Life Sciences, Vol . 24, pp . Printed in .the U .S .A .
1691-1698
Pergamon Press
SOLUBILIZATION OF MHMBRANH RHCHPTOR FOR HPIDHRMAL GROWTH FACTOR Graham Carpenter Departments of Biochemistry and Medicine (Dermatology) Vanderbilt University School of Medicine Nashville, Tennessee 37232 (Received in final form March 21,
1979)
Summary The membrane receptor for epidermal growth factor (HGF) has been eolubilized from A-431 tumor cells using Triton R-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1 .5 hrs and the requirement for polyethylene glycol precipitation to detect 1251-HGF :reProperties of the eolceptor complexes on membrane filters . ubilized HGF are characterized and compared to the properties The specific binding capacity of the particulate receptor . 125 1-HGF of the eolubilized HGF receptor was 8 .0 picomoles 60x of the binding capacbound per mg protein--approximately ity of particulate receptor preparations . Also, solubilization of the HGF receptor resulted in a 10-fold decrease in the affinity of the receptor for 12 5 1-EGF. The initial biochemical step by which eatracellular signals such as polypeptide hormones affect cellular proliferation and/or metabolism is the formation of specific hormone:receptor complexes on the plasma mem brane of sensitive cell populations . Understanding the biochemistry of this important interaction requires a system in which the molecular proThe perties of the two reactants, i .e . hormone and receptor, are known . biochemical properties of HGF (1), a small polypeptide (Mr ~ 6045) that enhances cell proliferation in vivo (1,2) and in cell culture (2-6), have Considerably less, however, is known concerning the nabeen described . ture and properties of the HGF receptor which has been difficult to solubilize from most tissues with retention of binding activity . The ability of lectins to block 125 1-EGF binding in intact cells (7) and the loos of binding capacity in a mutant cell line defective in glycoprotein syntheeis (B), suggest that the membrane receptor for EGF is a glycoprotein . Croaslinking reagents have been employed to produce covalent HGF:receptor complexes with molecular weights of about 190,000 in 3T3 cells (9) and a In the subunit molecular weight of 100,000 in rat liver membranes (10) . latter study crosalinked EGF:receptor complexes were reported at molecuIn intact cells it lar weights of 410,000 ; 200,000 ; 95,000 and 35,000 . has been demonstrated that EGF :receptor complexes are rapidly internalized by an endocytotic-like mechanism and eventually degraded is lysosomes (11,12) . The importance of the metabolism of the cell-bound growth factor to its biological activities hoe not, however, been ascertained.
0024-3205/79/181691-0702 .00/0
Copyright (c) 1979 Pergamon Press Ltd
1692
Receptor for Epidermal Growth Factor
Vol . 24, No . 18, 1979
Most cultured cells contain approximately 100,000 or leas EGF receptors per cell (9,13,4) ; however, the A-431 tumor cell line possesses approximately 2 x 10 receptors par cell (12) . This cell line, therefore, has been chosen to attempt the isolation of EGF receptors and to study the mechanism of action of EGF in a subcellular system . In a crude membrane fraction prepared from A-431 cells it was shown that high levels of specific receptors for EGF were present and that subsequent to the formation of EGF :receptor complexes in vitro the rate of protein phosphorylation was increased approximately three-fold (15) . As a first step in elucidating the nature of the EGF receptor and its interaction with membrane phosphorylation components, the solubilization of 125I-EGF binding activity from A-431 membranes is reported . Methode A-431 cells, a human epitheloid tumor cell line, were propagated in Falcon dishes containing Dulbecco's Modified Eagle Medium plus calf serum (10%), 20 mM HEPES buffer, pH 7 .4, and gentamycin . A crude membrane pre paration was prepared by the procedure described by Thom et al . (16) . The specific binding activity of the membrane preparation was 6-fold higher than that of intact cells and 81% of the total binding activity and 13% of the total cellular protein were recovered in the membrane preparation . Solubilization of the membranes was affected by incubating membranes (1 mg/ml) with Triton X-100 (1%) in 20 mM HEPES buffer, pH 7 .4 for 20 min at room temperature . The material was then centrifuged at 90,000 x g for 1 .5 hrs . The supernatant material was carefully decanted, kept at 5 ° , and used to measure 125 1-EGF binding . Protein was measured by the procedure of Bradford (17) using y-globulin as a standard . EGF, isolated from mouse submaxillary glands (18) and labeled with 1251 as previously described (11), was donated by Dr . Stanley Cohere, 125 1-EGF binding reactions were carried out in a Vanderbilt University . 200 ul volume which contained solubilized membrane protein (10-20 ug), 20 mM HEPES buffer, pH 7 .4, 0 .1% bovine serum albumin, Triton X-100 (0 .2%) and 125 1-EGF (4 .1 x 10 -8 M) . Non-specific binding was measured in collateral reaction mixtures which contained a 100-fold molar excess of unlabeled EGF . All results are expressed in terms of specific binding . Incubations were carried out at room temperature . To assay for the presence of soluble 125 1-EGF : receptor complexes, the polyethylene glycol precipitation technique described by Cuatrecasas (19) was employed . Tubes containing the reaction mixtures were kept at 25° and 0 .5 ml 0 .1% globulin in 0 .1 M phosphate buffer, pH 7 .4, and 0 .5 ml of 20 .4% polyethylene glycol 6000 (final concentration 8 .5%) were added . These mixtures were gently mixed and filtered within 5 min on EHWP Millipore filters (0 .5 u pore size) which had been presoaked in 0 .2% bovine serum albumin . The filters were washed with 6 ml of 8 .5% polyethylene glycol 6000 in 0 .1 M phosphate buffer, pH 7 .4 . The radioactivity retained on the filters was measured in a Nuclear Chicago gamma-spectrometer . Results and Discussion The objective of these experiments is to solubilize the EGF receptor and to measure its biochemical properties while in a soluble state . Detergent solubilized membrane proteins can be expected to aggregate if the concentration of detergent is decreased below a certain point which may vary depending upon the hydrophobic nature and concentration of individual proteins . In the experiment described in Fig . 1 the concentration of
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1693
_Ô X oU Z O m C) W
N
-PEG (Particulate Bindlnq)
TRITON X-100 (%) Fig. 1 Effect of Triton X-100 concentration on the assay of solubilized 1251-EGF:receptor complexes . Standard reaction mixtures containing 4 .1 x 10 -8 M 125 1-EGF (9667 cpm/ng), 10 .8 ug solubilized A-431 membrane pro tein, and Triton X-100 to yield the indicated concentrations were incubatOne-half the reaction mixtures were assayed for ~~ 5at 25° for 30 min. I-EGF binding by the addition of olyethylene glycol ("-") as described in Methods . The eatent of 1~5 I-EGF binding in the remaining tubes Binding in was determined without polyethylene glycol treatment (0-r0) . this latter set of reaction mixtures was measured by adding 0 .5 ml of O.1X y-globulin, 0.5 ml of water (minus polyethylene glycol), mixiag, filtering, and washing with 20 mM HEPES buffer, pH 7 .4
Triton X-100 present during the incubation of solubilized receptors with 125 1-EGF was varied and the retention of hormone:receptor complexes on 0 .5 yi filters was measured with or without polyethylene glycol precipitation . The results from this experiment show that at low concentrations of detergent (0 .05X) a large amount of binding activity (77X of the total) is retained on the filters in the absence of polyethylene glycol treatment . This is considered to reflect the aggregation of EGF receptors during the incubation period into particles large enough to be retained on the filter . At this reduced level of detergent only 23X of the total binding activity can be attributed to soluble 125 I-EGF:receptor complexes . If, however, the concentration of detergent present during the incubation is increased to 0.20X, no binding activity is retained on the filter un-
1694
Receptor for Epidermal Growth Factor
Vol . 24, No .
18, 1979
9
M
O î
a c~
0 z O
5
m u. c~
W
,~
N
3
Fig . 2 Effect of solubilized membrane protein concentration on 125 I_EGF binding . Standard reaction mixtures containing 1 .7 x 10 -8 M 1 25 1-EGF (12,385 cpm/ng), solubilized A-431 membrane protein at indicated concen trations, and Triton R-100 (0 .2X) were incubated at 25° for 60 min . The reaction mixtures were assayed for the extent of 125 1-EGF binding by polyethylene glycol treatment as described in Methods .
less the incubation mixtures are treated with polyethylene glycol to precipitate soluble hormone-receptor complexes . At this detergent concentration (0 .20%) the same results are also achieved using a smaller pore (0 .2 ru) membrane filter . Therefore, all subsequent incubations of solubilized EGF receptors with 125 1-EGF were carried out in reaction mixtures containing a final Triton R-100 concentration of 0 .2X . The data in Fig . 2 show that under these incubation conditions the extent of 125 1-EGF binding to solubilized receptor is linear between 4 and 64 tlg of solubilized protein . The effect of temperature on the time course of 125 1-EGF binding is shown in Fig . 3 . At each temperature an equilibrium level of binding
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1695
b
"o
X U O Z
W (~ W iy
TIME (MIN)
Fig . 3 Time course of 125 1-EGF binding to solubilized A-~31 membrane protein . Standard reaction mixtures containing 4 .1 a 10 - M 125 1-EGF (25,839 cpm/ng), 18 .7 pg solubilized A-431 membrane protein and Triton R-100 At the (0 .2%) were incubated at 0°, 25° or 37° for the indicated times . 125 1-EGF binding by times indicated reaction mixtures were assayed for polyethylene glycol treatment as described in Methods . of 125 1-EGF binding during was reached within 5-10 mina . The rate the first 30 sec at 25 ° was approximately Oî 13 picomoles of 1Z5I-EGF Previous studies of 25 I-EGF binding to particubound per sec per mg . late preparations of the EGF receptor from A-431 cells indicated as initial binding rate of 0.27 picomolea per sec per mg at 25° (Carpenter, The data in Fig . 3 also show Ring and Cohen, manuscript submitted) . that the level of equilibrium binding achieved with solubilized receptors The extent of is increased as the incubation temperature is raised . binding at 25 ° and 37 ° , respectively, was approximately 58x and 135x greater than that achieved at 0° . 125 1-EGF on the extent of The effect of increasing concentrations of EGF coacenligand binding to solubilized receptors is shown in ig . 4. trations of approximately 1 .6 x 10 -7 M and 1 .1 a 10-~ M were required for maximal and half-maximal binding, respectively . At these respective concentrations lOx and 1X of the total added 125 1-EGF was specifically bound . ~~gvious studies (Carpenter, Ring and Cohen, manuscript submitted) of I-EGF interaction with particulate preparations of EGF receptor from
1696
Receptor for Epidermal Growth Factor
Vol . 24, No . 18, 1979
N X O
En
0 z
O m w m w N
Fig . 4 Effect of increasing concentrations of 125 1-EGF on binding to soluStandard reaction mixtures containing bilized A-431 membrane protein . 18 .4 dug solubilized A-431 membrane protein, Triton X-100 (0 .27) and indicated concentrations of 125i2F~F (21,653 cpm/ng) were incubated at 25 ° 5 I-EGF binding was determined by polyethylfor 60 min . The extent of ene glycol treatment as described in Methods . A-431 cells showed that maximal an half-maximal binding were attained at ligand concentrations of 1 .6 x 10 - ~ and 1 .4 x 10 -9 M, respectively . Solubilization of the 8GF receptor with Triton X-100, therefore, produces an approximate 10-fold decrease in the affinity of the receptor for 125I_ EGF, A ScatChard plot of the binding data obtained with solubilized EGF receptors is curvilinear, as is the plot obtained with particulate receptor preparations . However, the available data do not indicate whether these results are due to heterogeneity of receptors or negative cooperativity . The maximum specific binding capacity of the soluble receptor, in the presence of saturating ligand concentrations at 25 ° , was 8 picomolea of 1 Z 5 I-EGF bound per mg protein . Under similar conditions, saturating ligand concentrations at 25 ° , particulate preparations of EGF receptors from A-431 cells bind 14 picomoles of 125 1-EGF per mg of protein (Carpenter, King and Cohen, manuscript submitted) . Triton Solubilization of the EGF receptor, therefore, not only lowers the affinity of the receptor for 1 25 1-EGF, but also decreases the specific binding capacity by by about 43i . These alterations of the binding capacity and affinity of the EGF receptor upon solubilization are not unexpected . Experiments
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1697
phoapholiare in progress to determine whether the addition of defined will restore its pids to the solubilized EGF receptor from A-431 cells binding characteristics. Acknowledgments This study was carried out with the technical assistance of Suzanne Rodell and the financial support of grant CA 24071 from the National Cancer Institute ; a Basil O'Connor Research Grant from the National Foua dation - March of Dimes; and Biomedical Research Support Grant RR-05424-16 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
S . COHEN (1962) J . Biol . Chem _237, 1555-1562 . G. CARPENTER and S. COHEN (1978) in Biochemical Actions of Hormones (G . Litwack, ed .) vol . V pp . 203-247, Academic Press, New York . M.D . HOLLENBSRG and P. CUATRECASAS (1973) Proc . Natl . Acad . Sci . U.S .A . 70, 2%4-2968 . G. CARPENTER and S. COHEN (1976) J. Cell . Phyaiol . 88, 227-238 . B. WESTSRMARR (1976) Biochem. Biophys. Res . Comet. 69,304-310 . D. GOSPODAROWICZ, G. GREENBURG, H . BIALECKI AND B. ZETTER (1977) In Vitro 14, 85-118 . G. CARPENTER and S. COHEN (1977) Biochem. Biophys . Res . Comet. 79, 545-552 . R.M. PRATT and I. PASTAN (1978) Nature 272, 68-70 . M. DAS, T. MIYARAWA, F.C . FOX, R.M. PRUSS, A. AHARONOV and H. HERSCHMAN (1977) Proc . Natl . Acad . Sci. U.S .A . 74, 2790-2794. N. SAHYOUN, R.A . HOCK and M.D . HOLLENBERG (1978) Proc . Natl . Acad . Sci . U.S .A . 75, 1675-1679 . G. CARPENTER and S. COHEN (1976) J . Cell Biol . 71, 159-171 . H. HAIGLER, J.F . ASH, S .J . SINGER and S . COHEN (1978) Proc . Natl . Acad . Sci . U.S .A . 75, 3317-3321. G. CARPENTER, R.J .LEMBACH, M. MORRISON and S. COHEN (1975) J . Biol . Chem . 250, 4297-4304 . M.D . HOLLENBERG and P . CUATRECASAS (1975) J . Biol . Chem . ~, 38453853 . G. CARPENTER, L . KING and S. COiD;N (1978) Nature 276, 409-410 . D. THOM, A.J . POWELL, C.W . LLOYD, D.A . REES (1977) Biochem. J. 168, 187-194. M.M. BRADFORD (1976) Anal . Biochem. 72, 248-254 . Biol . Chem . _247, 7609-7611 . C.R . SAVAGE, JR . and S . COHSN (1972) P. CUATRECASAS (1972) Proc . Natl . Acad . Sci . U .S .A . 69, 318-322 .
J.
1691-1698
Pergamon Press
SOLUBILIZATION OF MHMBRANH RHCHPTOR FOR HPIDHRMAL GROWTH FACTOR Graham Carpenter Departments of Biochemistry and Medicine (Dermatology) Vanderbilt University School of Medicine Nashville, Tennessee 37232 (Received in final form March 21,
1979)
Summary The membrane receptor for epidermal growth factor (HGF) has been eolubilized from A-431 tumor cells using Triton R-100. Operational criteria used to define solubilization include failure of the binding activity to be pelleted after centrifugation at 90,000 x g for 1 .5 hrs and the requirement for polyethylene glycol precipitation to detect 1251-HGF :reProperties of the eolceptor complexes on membrane filters . ubilized HGF are characterized and compared to the properties The specific binding capacity of the particulate receptor . 125 1-HGF of the eolubilized HGF receptor was 8 .0 picomoles 60x of the binding capacbound per mg protein--approximately ity of particulate receptor preparations . Also, solubilization of the HGF receptor resulted in a 10-fold decrease in the affinity of the receptor for 12 5 1-EGF. The initial biochemical step by which eatracellular signals such as polypeptide hormones affect cellular proliferation and/or metabolism is the formation of specific hormone:receptor complexes on the plasma mem brane of sensitive cell populations . Understanding the biochemistry of this important interaction requires a system in which the molecular proThe perties of the two reactants, i .e . hormone and receptor, are known . biochemical properties of HGF (1), a small polypeptide (Mr ~ 6045) that enhances cell proliferation in vivo (1,2) and in cell culture (2-6), have Considerably less, however, is known concerning the nabeen described . ture and properties of the HGF receptor which has been difficult to solubilize from most tissues with retention of binding activity . The ability of lectins to block 125 1-EGF binding in intact cells (7) and the loos of binding capacity in a mutant cell line defective in glycoprotein syntheeis (B), suggest that the membrane receptor for EGF is a glycoprotein . Croaslinking reagents have been employed to produce covalent HGF:receptor complexes with molecular weights of about 190,000 in 3T3 cells (9) and a In the subunit molecular weight of 100,000 in rat liver membranes (10) . latter study crosalinked EGF:receptor complexes were reported at molecuIn intact cells it lar weights of 410,000 ; 200,000 ; 95,000 and 35,000 . has been demonstrated that EGF :receptor complexes are rapidly internalized by an endocytotic-like mechanism and eventually degraded is lysosomes (11,12) . The importance of the metabolism of the cell-bound growth factor to its biological activities hoe not, however, been ascertained.
0024-3205/79/181691-0702 .00/0
Copyright (c) 1979 Pergamon Press Ltd
1692
Receptor for Epidermal Growth Factor
Vol . 24, No . 18, 1979
Most cultured cells contain approximately 100,000 or leas EGF receptors per cell (9,13,4) ; however, the A-431 tumor cell line possesses approximately 2 x 10 receptors par cell (12) . This cell line, therefore, has been chosen to attempt the isolation of EGF receptors and to study the mechanism of action of EGF in a subcellular system . In a crude membrane fraction prepared from A-431 cells it was shown that high levels of specific receptors for EGF were present and that subsequent to the formation of EGF :receptor complexes in vitro the rate of protein phosphorylation was increased approximately three-fold (15) . As a first step in elucidating the nature of the EGF receptor and its interaction with membrane phosphorylation components, the solubilization of 125I-EGF binding activity from A-431 membranes is reported . Methode A-431 cells, a human epitheloid tumor cell line, were propagated in Falcon dishes containing Dulbecco's Modified Eagle Medium plus calf serum (10%), 20 mM HEPES buffer, pH 7 .4, and gentamycin . A crude membrane pre paration was prepared by the procedure described by Thom et al . (16) . The specific binding activity of the membrane preparation was 6-fold higher than that of intact cells and 81% of the total binding activity and 13% of the total cellular protein were recovered in the membrane preparation . Solubilization of the membranes was affected by incubating membranes (1 mg/ml) with Triton X-100 (1%) in 20 mM HEPES buffer, pH 7 .4 for 20 min at room temperature . The material was then centrifuged at 90,000 x g for 1 .5 hrs . The supernatant material was carefully decanted, kept at 5 ° , and used to measure 125 1-EGF binding . Protein was measured by the procedure of Bradford (17) using y-globulin as a standard . EGF, isolated from mouse submaxillary glands (18) and labeled with 1251 as previously described (11), was donated by Dr . Stanley Cohere, 125 1-EGF binding reactions were carried out in a Vanderbilt University . 200 ul volume which contained solubilized membrane protein (10-20 ug), 20 mM HEPES buffer, pH 7 .4, 0 .1% bovine serum albumin, Triton X-100 (0 .2%) and 125 1-EGF (4 .1 x 10 -8 M) . Non-specific binding was measured in collateral reaction mixtures which contained a 100-fold molar excess of unlabeled EGF . All results are expressed in terms of specific binding . Incubations were carried out at room temperature . To assay for the presence of soluble 125 1-EGF : receptor complexes, the polyethylene glycol precipitation technique described by Cuatrecasas (19) was employed . Tubes containing the reaction mixtures were kept at 25° and 0 .5 ml 0 .1% globulin in 0 .1 M phosphate buffer, pH 7 .4, and 0 .5 ml of 20 .4% polyethylene glycol 6000 (final concentration 8 .5%) were added . These mixtures were gently mixed and filtered within 5 min on EHWP Millipore filters (0 .5 u pore size) which had been presoaked in 0 .2% bovine serum albumin . The filters were washed with 6 ml of 8 .5% polyethylene glycol 6000 in 0 .1 M phosphate buffer, pH 7 .4 . The radioactivity retained on the filters was measured in a Nuclear Chicago gamma-spectrometer . Results and Discussion The objective of these experiments is to solubilize the EGF receptor and to measure its biochemical properties while in a soluble state . Detergent solubilized membrane proteins can be expected to aggregate if the concentration of detergent is decreased below a certain point which may vary depending upon the hydrophobic nature and concentration of individual proteins . In the experiment described in Fig . 1 the concentration of
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1693
_Ô X oU Z O m C) W
N
-PEG (Particulate Bindlnq)
TRITON X-100 (%) Fig. 1 Effect of Triton X-100 concentration on the assay of solubilized 1251-EGF:receptor complexes . Standard reaction mixtures containing 4 .1 x 10 -8 M 125 1-EGF (9667 cpm/ng), 10 .8 ug solubilized A-431 membrane pro tein, and Triton X-100 to yield the indicated concentrations were incubatOne-half the reaction mixtures were assayed for ~~ 5at 25° for 30 min. I-EGF binding by the addition of olyethylene glycol ("-") as described in Methods . The eatent of 1~5 I-EGF binding in the remaining tubes Binding in was determined without polyethylene glycol treatment (0-r0) . this latter set of reaction mixtures was measured by adding 0 .5 ml of O.1X y-globulin, 0.5 ml of water (minus polyethylene glycol), mixiag, filtering, and washing with 20 mM HEPES buffer, pH 7 .4
Triton X-100 present during the incubation of solubilized receptors with 125 1-EGF was varied and the retention of hormone:receptor complexes on 0 .5 yi filters was measured with or without polyethylene glycol precipitation . The results from this experiment show that at low concentrations of detergent (0 .05X) a large amount of binding activity (77X of the total) is retained on the filters in the absence of polyethylene glycol treatment . This is considered to reflect the aggregation of EGF receptors during the incubation period into particles large enough to be retained on the filter . At this reduced level of detergent only 23X of the total binding activity can be attributed to soluble 125 I-EGF:receptor complexes . If, however, the concentration of detergent present during the incubation is increased to 0.20X, no binding activity is retained on the filter un-
1694
Receptor for Epidermal Growth Factor
Vol . 24, No .
18, 1979
9
M
O î
a c~
0 z O
5
m u. c~
W
,~
N
3
Fig . 2 Effect of solubilized membrane protein concentration on 125 I_EGF binding . Standard reaction mixtures containing 1 .7 x 10 -8 M 1 25 1-EGF (12,385 cpm/ng), solubilized A-431 membrane protein at indicated concen trations, and Triton R-100 (0 .2X) were incubated at 25° for 60 min . The reaction mixtures were assayed for the extent of 125 1-EGF binding by polyethylene glycol treatment as described in Methods .
less the incubation mixtures are treated with polyethylene glycol to precipitate soluble hormone-receptor complexes . At this detergent concentration (0 .20%) the same results are also achieved using a smaller pore (0 .2 ru) membrane filter . Therefore, all subsequent incubations of solubilized EGF receptors with 125 1-EGF were carried out in reaction mixtures containing a final Triton R-100 concentration of 0 .2X . The data in Fig . 2 show that under these incubation conditions the extent of 125 1-EGF binding to solubilized receptor is linear between 4 and 64 tlg of solubilized protein . The effect of temperature on the time course of 125 1-EGF binding is shown in Fig . 3 . At each temperature an equilibrium level of binding
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1695
b
"o
X U O Z
W (~ W iy
TIME (MIN)
Fig . 3 Time course of 125 1-EGF binding to solubilized A-~31 membrane protein . Standard reaction mixtures containing 4 .1 a 10 - M 125 1-EGF (25,839 cpm/ng), 18 .7 pg solubilized A-431 membrane protein and Triton R-100 At the (0 .2%) were incubated at 0°, 25° or 37° for the indicated times . 125 1-EGF binding by times indicated reaction mixtures were assayed for polyethylene glycol treatment as described in Methods . of 125 1-EGF binding during was reached within 5-10 mina . The rate the first 30 sec at 25 ° was approximately Oî 13 picomoles of 1Z5I-EGF Previous studies of 25 I-EGF binding to particubound per sec per mg . late preparations of the EGF receptor from A-431 cells indicated as initial binding rate of 0.27 picomolea per sec per mg at 25° (Carpenter, The data in Fig . 3 also show Ring and Cohen, manuscript submitted) . that the level of equilibrium binding achieved with solubilized receptors The extent of is increased as the incubation temperature is raised . binding at 25 ° and 37 ° , respectively, was approximately 58x and 135x greater than that achieved at 0° . 125 1-EGF on the extent of The effect of increasing concentrations of EGF coacenligand binding to solubilized receptors is shown in ig . 4. trations of approximately 1 .6 x 10 -7 M and 1 .1 a 10-~ M were required for maximal and half-maximal binding, respectively . At these respective concentrations lOx and 1X of the total added 125 1-EGF was specifically bound . ~~gvious studies (Carpenter, Ring and Cohen, manuscript submitted) of I-EGF interaction with particulate preparations of EGF receptor from
1696
Receptor for Epidermal Growth Factor
Vol . 24, No . 18, 1979
N X O
En
0 z
O m w m w N
Fig . 4 Effect of increasing concentrations of 125 1-EGF on binding to soluStandard reaction mixtures containing bilized A-431 membrane protein . 18 .4 dug solubilized A-431 membrane protein, Triton X-100 (0 .27) and indicated concentrations of 125i2F~F (21,653 cpm/ng) were incubated at 25 ° 5 I-EGF binding was determined by polyethylfor 60 min . The extent of ene glycol treatment as described in Methods . A-431 cells showed that maximal an half-maximal binding were attained at ligand concentrations of 1 .6 x 10 - ~ and 1 .4 x 10 -9 M, respectively . Solubilization of the 8GF receptor with Triton X-100, therefore, produces an approximate 10-fold decrease in the affinity of the receptor for 125I_ EGF, A ScatChard plot of the binding data obtained with solubilized EGF receptors is curvilinear, as is the plot obtained with particulate receptor preparations . However, the available data do not indicate whether these results are due to heterogeneity of receptors or negative cooperativity . The maximum specific binding capacity of the soluble receptor, in the presence of saturating ligand concentrations at 25 ° , was 8 picomolea of 1 Z 5 I-EGF bound per mg protein . Under similar conditions, saturating ligand concentrations at 25 ° , particulate preparations of EGF receptors from A-431 cells bind 14 picomoles of 125 1-EGF per mg of protein (Carpenter, King and Cohen, manuscript submitted) . Triton Solubilization of the EGF receptor, therefore, not only lowers the affinity of the receptor for 1 25 1-EGF, but also decreases the specific binding capacity by by about 43i . These alterations of the binding capacity and affinity of the EGF receptor upon solubilization are not unexpected . Experiments
Vol . 24, No . 18, 1979
Receptor for Epidermal Growth Factor
1697
phoapholiare in progress to determine whether the addition of defined will restore its pids to the solubilized EGF receptor from A-431 cells binding characteristics. Acknowledgments This study was carried out with the technical assistance of Suzanne Rodell and the financial support of grant CA 24071 from the National Cancer Institute ; a Basil O'Connor Research Grant from the National Foua dation - March of Dimes; and Biomedical Research Support Grant RR-05424-16 . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
S . COHEN (1962) J . Biol . Chem _237, 1555-1562 . G. CARPENTER and S. COHEN (1978) in Biochemical Actions of Hormones (G . Litwack, ed .) vol . V pp . 203-247, Academic Press, New York . M.D . HOLLENBSRG and P. CUATRECASAS (1973) Proc . Natl . Acad . Sci . U.S .A . 70, 2%4-2968 . G. CARPENTER and S. COHEN (1976) J. Cell . Phyaiol . 88, 227-238 . B. WESTSRMARR (1976) Biochem. Biophys. Res . Comet. 69,304-310 . D. GOSPODAROWICZ, G. GREENBURG, H . BIALECKI AND B. ZETTER (1977) In Vitro 14, 85-118 . G. CARPENTER and S. COHEN (1977) Biochem. Biophys . Res . Comet. 79, 545-552 . R.M. PRATT and I. PASTAN (1978) Nature 272, 68-70 . M. DAS, T. MIYARAWA, F.C . FOX, R.M. PRUSS, A. AHARONOV and H. HERSCHMAN (1977) Proc . Natl . Acad . Sci. U.S .A . 74, 2790-2794. N. SAHYOUN, R.A . HOCK and M.D . HOLLENBERG (1978) Proc . Natl . Acad . Sci . U.S .A . 75, 1675-1679 . G. CARPENTER and S. COHEN (1976) J . Cell Biol . 71, 159-171 . H. HAIGLER, J.F . ASH, S .J . SINGER and S . COHEN (1978) Proc . Natl . Acad . Sci . U.S .A . 75, 3317-3321. G. CARPENTER, R.J .LEMBACH, M. MORRISON and S. COHEN (1975) J . Biol . Chem . 250, 4297-4304 . M.D . HOLLENBERG and P . CUATRECASAS (1975) J . Biol . Chem . ~, 38453853 . G. CARPENTER, L . KING and S. COiD;N (1978) Nature 276, 409-410 . D. THOM, A.J . POWELL, C.W . LLOYD, D.A . REES (1977) Biochem. J. 168, 187-194. M.M. BRADFORD (1976) Anal . Biochem. 72, 248-254 . Biol . Chem . _247, 7609-7611 . C.R . SAVAGE, JR . and S . COHSN (1972) P. CUATRECASAS (1972) Proc . Natl . Acad . Sci . U .S .A . 69, 318-322 .
J.
