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Monoclonal antibody studies of epidermal growth factor receptor
Cell Biohgy
International
Reports,
MONCKLONAL ANTIBODY
STUDIES
OF EPIDERMAL
M. Gregoriou and A.R. Laboratory of Molecular Biophysics, Department of Zoology, Oxford, The receptor for epidermal growth factor, a potent mitogen, is a glycoprotein of about 17O,CC-180,ooO daltons found in the plasma membranes of a large number of epidermoid and non-epidermoid cell types. The receptor has intrinsic protein kinase activity which is activated by EGF and, depending on the cell type, is directed at the phosphorylation of ser, thr and tyr residues of the receptor itself and of many other cellular proteins. We have raised monoclonal antibodies against the EGFR as part of a study of the receptor structure and to aid a continuing study of the ontogeny of this receptor Balb/c mice during embryonic development. were immunised with A431 (a human cervical carcinoma-derived cell line) cells or purified membranes and fusions were carried NSl myeloma cells. Clones were out using screened for binding to A431 cells and the positive hybridomas were taken through a three-step secondary screening procedure: inhibition of binding of 1251-EGF (a) to A-431 monolayers (b)
differential binding of antibody (using 1251-(Fab)i rabbit antimouse immunoglobulin) to untreated and EGF down-regulated A-431 cells.
(Cl
immunoprecipitation labelled receptor.
of
539
Vol. 7, No. 7, July 1983
radio-
Using this procedure it is possible to detect antibodies directed both against the binding site and other determinants. The latter type of monoclonal may be important for studies of species differences or stage-specific changes in the receptor during development (1). A monoclonal IgG, G49, obtained by these procedures had particularly interesting properties. When tested for its ability to precipitate the EGF receptor from Triton-solubilised cell membrane it was extremely efficient for the A-431 receptor but we have been unable to immunoprecipitate the placental receptor using this monoclonal (see Fig. 1). This result led us to suspect that the difference between these two receptors, suggested previously by their differential stabilities in detergents (2) may actually be a structural difference.
Fig.
1.
GROWTH FACTOR RECEPTOR Rees University OX1 3PS,
of Oxford, U.K.
Autoradiograms of EGFR after immunoprecipitation with G49. Tracks: 1 - A-431 1251-labelled; 2 - placental membranes 1251labelled; 3 - 32P-labelled A-431 receptor after EGF stimulation in the presence of (y-32P)ATP; 4 as 3 but in the absence of EGF; 5 - non-precipitated material from 3; 6 - as 5 but in the absence of EGF.
When tested for binding of other cell lines the table 1 were obtained:
against results
a number shown in
Table 1 Binding of G49 to monolayer cultures measured using 1251-rabbit anti-mouse immunoglobulin (F(ab); fragment). Cell
line
K562 mc-5
EJ 389
Predictedt binding
Binding* % 1
human
3T3
mouse
-1 -1
-
10 10
NRK RAT-l MMC-E
rat
-
10 10
-
20
ST0
* as percentage of ?' based on estimated
unknown
A-431 binding receptor numbers
The cell line MMC-E has elevated numbers (20% that of A-431) while has more normal receptor numbers
0
1983
Academic
%
1OW - 10 5-10
Press
Inc.
receptor MRC-5 (5-10%
(London)
Ltd.
Cell Biology
540
International
that of A-431). We might therefore have expected a much higher percentage binding than was observed. We do not know whether the distinctive feature of the A-431 receptor resides in the carbohydrate or the protein but this is under investigation. Since G49 is able to recognise the receptor after SDS denaturation and Western blotting we suspect a carbohydrate determinant either as part of or tightly associated with the receptor. Inhibition of EGF binding is only shown when G49 is preincubated with membranes prior to EGF. The inhibition curvf? obtained suggests that the antibody is not a competitive inhibitor since EGF binding is not completely abolished. To investigate this further a series of binding curves were obtained at different G49 concentration (Fig. 2).
Reports,
Vol. 7, No. 7, July 1983
FDGF, Vasopressin and TPA (see 4) accompanied by a reduction in the affinity of EGF for its own receptor. We would argue therefore that receptor clustering is part of a down-regulation process and results in conversion of EGFR to a low affinity state (see 5). It has been proposed, however, that clustering is important for development of the mitogenic signal (6). There are numerous data which make this a difficult proposition to sustain unless there are two clustering processes, only one of which gives rise to the correct signal. Stroschek & Carpenter have produced antibodies which are able to induce clustering and internalisation of EGFR both in the bivalent (F(ab);) and the monovalent (Fab) forms but which are not mitogenic. the clustering and internAlso, alisation of EGFR is a very fast process (of the order of minutes) yet for a mitogenie effect EGF must be present at the cell surface for at least 8 hours. At the present time it is difficult to reconcile all these observations but it may be that the existence of high and low affinity populations of EGFR whose relative levels can be perturbed by receptors for other growth factors and by receptor antibodies is a key factor in the response status of a cell. In conclusion, it seems to us that clustering per se is not a sufficient signal for mitogenic stimulation since it leads to conversion of EGF receptors to a low affinity state followed by rapid internalisation and degradation. Acknowledgements
Fig.
2.
Scatchard analysis of EGF binding to A-431 monolayers after preincubation with various concentrations Both incubations were of G49. carried out at 4%.
The Scatchard analyses show that G49 induces site-heterogeneity, producing a high and a low affinity population of receptors while not affecting the receptor number. Since G49 does not bind to the EGF binding site (it efficiently precipitates the receptor-EGF covalent complex) it is likely that some sort of allosteric modulation of This may be the receptor is occurring. via a classical allosteric effect where a monovalent (Fab) interaction with the receptor modifies its affinity for EGF or via a bivalent interaction where the antibody cross-links two receptors or groups of receptors with a resulting affinity change. We favour the latter interpretation for a It has been shown (3) number of reasons. that PDGF is able to induce the down-regulation of EGF receptors by, presumably, a PDGF-dependent co-clustering process. Similar effects have been observed with
We would like to thank the MedicalResearch Council for a grant and Sharon Smith for expert technical assistance. References 1.
Rees, A.R., Adamson, (1979) Nature 281,
E.D. & Graham,C.F. 309-311.
2.
Hock, R.A., Nexo, E. (1979) Nature 277,
& Hollenberg, 403-405.
3.
Wrann, 210,
El. & FOX,
C.Fred.
(1980)
M.D. Science
1363-1365.
4.
Rozengurt, E., Collins, & Pettican, P. (1982)
5.
Adamson, E.D. & Rees, & Cellular Biochem.
6.
Schecter, Y., Hernaez, L., Schlessinger. J. & cuatrecasas, P. (1979) Nature
257,
278,
M., Brown, K.D. J. Biol. Chem.
3680-3686.
835-838.
A.R. (1981) 34, 129-152.
Molec.
International
Reports,
MONCKLONAL ANTIBODY
STUDIES
OF EPIDERMAL
M. Gregoriou and A.R. Laboratory of Molecular Biophysics, Department of Zoology, Oxford, The receptor for epidermal growth factor, a potent mitogen, is a glycoprotein of about 17O,CC-180,ooO daltons found in the plasma membranes of a large number of epidermoid and non-epidermoid cell types. The receptor has intrinsic protein kinase activity which is activated by EGF and, depending on the cell type, is directed at the phosphorylation of ser, thr and tyr residues of the receptor itself and of many other cellular proteins. We have raised monoclonal antibodies against the EGFR as part of a study of the receptor structure and to aid a continuing study of the ontogeny of this receptor Balb/c mice during embryonic development. were immunised with A431 (a human cervical carcinoma-derived cell line) cells or purified membranes and fusions were carried NSl myeloma cells. Clones were out using screened for binding to A431 cells and the positive hybridomas were taken through a three-step secondary screening procedure: inhibition of binding of 1251-EGF (a) to A-431 monolayers (b)
differential binding of antibody (using 1251-(Fab)i rabbit antimouse immunoglobulin) to untreated and EGF down-regulated A-431 cells.
(Cl
immunoprecipitation labelled receptor.
of
539
Vol. 7, No. 7, July 1983
radio-
Using this procedure it is possible to detect antibodies directed both against the binding site and other determinants. The latter type of monoclonal may be important for studies of species differences or stage-specific changes in the receptor during development (1). A monoclonal IgG, G49, obtained by these procedures had particularly interesting properties. When tested for its ability to precipitate the EGF receptor from Triton-solubilised cell membrane it was extremely efficient for the A-431 receptor but we have been unable to immunoprecipitate the placental receptor using this monoclonal (see Fig. 1). This result led us to suspect that the difference between these two receptors, suggested previously by their differential stabilities in detergents (2) may actually be a structural difference.
Fig.
1.
GROWTH FACTOR RECEPTOR Rees University OX1 3PS,
of Oxford, U.K.
Autoradiograms of EGFR after immunoprecipitation with G49. Tracks: 1 - A-431 1251-labelled; 2 - placental membranes 1251labelled; 3 - 32P-labelled A-431 receptor after EGF stimulation in the presence of (y-32P)ATP; 4 as 3 but in the absence of EGF; 5 - non-precipitated material from 3; 6 - as 5 but in the absence of EGF.
When tested for binding of other cell lines the table 1 were obtained:
against results
a number shown in
Table 1 Binding of G49 to monolayer cultures measured using 1251-rabbit anti-mouse immunoglobulin (F(ab); fragment). Cell
line
K562 mc-5
EJ 389
Predictedt binding
Binding* % 1
human
3T3
mouse
-1 -1
-
10 10
NRK RAT-l MMC-E
rat
-
10 10
-
20
ST0
* as percentage of ?' based on estimated
unknown
A-431 binding receptor numbers
The cell line MMC-E has elevated numbers (20% that of A-431) while has more normal receptor numbers
0
1983
Academic
%
1OW - 10 5-10
Press
Inc.
receptor MRC-5 (5-10%
(London)
Ltd.
Cell Biology
540
International
that of A-431). We might therefore have expected a much higher percentage binding than was observed. We do not know whether the distinctive feature of the A-431 receptor resides in the carbohydrate or the protein but this is under investigation. Since G49 is able to recognise the receptor after SDS denaturation and Western blotting we suspect a carbohydrate determinant either as part of or tightly associated with the receptor. Inhibition of EGF binding is only shown when G49 is preincubated with membranes prior to EGF. The inhibition curvf? obtained suggests that the antibody is not a competitive inhibitor since EGF binding is not completely abolished. To investigate this further a series of binding curves were obtained at different G49 concentration (Fig. 2).
Reports,
Vol. 7, No. 7, July 1983
FDGF, Vasopressin and TPA (see 4) accompanied by a reduction in the affinity of EGF for its own receptor. We would argue therefore that receptor clustering is part of a down-regulation process and results in conversion of EGFR to a low affinity state (see 5). It has been proposed, however, that clustering is important for development of the mitogenic signal (6). There are numerous data which make this a difficult proposition to sustain unless there are two clustering processes, only one of which gives rise to the correct signal. Stroschek & Carpenter have produced antibodies which are able to induce clustering and internalisation of EGFR both in the bivalent (F(ab);) and the monovalent (Fab) forms but which are not mitogenic. the clustering and internAlso, alisation of EGFR is a very fast process (of the order of minutes) yet for a mitogenie effect EGF must be present at the cell surface for at least 8 hours. At the present time it is difficult to reconcile all these observations but it may be that the existence of high and low affinity populations of EGFR whose relative levels can be perturbed by receptors for other growth factors and by receptor antibodies is a key factor in the response status of a cell. In conclusion, it seems to us that clustering per se is not a sufficient signal for mitogenic stimulation since it leads to conversion of EGF receptors to a low affinity state followed by rapid internalisation and degradation. Acknowledgements
Fig.
2.
Scatchard analysis of EGF binding to A-431 monolayers after preincubation with various concentrations Both incubations were of G49. carried out at 4%.
The Scatchard analyses show that G49 induces site-heterogeneity, producing a high and a low affinity population of receptors while not affecting the receptor number. Since G49 does not bind to the EGF binding site (it efficiently precipitates the receptor-EGF covalent complex) it is likely that some sort of allosteric modulation of This may be the receptor is occurring. via a classical allosteric effect where a monovalent (Fab) interaction with the receptor modifies its affinity for EGF or via a bivalent interaction where the antibody cross-links two receptors or groups of receptors with a resulting affinity change. We favour the latter interpretation for a It has been shown (3) number of reasons. that PDGF is able to induce the down-regulation of EGF receptors by, presumably, a PDGF-dependent co-clustering process. Similar effects have been observed with
We would like to thank the MedicalResearch Council for a grant and Sharon Smith for expert technical assistance. References 1.
Rees, A.R., Adamson, (1979) Nature 281,
E.D. & Graham,C.F. 309-311.
2.
Hock, R.A., Nexo, E. (1979) Nature 277,
& Hollenberg, 403-405.
3.
Wrann, 210,
El. & FOX,
C.Fred.
(1980)
M.D. Science
1363-1365.
4.
Rozengurt, E., Collins, & Pettican, P. (1982)
5.
Adamson, E.D. & Rees, & Cellular Biochem.
6.
Schecter, Y., Hernaez, L., Schlessinger. J. & cuatrecasas, P. (1979) Nature
257,
278,
M., Brown, K.D. J. Biol. Chem.
3680-3686.
835-838.
A.R. (1981) 34, 129-152.
Molec.