (eds.), Oxford University Press, New York, 1973, in press. Vale, W., G. Grant, 3. Rivier, M. Monahan, M. Amoss, R. Blackwell, R. Burgus and R. Guillemin, Science, 176, 933, 1972. McCann, S., in Frontiers in Neuroendocrinology, L. Martini and W. Ganong (eds.), Oxford University Press, New York, 1971, p. 209. Grant, G., W. Vale and R. Guillemin, Biochem. Biophys. Res. Commun., 66, 28, 1972. Labrie, F., N. Barden, G. Poirier and A. DeLean, Proc. Nat. Acad. Sci. &A, tY& 283, 1972. Guillemin, R., R. Burgus and W. Vale, in Vitamins and Hormones, 29, P. Munson (ed.), Academic Press, New York, 1971, p. 1. Grant, G. and W. Vale, Nature (New Biology), 237, 182, 1972.

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Vol. 50, No. 3, 1973

8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vale, W., G. Grant, El. Amoss, R. Blackwell and R. Guillemin, Endocrinology, 9l, 562, 1972. Rivier, J., IL Monahan, W. Vale, G. Grant, M. Amoss, R. Blackwell, R. Guille& and R. Burgus, Chimia, 2, 300, 1972. Monahan, M. and J. Rivier, Biochem. Biophys. Res. Commun.,48, 1100, 1912. Grant, G., W. Vale and R. Guillemin, Endocrinology, submitted for publication. Borkaert, J., M. Imhert, S. Jard and F. Morel, Molecular Pharm., 8, 230, 1972. Freychet, P., in Proceedings of the IVth International Congress of Endocrinology, Excerpta Medica, Amsterdam, in press.

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