Vol. January

166,

No. 30,

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

Epidermal Growth Induce Differential

615-621

Factor and Transforming Growth Factor-alpha Processing of the Epidermal Growth Factor Receptor Stuart

J. Decker

Rockefeller University 1230 York Ave. New York. NY 10021 Received

December

7, 1989

The capacity of epidermal growth factor (EGF) or transforming SUMMARY: growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr=125,000 aminoterminally truncated degradation product from the mature EGF receptor (EGFdependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGFalpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [1251]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [1251]-EGF treatment did not appear to require protein binding after TGF-alpha Tyrosine phosphorylation of EGF receptor from cells treated with synthesis. TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpho allows receptor recylcing prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing. 0 1990 Academic Press, Inc.

Transforming sequence to bind

similarity

with

to and activate

transmembrane

receptor

kinase

and

Despite reported

more potent induction prostacyclin

can result (5,6),

growth

factor (1).

(2,3,4). in activation

the biological

activity

production

(q), (12).

megacolony

TGF-alpha

receptor

are believed from

tyrosine

and c-myc

larger

of these

ligands specific

(7.8)

(9.10). of instances have been has been shown to be

is more effective of wound

stimulation formation

and both

of either

a number of TGF-alpha

promotion

significant

are processed

of EGF

division

characteristics,

than that of EGF.

Both

of c-for

of cell

shares

(EGF)

Binding

common

of angiogenesis

and in inducing

(TGF-ufphu)

stimulation

stimulation

these

in which

epidermal precursors

activity

transcription,

factor-alpha

the EGF receptor

protein

to the EGF protein

growth

of calcium in keratinocytes

healing release (14).

than EGF in (ll),

stimulation

from

bone

The

basis

(13), for

0006-291x/90 615

of

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

166,

No.

2, 1990

the different have

BIOCHEMICAL

potencies

compared

of the EGF

AND

of the two

the effects

ligands

BIOPHYSICAL

RESEARCH

In this

is not known.

of TGF-alpha

and EGF

COMMUNICATIONS

report,

on degradation

we

and recycling

receptor.

MATERIALS

AND METHODS

Materials--TranS35-label (a mixture of [35S] methionine and cysteine) was from ICN, gamrn~-[~~P]-ATP from Amersham and [1251]-EGF was from NEN. EGF was from Collaborative Research or Amgen and recombinant TGF-alpha was from Bachem. Cell culture and Metabolic Labeling--NIH-3T3 cells expressing the human EGF receptor (15) were used throughout this work. Cells were cultured in DMEM containing 5% calf serum. Cells were [35S]-labeled for 16 hours and EGF receptor was immune precipitated from labeled cells using antibody against the human EGF receptor as previously described (16). Immune complex kinase assays and assay of [1251]-EGF binding were performed as described (16,17). As previously reported (18) we found TGFalpha to be about half as effective as EGF in competing for binding of [125I]EGF. Therefore, twice as much TGF-alpha as EGF was used in most experiments to ensure equal receptor occupancy. Ligand-induced EGF receptor internalization was determined by measuring the amount of bound [*251]-EGF (after cells were incubated with 50 rig/ml [ *251]-EGF for 30 minutes at 37OC) resistant to washing with 50 mM glycine,lOO mM NaCl, pH 3.0 (19). Analysis of EGF receptor tyrosine phosphorylation by immunoblotting was performed according Towbin et. al. (20) using antiserum against phosphotyrosine prepared as described by Ek and Heldin (21).

RESULTS We had the human products

EGF

found

receptor

with

of the receptor

(16).

the amino a minor

previously

terminus product

than

EGF

1).

When

labeled

for 12 hours,

the Mr=125,000

cells.

of the mature treated

sensitive, labeled

To abilities labeled

cells.

degradation In the immune from

of EGF

chased

or TGF-alpha.

in

a basis

for this

to generate

experiments normal

Both

were kinase

receptor

EGF-treated

of EGF only similar

degradation from or TGF-alpha in EGF

treated

in EGF

and

are heavily

but present

with

is much

assay, which

forms cells,

at

is more [32P]-

at about

lo-

treated cells. Again, labeling of the mature in assays from EGF and TGF-alpha treated cells.

and TGF-alpha

pulse-chase

receptor

is detected

receptor

truncated

precipitated

amounts

complex

and Mr=lOO,OOO

were

degradation

that TGF-alpha

immune

product EGF

expressing

was Mr=125,000

of these

saturating

Mr=170,000

to establish

products

formation

with

cells

of discrete

product

Here, we report were

treated

precipitates

attempt

and

degradation

and the major

receptors

levels in TGF-alpha receptor was similar

we performed EGF

cells

the Mr=125,000 in immune

fold lower M,=170,000

These

EGF

of NIH-3T3

led to the generation

in inducing

metabolically

TGF-alpha

incubation

of about Mr=lOO,OOO.

less effective

Levels

EGF

of the receptor

products

(Fig.

that

ligands

(Fig.

medium markedly 616

apparent

difference

in the

these receptor degradation products, Cells were metabolically

2).

containing stimulated

saturating turnover

concentrations of the EGF

of

Vol.

166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

ABCDEF

RESEARCH COMMUNICATIONS

170125

200-

9768-

01

02

u. Effects of EGF and TGF-alpha on generation of a M,=125,000 proteolytic degradation product of the EGF receptor. In A, B, and C cells were [35S]-labeled for 16 hours with in the presence of vehicle (A), 200 rig/ml EGF (B), or 400 rig/ml TGF-alpha (C). Lysates were then immune precipitated with antibody against the human EGF receptor as described in “Materials and Methods”. For D through F normally growing cells were incubated with vehicle (D), 400 rig/ml TGF-alpha (E), or 200 rig/ml EGF (F) for the in vitro kinase assay. Lysates were prepared and immune precipitated with antibody against the EGF receptor and precipitated receptor forms autophosphorylated with gamma-[32P]-ATP as described in “Materials and Methods”. Positions of molecular weight standards (x 103) are indicated. w. EGF and TGF-alpha -induced degradation of the EGF receptor. Cells were [J5S]-labeled for 16 hours for pulse-chase experiments as described in “Materials and Methods”. Labeling medium was removed and cells were lysed as zero time controls (A and F) or chased for 2 hours (B and G), 6 hours (C and H), 12 hours (D and I) or 24 hours (E, I, and K) in normal medium (K), &ma1 medium containing 200 n&ml EGF (B-E), or normal medium containing 400 rig/ml TGF-u/phi (G-J). -Cells were lysed and EGF receptor immune precipitated as described in “Materials and Methods”. Positions of the M,=l70,000 mature EGF receptor and Mr=125,000 receptor degradation product are shown. Results of a representative experiment are shown.

receptor, degree

although than

EGF

TGF-alpha

consistently

reduced

(compare

receptor

Particularly noticable two ligands). receptor to chase into the Mr=125,000 EGF. A relatively minor fraction M,=12.5,000 form when TGF-alpha Since of the EGF recycling cells

these

data

receptor

in cells exposed

were

incubated

with

minutes,

washed

extensively

binding

measured

at various

virtually

no recovery

whereas

TGF-alpha

was the

increased

alternate

saturating

bound,

unbound

of the mature into

3).

intracellular

routing

EGF

receptor

For these experiments,

of EGF

or TGF-ulphu

and recovery

for 30 of EGF

after the wash.

Cells exposed

to EGF

after removal

of EGF,

617

SO-70%

of

the

for up to 1 hour recovered

the

in the presence

of

ligand,

a greater

of chase with

we compared

(Fig.

concentrations

to remove

cells

to

tendency

product

pathways

to EGF of TGF-alpha

times

at 6 hours

degradation

on the ligand

of binding treated

levels

half-life

of the mature receptor chased was included in the chase.

suggested

dependent

receptor

of lost

binding

showed

activity

Vol. 166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

P if? i x 2 f 8

ABCDE

03

FGH

MINUTES u. Recovery of [1251]-EGF binding following incubation of cells with saturating amounts of EGF or TGF-n&ha . Cells were incubated with 200 r&n1 EGF (squares) or 400 rig/ml TGF-alpha (triangles, x--x) for 30 minutes at which time samples were assayed for EGF binding or washed 4 times with normal medium and incubated at 37OC for various times prior to assay of [1251]-EGF binding. For data from samples represented by the open symbols, cells were treated with 50 ug/ml cycloheximide for 90 minutes prior to and throughout the experiment. For another group of samples, 10 mM methylamine was added 30 minutes prior to TGF-alpha addition and included throughout the remainder of the experiment (x--x). For binding assays, cells were incubated with 10 rig/ml [1251]-EGF for 4 hours at 4eC as described in “Materials and Methods”. u. EGF and TGF-alpha-induced tyrosine phosphorylation of the EGF Cultures of NIH-3T3 cells expressing the human EGF receptor were receptor. incubated for 30 minutes in normal growth medium containing vehicle (A, E), 100 @ml EGF (B,C,D), or 200 rig/ml TGF-alpha (F,G,H). Cells were then lysed in sample buffer (A,B,E,F) or washed 4 times with growth medium (37OC) and incubated for 15 minutes (C,G) or 45 minutes (D,H) prior to lysis. Proteins from lysates were separated on a 7.5% SDS gel and immunoblotting with antiphosphotyrosine antibody performed as described in “Materials and Methods”. The experiment was repeated 3 times with similar results. The position of the Mr=170,000 EGF receptor is indicated. during

this

period.

by greater

synthesis

intravesicular recovery

acid

pH

(which

inhibited

protein

of endosomes

with

and

lysosomes

(22,23),

by TGF-alpha

treated

or TGF-alpha

led to internalization

EGF

completely

inhibited

The initial 30 minute

cells.

of 70% to 80% of cell

receptors as judged by susceptibility of bound [1251]-EGF to removal by

wash

(19)

Since deficient

of cells

than

of binding

incubation

treatment

95%) had no effect on recovery of EGF binding by treated cells. However, methylamine, which perturbs the

TGF-alpha

surface

Cycloheximide

(not

EGF

EGF

shown).

does not increase

receptor

mutants

and

the rate of degradation such receptors

appear

of tyrosine to recycle

kinase in the

presence of EGF (24,25), we examined tyrosine phosphorylation of the EGF receptor by

following

immunoblotting

treatment with

of cells

with

EGF or TGF-alpha

anti-phosphotyrosine

618

antibodies,

(Fig. 4). treatment

As judged of

cells

Vol.

166, No. 2, 1990

for

30 minutes

BIOCHEMICAL

with

either

ligand

autophosphorylation

of the EGF

when

ligand

extracellular

phosphorylation In this 1047

occurred

experiment cpm

counting.

was

respectively Lanes

Despite

bands

receptor

metabolism, to EGF

[3H]-thymidine

to about in cells

from

lanes

excised

and

we

were

not

RESEARCH COMMUNICATIONS

specific the same extent. loss of

initially

exposed

quantitated

(not

to TGF-alpha. 1161 cpm

by liquid

to detect

and

scintillation

of these

ligands

any difference

by these cells as judged

incorporation

tyrosine

178, and 134 respective

on the effects

able

However,

receptor

A and F contained

371, 253,

differences

or TGF-alpha

tyrosine

by washing,

and H contained

the observed

response

receptor rapidly

when

C,D,G,

induced

removed

more

receptor

AND BIOPHYSICAL

cpm. on

in mitogenic

by soft agar growth

or

shown).

DISCUSSION These induce

results

indicate

for the first time that EGF and TGF-alpha

processing

of the EGF

receptor

through

altered

degradation

of the EGF

receptor

in TGF-alpha

rapid,

cycloheximide

TGF-alpha

insensitive

binding

surface

suggest

following

bound routing

membrane.

in

these

Although difference

treated

to a degradative

cells

recycle

prior

binding

to the cells initially

to recylcing

results

pathway

with

of receptor

in a more

little

both

ligands

degradative

approximately

greatly

relative

reduce abilities

intermediate

equivalent

It is possible

receptor. degradation

of

the

of the EGF

receptor

mechanisms

responsible

are not clear. be necessary mutant line,

receptors binding

pH range TGF-alpha receptor occur

that

receptor,

receptor

apparent

half-life,

a striking

to cause accumulation

of the EGF

for

receptor.

of a

Cells

appear

to inducing binding

kinase

as for the kinase

degradative

continuously

degradation

following deficient

This

EGF

receptor

and kinase

could Our

of EGF occurs

(24.25).

over

for more

lead to more

internalization,

619

of the

The

of the receptor

allowing

mutant.

of much

has been shown

to the EGF receptor vesicles.

slower

in the routing

of the receptor

in the presence

(6), perhaps

mature

than does EGF.

processing

activity

higher levels while each

M,=170,000

somewhat

results

pathway

differential

kinase

to recycle

to EGF

in endocytic tyrosine

in addition

induced

of TGF-alpha

of the mature

TGF-alpha this

tyrosine

EGF

compared

amounts

to a different

The for

direct

cells.

exposed to saturating amounts of EGF accumulate about 10 times of this M,=125,000 receptor form than cells exposed to TGF-u2phu., possess

and the following

Evidently,

internalization. EGF

The

binding

effeciently

the receptor

In contrast,

is seen in their

Mr=125,000

from

pathways.

of [ 1 251]-EGF

receptors

induced

dissociates

of the receptor

recycling

that

TGF-alpha

TGF-alpha

to the plasma

recovery

different

can

rapid

allowing

deficient

Along

this

a much narrower

rapid

data examining

to

dissociation inactivation

recycling

to

the rate of

of of

Vol. 166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

dephosphorylation of tyrosine residues of the EGF receptor following exposure of cells to EGF versus TGF-alpha support this concept. Recycling could provide a mechanism whereby receptor signaling events which occur at the plasma membrane are amplified, perhaps explaining some of the results in which TGF-alpha functions as a superagonist relative to EGF.

Many of the responsesfor which TGF-alpha is a stronger

agonist than EGF such as induction of cell surface ruffling (26), induction of calcium release from bone cultures (13), and stimulation of arterial blood flow (27) probably involve processesat the plasma membrane and may not result in mitogenesis.

ACKNOWLEDGMENTS Thanks to Kathryn Malakom for technical assistance and Alan Saltiel and Antony Rosen for helpful suggestions. This work was supported by research grant CA37754 from the National Cancer Institute.

REFERENCES 1. Derynck, R. (1988) Cell 54, 593-595. 2. Gray, A., Dull, T.J., and Ullrich, A. (1983) Nature 303, 722-725. 3. Scott, J. Urdea, M., Quiroga, M., Sanchez-Pescador,R., Fong, N., Selby, M., Rutter, W.J., and Bell, G.I. (1983) Science 221, 236-240. 4. Derynck, R., Roberts, A.B., Winkler, M.E., Chen, E.Y., and Goeddel, D.V. (1984) Cell 38, 287-297. 5. Pike, L.J., Marquardt, H., Todaro, G.J., Gallis, B., Casnellie,J,E., Bomsten, P., and Krebs, E.G. (1982) J. Biol. Chem. 257, 14628-14631. 6. Massague,J. (1983) J. Biol. Chem. 258, 13614-13620. 7. Ran, W., Dean, M., Levine, R.A., Henkle, C., and Campisi,J. (1986) Proc. Natl. Sci. USA 83, 2228-2232. 8. Cutry, A.F., Kinniburgh, A.J., Twardzik, D.R., and Wenner, C.E. (1988) Biochem. Biophys. Res. Commun. 152, 216-222. 9. Schreiber, A.B., Winker, M.E., and Derynck, R. (1986) Science 232, 12501253. 10. Anzano, M.A.,Roberts, A.B., Smith, J.M., Spom, M.B., and DeLarco, J.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6264-6268. 11. Schlutz, B.S., White, M., Mitchell, R., Brown, G., Lynch, J., Twardzik, D.R., and Todaro, G.J. (1987) Science 235, 350-352. 12. Ristimaki, A. (1989) Biochem. Biophys. Res. Commun. 160, 1100-1105. 13. Ibbotson, K.J., Harrod, J., Gowen, M., D’Souza, S., Smith, D.D., Winkler, M.E., Derynck, R., and Mundy, G.R. (1986) Proc. Acad. Sci. U.S.A. 83, 2228-2232. 14. Barrandon, Y., and Green, H. (1987) Cell 50, 1131-1137. 15. Velu, T.J., Beguinot, L., Vass, W.C., Willingham, M.C., Merlino, G.T., Pastan, I, and Lowy, D. (1987) Science 238, 1408-1410. 16. Decker, S.J. (1989) J. Biol. Chem. 264, 17641-17644(Oct. 25, 1989 issue). 17. Decker, S.J., and Harris, P. (1989) J. Biol. Chem. 264, 92049209. 18. Winkler, M.E., Bringman, T., and Marks, B.J. (1987) J. Biol. Chem. 261, 1383813843. 19. Ascoli, M. (1983) J. Biol. Chem. 260, 9613-9617. 20. Towbin, H., Staehilin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354.

21. Ek, B., and Heldm, C. (1984) J. Biol. Chem. 259, 11145-11152. 620

Vol.

166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

22. Maxfield, F.R. (1982) J. Cell Biol. 95, 676-684. 23. King, A.C., Hemaez-Davis, N., and Cuatrecasas,P. (1981) Proc. Natl. Acad. Sci U.S.A. 78, 717-721. 24. Glenney, J.R., Jr., Chen, W.S., Lazar, C.S., Walton, G.M., Zokas, L.M., Rosenfeld,M.G., and Gill, G.N. (1988) Cell 52, 675-684. 25. Honegger, A.M., Dull, T.J., Felder, S., Obberghen,E.V., Bellot, F., Szapary, D., Schmidt, A., Ullich, A., and Schlessinger,J. (1987) Cell 51, 199-209. 26. Myrdal, S.E., Twardzik, D.R., and Auersperg, N. (1985) J. Cell Biol. 102, 12301234. 27. Gan, B.S., Hollenberg, M. D., MacCannell, K.C. Lederis, K., Winkler, M.E., and Derynck, R. (1987) J. Pharm. Exp. Ther. 242, 331-337.

621