cholecystokinin (CCK) releasing-peptide (monitor peptide) with epidermal growth factor for binding to 3T3 fibroblasts

Vol. 145, No. 2, 1987 June 15, 1987

BIOCHEMICAL

COMPETITION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 646-650

OF A GROWTH STIMULATING-/CHOLECYSTOKININ

RELEASING-PEPTIDE

(MONITOR PEPTIDE)

(CCK)

WITH EPIDERMAL

GROWTH FACTOR FOR BINDING TO 3T3 FIBROBLASTS Shin-Ichi

Fukuoka,

+&Institute of Agriculture,

Faculty April

Fushiki, Yasuo Kitagawa", Kazuo Iwail

Etsuro

Laboratory of Nutritional Chemistry, of Food Science and Technology, Faculty Kyoto University, Kyoto 606, Japan

Department

Received

Tohru

20,

for

Sugimoto*,

and

of Agriculture,

Biochemical Regulation, Nagoya University, Nagoya

464,

Japan

1987

SUMMARY: The growth stimulating-/cholecystokinin releasing-peptide (CCK) (monitor peptide) is a peptide purified from rat bile-pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. Its multiple functions and peptide sequence suggested that it is distinct from epidermal growth factor (EGF). However, we found that the peptide competes with ['=I]-EGF in the binding to Swiss 3T3 fibroblast cells to almost the same extent as unlabeled EGF does. [1251]-EGF binding was inhibited by 50% by the peptide at 82.8 rig/ml and by unlabeled EGF at 71.4 rig/ml. This suggests that the growth stimulating effect of the peptide on 3T3 fibroblasts is mediated via the EGF receptor, and also suggests that the partial homologous sequence between monitor peptide and EGF is required for the receptor binding, or that o 1987 Academic Press, Inc. the EGF receptor has a broad ligand specificity.

We previously about

6,500,

activity

that

response

this

juice

enzyme

activity,

activity

the

i.ntestine.

Swiss

should

$1.50

0 1987 by Academic Press, of reproduction in any form

Inc. reserved.

646

is

its

found

rat

intestine(Z). enzyme

also with

Professor,

We

by sereval

to

its

Kyoto

lines from

its

CCK-releasing

exhibits

almost

to

secretion

peptide"(5)

addition

of

stimulatory was

supported

peptide

weight

peptide

the

as "monitor

3T3 fibroblasts

be addressed;

of

of pancreatic

In

monitor

molecular

basis

in

and it

peptide

a

The

as a mediator

that

toward

on the

secretion(l).

intake(l),

the

demonstrated

To whom correspondence

0006-291X/87

acts

protein

in

with

activity

We designated role

we

stimulating

peptide

to dietary

physiological

Conright All rights

a peptide,

bile-pancreatic

pancreatic

of evidence(3,b).

1

rat

and purified

cholecystokinin(CCK)-releasing

postulate in

from

toward

exhibit

found

the

a

growth

same

potency

University.

BIOCHEMICAL

Vol. 145, No. 2, 1987 as epidermal complete the

growth

amino

peptide

that

acid

inhibitor

monitor

peptide

other.

It

inhibitor

growth

of monitor

61 amino that

acid of

(pancreatic is

suggests

and has

highly

conserved

related

to other

that

monitor

peptide

to 3T3 fibroblasts activity

trypsin

is

competition

to be

was investigated peptide

an

indicated

amino

acid

region

in

than

they

that

Kazal

type

However,

are

into

the

sequence

PSTI).

classified

to

Kazal

each type

peptide(5).

monitor

to elucidate is

determine

results

PST&

related by

to

inhibitor,

mammalian

PST1 or its

of this

and the

residues

less

communication,

stimulating

a

RESEARCH COMMUNICATIONS

we succeeded

peptide,

secretory

and may be one of rat In this

binding

sequence

resembles

trypsin

Recently

factor(EGF)(6).

comprises

closely

AND BIOPHYSICAL

mediated

peptide

in

whether via

['%I]-EGF or

not

the

the EGF receptor.

MATERIALS AND METHODS: Purification of the growth stimulating-/CCK releasing-peptide (monitor peptide) was carried out by the method described elsewhere(5,6). The purity of the peptide was demonstrated by the following lines of evidence: The sharp single peak seen on high performance liquid chromatography (HPLC) with a reverse phase column (Bio-Rad RP 304), as shown in Fig.1, the single band seen on SDS-PAGE(l), and the N-terminal amino acid sequencing results(S). Mouse EGF was purchased from Wako Pure Chemicals (Japan). This EGF preparation was purified by the method described(7), and its purity was investigated by SDS-PAGE. EGF and monitor peptide were separated as distinct peaks on reverse phase HPLC (Fig.1). [lzI]-EGF was obtained from Amersham. Other chemicals used were of the highest purity available from commercial sources.

THE PURIFIED PEPTIDE

50

0

T I M E (min) Fig.1. Reverse phase HPLC analysis of EGF and the growth stimulating-/ EGF (100ng) and/or cholecystokinin (CCK) releasing-peptide (monitor peptide). x 250 monitor peptide (100ng) were applied onto a Bio-Rad RP 304 column (4.6 mm) in a HPLC system with a Model 660 programmer and a Model 550 pumps (Waters). Elution was carried out with a gradient of acetonitrile, as indicated in the figure by the broken line. 647

BIOCHEMICAL

Vol. 145, No. 2, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Cell culture: The stock cultures of Swiss 3T3 fibroblast cells were maintained in 6Omm dishes (NUNC, Denmark) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, M.A.Bioproducts), penicillin (50units/ml) and streptomycin (25&ml) (the standard medium) at 37°C under a humidified atmosphere of 5% CO2 and 95% air (8). To prepare cultures for [i251]-EGF binding 0.3 ml of a cell suspension (5x104cells/ml) was inoculated and into each well of 'a 24-well (16mm diameter) microtest plate (NUNC), followed by incubation at 37°C under the above conditions for 24 hr. ['%I]-EGF binding radioreceptor assay: The standard medium was removed 0.3 ml and the surface of cultures was washed once with serum-free DMEM. Then amounts of DMEM containing ['%I]-EGF (50ng/ml, 2346 cpm/ng) and the indicated of unlabeled EGF or the purified monitor peptide were added to each well. After incubation at 37°C for 1 hr, unbound [I25 I]-EGF was removed by washing the of 3 ml of phosphate-buffered saline cells five times with a total volume (O.l5M, pH 7.4) containing 0.1% bovine serum albumin. The cells in each well the radioactivity of the cell were lysed with 0.3ml of 0.5N NaOH, and then lysate was measured with an Aloka autowell gammacounter. RESULTS AND DISCUSSION The EGF receptor replacement

of

replacement

with

15-500

rig/ml.

however,

about

(Fig.2).

The

on 3T3 fibroblasts

bound

['251]-EGF

unlabeled

remaining

with

over

EGF was added

the

initial

radioactivity

UNLABELED

demonstrated

unlabeled

EGF was observed

When unlabeled 25 % of

can be

EGF. the

in

FACTOR

the

binding presence

ADDED

In

the

our

specific

range

up to 2,500 was of

the

study,

concentration

at concentrations

[1251]-EGF

by

still excess

of

rig/ml, observed EGF

(rig/ml)

Fig.2.

Competition by unlabeled EGF (0) and the purified growth stimulating-/ cholecystokinin(CCK) releasing-peptide (monitor peptide) (0) in ['25Il-EGF binding to Swiss 3T3 fibroblasts. The indicated amounts of each unlabeled factor and 15 ng of ['=I]-EGF (2346 cpm/ng) were added simultaneously culture wells containing 0.3ml of the standard binding medium. Binding of incubation at 37'c, ['26I]-EGF to fibroblasts was determined after 1 hr described under "MATERIALS AND METHODS". Each point represents the average duplicate determinations.

to the as for

was

BIOCHEMICAL

Vol. 145, No. 2, 1987 considered

to

subtracting

be

this

50% inhibition rig/ml.

'nonspecific'.

This

The

nonspecific of the

AND BIOPHYSICAL

value.

specific

'specific'

binding

The concentration

['%I]-EGF

concentration

RESEARCH COMMUNICATIONS

shows

calculated

of unlabeled

binding

good

was

was

agreement

EGF

estimated

with

by

to

causing be

previously

71.4

reported

values(9,lO). The purified competed equal

growth

in the

to that

binding

['% I]-EGF

peptide

of excess initial

excess

EGF was added.

almost

the

The peptide juice

described

as a substance

recently

found

points(6):

Monitor

inhibitory

activity

peptide that

(mouse

interstingly, 5-18

explain in

the

EGF binding

curves

for

These

EGF receptor was

are

toward

from

peptide

to

EGF or alpha-tumor region

(-19

were

ref.5). EGF-like to the

14 amino

This

partial

growth

that

the

22% when

EGF

have

affinities

of

rat

bile-pancreatic

secretion,

but

[3 HI-thymidine

are about

was

incorporation,

3T3 fibroblasts

peptide of

it

with

similar

in

6,500

a

some

and

shows

EGF has a

molecular

weight

trypsin-like

enzyme.

However,

monitor

(transforming) does

growth

not

peptide (57.1%)

homology

cell.

649

peaks are

(a-TGF)

of the

peptide peptide

with

with and

that EGF

Whereas, with

to

in

column,

(Fig.1).

comprared

can be seen

of monitor

activity

phase

a reverse

as distinct

acids

factor

show any homology

with

of monitor

stimulating

and

the

Similarly,

separated

8 of

peptide

from

weight

residues)

20-33

seen

of Swiss

On HPLC analysis clearly

the same as that this

EGF and this

a

about

enzyme

and proliferation

binds

In

was

purified

stimulates

trypsin(l,5).

['=I]-EGF

rig/ml.

binding

indicate

pancreatic

has a molecular

specific

almost

similar.

first

also

peptide)

a potency

of 82.8

nonspecific

results

peptide

when residues

3, from

almost

of EGF(6).

of mouse EGF(7),

(Fig.

being

peptide

EGF or (Y -TGF(S).

and monitor

this

to that

N-terminal

of either

binding,

stimulated

EGF) and

differs its

the

incorporation

comparable

of 6,000

this

the

at a concentration

r&ml)

here

that

that

[35S]-methionine potency

the

of

(2,500

(Fig.2).

and EGF for

(monitor showing

Inhibition

The dose-response

same profile

peptide

50%

was observed

['251]-EGF

releasing-peptide

to 3T3 fibroblasts,

EGF.

peptide

of the

the

binding

of unlabeled

by this

presence

stimulating-/CCK

be

residues identical EGF

might

competition

Vol. 145, No. 2, 1987 Monitor

peptide

Mouse EGF(5-18)

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

GNPPAEVNGKTPNCPKQIMGCPRIYDPVCGTNGITYPSECSLCFENRKFGTSIHIQRRGGC .;i+>*c $f+F -:t +;* -GCPSSYDGYCLNNG-

Fig.3. Comparison of amino acid sequence of the growth stimulating-/ cholecystokinin (CCK) releasing-peptide (monitor peptide) with mouse EGF. The one letter amino acid abbreviations are used: A;Ala, C;Cys, D;Asp, E;Glu, F;Phe, G;Gly, H;His, I;Ile, K;Lys, L;Leu, M;Met, N;Asn, P;Pro, Q;Gln, R;Arg, S;Ser, T;Thr, V;Val, Y;Tyr. Identical amino acid residues are indicated by These results are from ref.5. (i').

The present activity It

of the peptide

remains

between

results

toward

to be further

monitor

or whether

support

the

peptide

the

3T3 fibroblasts

determined and EGF is

EGF receptor

hypothesis

required

the for

ligand

ACKNOWLEDGEMENT: This work was supported Research (No.61108006 to K-1.) from the Culture of Japan.

the

is mediated

whether

has a broad

that

the

partial binding

growth via

the

stimulating EGF receptor.

homologous to the

sequence

EGF receptor,

specificity.

by a Ministry

Grant-in-Aid of Education,

for

Scientific Science and

REFERENCES 1. Fushiki,T., Fukuoka,S. and Iwai,K. (1984) Biochem. Biophys. Res. Commun. 118, 532-537 2. Iwai,K., Fukuoka,S., Fushiki,T., Kodaira,T. and Ikei,N. (1986) Biochem. Biophys. Res. Commun. 136, 701-706 3. Fushiki,T., Fukuoka,S. and Iwai,K. (1984) Agric. Biol. Chem. 48, 1867-1874 Fushiki,T. and Iwai,K. (1986) J. Nutr. 116, 15404. Fukuoka,S., Tsujikawa,M., 1546 5. Iwai,K., Fukuoka,S., Fushiki,T., Tsujikawa,M., Hirose,M., Tsumasawa,S., and Sakiyama,F. (1987) J. Biol. Chem. in the press. Sugimoto,E. and Iwai,K. (1986) Biochem. 6. Fukuoka,S., Fushiki,T., Kitagawa,Y., Biophys. Res. Commun. 139, 545-550 7. Carpenter,G. and Cohen,S. (1979) Ann. Rev. Biochem. 48, 193-216 8. Morita,A., Aratani,Y., Sugimoto,E. and Kitagawa,Y. (1984) J. Biochem. 95, 743-750 9. O'Keefe,E., Hollenberg,M.D. and Cuatrecasas,P. (1974) Arch. Biochem. Biophys 164, 518-526 10. Carpenter,G., Lembach,K.J., Morrison,M.M. and Cohen,S. (1975) J. Biol. Chem. 250, 4297-4304

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