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Dimerization of the Extracellular Domain of the Receptor for Epidermal Growth Factor Containing the Membrane-spanning Segment in Response to Treatment with Epidermal Growth Factor

1999; Elsevier BV; Volume: 274; Issue: 50 Linguagem: Inglês

10.1074/jbc.274.50.35985

1083-351X

Kirk Tanner, Jack Kyte,

Cell Adhesion Molecules Research

A recombinant fragment of the human receptor for epidermal growth factor containing both its extracellular domain and its membrane-spanning segment, when dissolved with Triton X-100, was observed to dimerize in response to addition of epidermal growth factor (EGF) even at the lowest concentration of this fragment that could be assayed (4 nm). Consequently, the dissociation constant for the dimer of this fragment is at least 10,000-fold smaller than that for dimers of soluble, recombinant forms of the extracellular domain lacking the membrane-spanning segment. The second-order rate constant for dimerization of the fragment containing the extracellular domain and the membrane-spanning segment was estimated to be greater than 0.3 nm−1min−1, more than 10-fold that of the native enzyme under the same conditions. This result suggests that the cytoplasmic domain of the intact enzyme sterically hinders its dimerization. When EGF is removed from the dimer of the fragment, the rate constant for its dissociation is greater than 0.2 min−1, more than 40-fold that of the native enzyme. This result suggests that interfaces between cytoplasmic domains of intact EGF receptor impart significant stabilization to the dimer of the enzyme. A recombinant fragment of the human receptor for epidermal growth factor containing both its extracellular domain and its membrane-spanning segment, when dissolved with Triton X-100, was observed to dimerize in response to addition of epidermal growth factor (EGF) even at the lowest concentration of this fragment that could be assayed (4 nm). Consequently, the dissociation constant for the dimer of this fragment is at least 10,000-fold smaller than that for dimers of soluble, recombinant forms of the extracellular domain lacking the membrane-spanning segment. The second-order rate constant for dimerization of the fragment containing the extracellular domain and the membrane-spanning segment was estimated to be greater than 0.3 nm−1min−1, more than 10-fold that of the native enzyme under the same conditions. This result suggests that the cytoplasmic domain of the intact enzyme sterically hinders its dimerization. When EGF is removed from the dimer of the fragment, the rate constant for its dissociation is greater than 0.2 min−1, more than 40-fold that of the native enzyme. This result suggests that interfaces between cytoplasmic domains of intact EGF receptor impart significant stabilization to the dimer of the enzyme. epidermal growth factor Ligand-induced activation and dimerization of receptors in the plasma membranes of cells has received much attention (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar, 2Siegel P.M. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8878-8883Crossref PubMed Scopus (92) Google Scholar, 3Philo J. Aoki K.H. Arakawa T. Narhi L.O. Wen J. Biochemistry. 1996; 35: 1681-1691Crossref PubMed Scopus (181) Google Scholar, 4Cohen B.D. Green J.M. Foy L. Fell H.P. J. Biol. Chem. 1996; 271: 4813-4818Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar). The dimerization of a monomeric form of the receptor for epidermal growth factor (EGF)1 is an inescapable step (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar) in the signal transduction initiated by this enzyme. The receptor for epidermal growth factor is composed of three domains (6Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L.V. Whittle N. Waterfield M.D. Seeburg P.H. Nature. 1984; 309: 418-425Crossref PubMed Scopus (1996) Google Scholar): an extracellular domain that binds (7Lax I. Mitra A.K. Ravera C. Hurwitz D.R. Rubinstein M. Ullrich A. Stroud R.M. Schlessinger J. J. Biol. Chem. 1991; 266: 13828-13833Abstract Full Text PDF PubMed Google Scholar) the EGF, a membrane-spanning segment, and an intracellular domain responsible for its protein tyrosine kinase activity (8Wedegaertner P.B. Gill G.N. J. Biol. Chem. 1989; 264: 11346-11353Abstract Full Text PDF PubMed Google Scholar). It is of interest to determine the role of each of these domains in the dimerization of the intact protein. In the experiments reported here, a recombinant form of the extracellular domain of human EGF receptor also containing the membrane-spanning segment (9Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C. Walton G.M. Channing J. Der H. Wiley S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (260) Google Scholar), dissolved in a solution of detergent, was assayed for its ability to undergo dimerization induced by EGF. Comparisons of the behavior of this extracellular domain of EGF receptor containing the membrane-spanning segment with that of the native, intact EGF receptor and with that of soluble, recombinant forms of the extracellular domain provide insight into the participation of the three domains in the process of dimerization. Cells of the murine B82 line expressing a recombinant form of EGF receptor that is missing its cytoplasmic domain and contains only the extracellular domain and the membrane-spanning segment (9Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C. Walton G.M. Channing J. Der H. Wiley S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (260) Google Scholar) were graciously provided by Dr. Gordon Gill, Department of Medicine, University of California at San Diego. These cells were grown to confluence, the plates were scraped to release the cells, and the resulting suspension was spun at 3,000 rpm for 30 min in a Sorvall SS-34 rotor at 4 °C. The pelleted cells were lysed (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar) by the addition of 4-fold of the pelleted cell volume of 1 mm EDTA, 5 mm ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, 5 mm 2-mercaptoethanol, 2 mmbenzamidinium chloride, 0.1 mm phenylmethanesulfonyl fluoride, 2.5 μg ml−1 aprotinin, 5 μg ml−1 leupeptin, and 20 mm HEPES, pH 7.4. An equal volume of 2% Triton X-100, 20% glycerol, and 30 mmHEPES, pH 7.4, was added to the cell lysates, and the mixture was homogenized with 15–20 strokes in a Dounce homogenizer at 0 °C and then clarified by centrifugation at 100,000 rpm for 30 min at 4 °C in a TLA-100.2 rotor in a Beckman T1-100 table top ultracentrifuge. The resulting extracts are referred to as detergent extracts of B82 cells. They were either used immediately or stored for future use at −70 °C. All dilutions of the detergent extract were made in 1% Triton X-100, 30 mm HEPES, pH 7.4. The states of dimerization of the two truncated forms of EGF receptor were determined by subjecting solutions containing these proteins to quantitative cross-linking with glutaraldehyde (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar). Solutions of the purified soluble, recombinant extracellular domain of human EGF receptor (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar) in 0.1 mNaCl, 0.5 mm dithiothreitol, 20 mm HEPES, pH 7.4, were graciously provided by Dr. Deborah Cadena, Department of Medicine, University of California at San Diego. These solutions were diluted to the desired concentration of protein with 30 mmHEPES, pH 7.4. Samples (0.3 ml) of the soluble, recombinant extracellular domain or of detergent extracts of B82 cells were mixed at room temperature with 0.15 ml of EGF in 150 mm NaCl, 0.1 mm EDTA, and 20 mm sodium phosphate, pH 7.2 (phosphate-buffered saline) or with phosphate-buffered saline alone to produce the noted final concentrations. The samples then stood at room temperature. Portions were removed at the noted times and cross-linked with glutaraldehyde (final concentration of 80 mm) for 1 min prior to quenching the cross-linking reaction with glycine (final concentration of 0.2 m). The proteins in the samples were unfolded in a solution of recrystallized SDS at 5 g SDS (g protein)−1 for 1 min at 100 °C prior to submitting them to electrophoresis on 6% polyacrylamide gels cast in a solution of 0.1% SDS (11Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The cross-linked polypeptides were then electrotransferred (12Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar) to membranes of polyvinylidene difluoride and immunostained (13Blake M.S. Johnston K.H. Russell-Jones G.J. Gotschlich E.C. Anal. Biochem. 1984; 136: 175-179Crossref PubMed Scopus (1631) Google Scholar). For immunostaining of blots of detergent extracts of B82 cells, a mouse monoclonal immunoglobulin (Genosys Biotechnologies, Inc.) raised against the synthetic peptide IQCAHYIDGPHC, corresponding to a region near the juxtamembrane section of the extracellular domain of EGF receptor, was used as the primary immunoglobulin. The intensity of the staining on immunoblots was quantified by scanning them with a reflectance densitometer (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar). The assays for the concentration of binding sites for EGF were performed as described by Sherrill & Kyte at a concentration of EGF of 3.3 μm (14Sherrill J.M. Kyte J. Biochemistry. 1996; 35: 5705-5718Crossref PubMed Scopus (47) Google Scholar). Several different concentrations of protein were assayed as well as a control with no protein. The counts per minute of specifically bound EGF were usually 10-fold greater in the samples with the highest concentration of protein than those for nonspecifically bound EGF in the control samples. When125I-EGF was used as the radioactive tracer, the supernatant fluids were removed, and both the pellets and the supernatants were submitted to γ-counting. In some samples, unlabeled EGF was added at a higher concentration than that of the125I-EGF to demonstrate that the bound radioactivity could be chased by unmodified ligand. A recombinant extracellular domain of human EGF receptor containing the membrane-spanning segment has been expressed in murine B82 cells (9Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C. Walton G.M. Channing J. Der H. Wiley S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (260) Google Scholar). This truncated version of EGF receptor contains the amino-terminal portion of the protein through arginine 647. It includes the entire membrane-spanning segment of the protein but does not include more than about six residues of the cytoplasmic domain. It was dissolved in a solution of Triton X-100 and tested to see if it could undergo dimerization in a manner dependent on the concentration of EGF as does the intact enzyme (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar). Samples of a detergent extract of B82 cells stood at room temperature with the indicated concentrations of EGF for 20 min prior to subjecting each sample to quantitative cross-linking with glutaraldehyde. The polypeptides in the samples were then separated by electrophoresis on polyacrylamide gels, electrotransferred to a membrane of polyvinylidene difluoride, and immunostained. The recombinant extracellular domain of EGF receptor containing the membrane-spanning segment was observed to dimerize readily in response to EGF (Fig. 1). In lanes 5, 6,7, and 8 of Fig. 1, the fractions of the protein that had dimerized were 0.09, 0.33, 0.44, and 0.55, respectively. The fraction of the protein that had dimerized was plotted as a function of the concentration of EGF for all of the data from experiments of this type, and the combined data were fit with a rectangular hyperbola. The apparent dissociation constant for EGF, as judged by the concentration of EGF producing half maximum dimerization, was found to be 500 ± 150 nm. In all of the experiments examining the dimerization of the recombinant extracellular domain containing the membrane-spanning segment, the maximum extent of dimerization observed at saturation with EGF was about 0.45 to 0.50. This result indicates that in the B82 cells, the expressed protein is present in at least two forms, one competent to dimerize, the other incompetent. Experiments were performed to monitor the dimerization of the extracellular domain of EGF receptor containing the membrane-spanning segment as a function of time. Samples of detergent extracts of B82 cells were mixed at room temperature with either phosphate-buffered saline or a saturating concentration of EGF in phosphate-buffered saline. At the noted times, samples were assayed for dimerization. The extracellular domain of EGF receptor containing the membrane-spanning segment in undiluted detergent extracts of B82 cells dimerized to the maximum extent within the 1 min resolution of the cross-linking reaction (Fig. 2). The fractions of the protein that had dimerized in the experiment presented in Fig. 2 were 0.47, 0.45, 0.46, and 0.45 at 1 min, 3 min, 5 min, and 10 min, respectively. Detergent extracts of B82 cells were diluted in an attempt to slow the presumably second-order dimerization of the extracellular domain of EGF receptor containing the membrane-spanning segment. Several dilutions (3-fold, 5-fold, 10-fold, and 20-fold) of the detergent extract of B82 cells were attempted. In each case, the dimerization of the extracellular domain of EGF receptor containing the membrane-spanning segment still proceeded too rapidly. At a dilution of 20-fold, the fractions of the protein that had dimerized were 0.44, 0.51, 0.50, and 0.49 at 1 min, 3 min, 5 min, and 10 min, respectively (Fig. 3). The molar concentration of extracellular domain of EGF receptor containing the membrane-spanning segment in the undiluted detergent extract of B82 cells used in this last experiment was estimated to be 150 nm on the basis of an assay for the binding of EGF (14Sherrill J.M. Kyte J. Biochemistry. 1996; 35: 5705-5718Crossref PubMed Scopus (47) Google Scholar). If it is assumed that the dimerization is greater than 50% complete in less than 1 min (Fig. 3) at a concentration of protein competent to dimerize of 2.5 nm, then the second-order rate constant for the dimerization of the protein must be greater than 0.3 nm−1 min−1. Under identical conditions of pH, temperature, and concentrations of glycerol and Triton X-100, the rate constant for the dimerization of intact native EGF receptor is 0.03 nm−1 min−1(1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar, 14Sherrill J.M. Kyte J. Biochemistry. 1996; 35: 5705-5718Crossref PubMed Scopus (47) Google Scholar). Therefore, the extracellular domain of EGF receptor containing the membrane-spanning segment dimerizes at a rate more than 10-fold faster than that for the dimerization of native, intact EGF receptor. In an attempt to slow the dimerization of the extracellular domain of EGF receptor containing the membrane-spanning segment even further, EGF was added to samples of a detergent extract of B82 cells at 0 °C. Even at the lower temperature, undiluted samples of the extract still dimerized completely within 1 min of the cross-linking reaction. Epidermal growth factor was then added to a sample of a detergent extract of B82 cells that had been diluted 10-fold and brought to 0 °C. The rate of dimerization of the extracellular domain of EGF receptor containing the membrane-spanning segment in these samples standing on ice was decreased sufficiently to observe the dimerization proceed over the course of 20 min (Fig. 4). In a previous study (15Tanner K. Biochemistry. 1997; 36: 14889-14896Crossref PubMed Scopus (5) Google Scholar), it was found that when intact, native EGF receptor was activated with saturating EGF to produce a fully dimerized and enzymatically active state and then passed over a column containing an immunoadsorbent made from anti-EGF immunoglobulins that could remove both the EGF in solution and any bound EGF, the unoccupied EGF receptor nevertheless remained dimerized and enzymatically active over extended periods of time. The rate of dissociation the dimerized recombinant extracellular domain of EGF receptor containing the membrane-spanning segment was assessed in undiluted detergent extracts of B82 cells that had been exposed to EGF for 5 min at room temperature prior to removing the EGF by passing the sample over the immunoadsorbent for EGF. Samples of a pool of the fractions of the eluate from the immunoadsorbent containing the protein were subjected to quantitative cross-linking with glutaraldehyde at various times over a period of 80 min (Fig. 5). The dimerized extracellular domain of EGF receptor containing the membrane-spanning segment had reverted completely to its monomeric form within 5 min following the removal of the EGF (lane 1). The readdition of EGF after its removal, however, was able to return the protein to its dimeric form (Lanes 5–8). This control shows that the immunoadsorbent had not affected the ability of the protein to dimerize but had merely removed the EGF. From these results, it can be estimated that the rate constant for the monomerization of the dimeric extracellular domain containing the membrane-spanning segment, following removal of the EGF, is greater than 0.2 min−1. This value is more than 50-fold greater than that of the rate constant (0.26 h−1) for the monomerization of dimeric native EGF receptor following the removal of EGF by the same immunoadsorbent under the same conditions (15Tanner K. Biochemistry. 1997; 36: 14889-14896Crossref PubMed Scopus (5) Google Scholar). In previous reports (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar), it has been demonstrated that soluble, recombinant forms of the extracellular domains of EGF receptor must be at high concentrations (40 μm or greater) to display dimerization. It was possible, however, that the conditions we were using to examine the dimerization of the extracellular domain containing the membrane-spanning segment, which occurred readily even at very low concentrations (2.5 nm), somehow dramatically enhanced the dimerization of the extracellular domain. Therefore, experiments were performed to assess the ability of a soluble, recombinant extracellular domain of EGF receptor to dimerize in response to treatment with EGF under the same conditions of pH, detergent concentration, and temperature as the experiments just described. The soluble, recombinant extracellular domain used in these experiments was the one expressed in Chinese hamster ovary cells by Cadena & Gill (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar), which included the amino-terminal domain of human EGF receptor through threonine 624. This protein was a gift of Dr. Deborah Cadena, who had purified it by chromatography on Sepharose to which wheat germ agglutinin had been attached. Each preparation had been routinely assayed by Dr. Cadena for the binding of EGF which displayed an apparent dissociation constant of 500 ± 200 nm (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar). The concentrations of the soluble, recombinant extracellular domain of EGF receptor used in these assessments of its ability to dimerize spanned a range of 1,000-fold (5 nm to 5 μm). In some experiments the concentration of the soluble, recombinant extracellular domain of EGF receptor was high enough (5 μm) to visualize it on polyacrylamide gels by staining them with Coomassie Brilliant Blue. Epidermal growth factor was incubated with the soluble, recombinant extracellular domain of EGF receptor at final concentrations as high as 30 μm, which is 60-fold higher than its apparent dissociation constant. Both quantitative cross-linking with glutaraldehyde (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar) and cross-linking with disuccinimidyl suberate (7Lax I. Mitra A.K. Ravera C. Hurwitz D.R. Rubinstein M. Ullrich A. Stroud R.M. Schlessinger J. J. Biol. Chem. 1991; 266: 13828-13833Abstract Full Text PDF PubMed Google Scholar) were performed. Most of these experiments were carried out in the absence of detergent, but in two experiments, one at 5 μm in soluble, recombinant extracellular domain, Triton X-100 and glycerol were added to 1 and 10%, respectively, to mimic more closely the conditions under which the detergent extracts of B82 cells expressing the soluble, recombinant extracellular domain of EGF receptor containing the membrane-spanning segment were examined. Experiments were also performed in which the cross-linking reaction with glutaraldehyde was extended from 1 to 10 min. No dimerization of the soluble, recombinant extracellular domain of EGF receptor induced by the addition of EGF was detected under any of these conditions. The concentration of EGF that was incubated with the soluble, recombinant extracellular domain of EGF receptor in these studies was always well above the reported dissociation constant of 500 nm. Using the same preparation of soluble, recombinant extracellular domain of EGF receptor, Cadena & Gill (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar) also could not detect any EGF-induced dimerization, as determined by cross-linking with glutaraldehyde, at 1.5 μm protein and 1.7 μm EGF. Neither our results nor those of Cadena & Gill (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar) are surprising, however, because concentrations of 45 μm or greater of a similar recombinant extracellular domain of EGF receptor were needed to observe dimerization in the experiments of Lemmon et al. (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar). To define more precisely the regions of EGF receptor that may form contacts that are important in its dimerization, a recombinant form of the extracellular domain of EGF receptor containing the membrane-spanning segment of the protein (9Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C. Walton G.M. Channing J. Der H. Wiley S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (260) Google Scholar) dissolved in a solution of Triton X-100 was examined to see if it would dimerize in response to addition of EGF. Not only did this fragment of the protein dimerize in response to treatment with EGF, but it did so at a rate more than 10-fold greater than that for the dimerization of intact native EGF receptor when the two proteins are assayed at the same concentration of EGF. This result suggests that regions of the cytoplasmic domain of native, intact EGF receptor interfere with its dimerization. It has been difficult to detect dimerization of soluble, recombinant forms of the extracellular domain of EGF receptor lacking the membrane-spanning domain. Several other investigators have had experiences similar to ours in being unable to detect dimerization of soluble, recombinant extracellular domains of human EGF receptor (20Günther N. Betzel C. Weber W. J. Biol. Chem. 1990; 265: 22082-22085Abstract Full Text PDF PubMed Google Scholar,21Greenfield C. Hils I. Waterfield M.D. Federwisch M.L. Wollmer A. Blundell T.L. McDonald N. EMBO J. 1989; 8: 4115-4123Crossref PubMed Scopus (140) Google Scholar). In other studies, however, it has been concluded that EGF is able to promote dimerization of the soluble, recombinant extracellular domain of EGF receptor when that domain is present at high concentrations. Hurwitz et al. (22Hurwitz D.R. Emanuel S.L. Nathan M.H. Sarver N. Ullrich A. Felder S. Lax I. Schlessinger J. J. Biol. Chem. 1991; 266: 22035-22043Abstract Full Text PDF PubMed Google Scholar) were able to detect small amounts of dimerization of a soluble, recombinant extracellular domain of EGF receptor produced in baculovirus by cross-linking with disuccinimidyl suberate. Under these conditions an almost equivalent amount of trimer and detectable amounts of tetramer were also observed (7Lax I. Mitra A.K. Ravera C. Hurwitz D.R. Rubinstein M. Ullrich A. Stroud R.M. Schlessinger J. J. Biol. Chem. 1991; 266: 13828-13833Abstract Full Text PDF PubMed Google Scholar, 22Hurwitz D.R. Emanuel S.L. Nathan M.H. Sarver N. Ullrich A. Felder S. Lax I. Schlessinger J. J. Biol. Chem. 1991; 266: 22035-22043Abstract Full Text PDF PubMed Google Scholar). The long cross-linking reaction with disuccinimidyl suberate at the high concentrations of EGF (0.1 mg ml−1) may have resulted in the cross-linking of the EGF itself, forming dimers, trimers, and tetramers, and these multivalent forms of the hormone may have led to the higher oligomers of the extracellular domain that were observed. In a separate study, Brown et al. (23Brown P.M. Debanne M.T. Grothe S. Bergsma D. Caron M. Kay C. O'Connor-McCourt M.D. Eur. J. Biochem. 1994; 225: 223-233Crossref PubMed Scopus (57) Google Scholar) presented data interpreted as demonstrating that the binding of EGF could induce dimerization of a soluble, recombinant extracellular domain of EGF receptor, but the concentration of protein used (170 μm) was so high that significant amounts of dimer were detected even in the absence of EGF, whereas incubation with high concentrations of EGF (340 μm) were required to produce increases in the yield of dimer and even then the monomer still predominated. The most striking experimental observation in all of these studies was that the concentrations of EGF required to observe significant amounts of the dimeric recombinant extracellular domain were anywhere from 100-fold to more than a 1,000-fold higher than the reported dissociation constants (22Hurwitz D.R. Emanuel S.L. Nathan M.H. Sarver N. Ullrich A. Felder S. Lax I. Schlessinger J. J. Biol. Chem. 1991; 266: 22035-22043Abstract Full Text PDF PubMed Google Scholar, 23Brown P.M. Debanne M.T. Grothe S. Bergsma D. Caron M. Kay C. O'Connor-McCourt M.D. Eur. J. Biochem. 1994; 225: 223-233Crossref PubMed Scopus (57) Google Scholar). One reason that such high amounts of EGF had to be used in some of these experiments was that the soluble, recombinant extracellular domain had to be at high concentrations to observe the dimerization. Nevertheless, one is left with the impression that the oligomerization that is occurring during the long times involved in the cross-linking of EGF receptor performed in many of these experiments is only indirectly related to the binding of EGF. The recent report by Lemmon et al. (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar), however, is significantly more convincing. They report that EGF induces the quantitative formation of dimers of a soluble, recombinant extracellular domain of EGF receptor, as assayed by small angle x-ray scattering, but only at high concentrations of the protein. This has been the only report that has demonstrated a quantitative dimerization of a soluble, recombinant extracellular domain of EGF receptor. Dimerization of the extracellular domain containing the membrane-spanning segment proceeded to completion at concentrations of competent protein as low as 2.5 nm (Fig. 3). Under identical conditions of pH, ionic strength, temperature, and concentrations of glycerol and Triton X-100, intact, native EGF receptor at concentrations as low as 2 nm also dimerizes completely upon addition of EGF (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar, 14Sherrill J.M. Kyte J. Biochemistry. 1996; 35: 5705-5718Crossref PubMed Scopus (47) Google Scholar). In contrast, to observe quantitative yields of dimerization of a soluble, recombinant extracellular domain lacking the membrane-spanning segment, Lemmonet al. (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar) used concentrations of the domain of 45 μm or greater. Consistent with their results, we and Cadena & Gill (10Cadena D.L. Gill G.N. Protein Expression Purif. 1993; 4: 177-186Crossref PubMed Scopus (13) Google Scholar) were unable to observe any dimerization of a similar soluble, recombinant extracellular domain at concentrations as high as 5 μm. The fact that the apparent dissociation constant for EGF of the extracellular domain containing the membrane-spanning segment, as judged by its effect on the yield of dimer (Fig. 1), and the apparent dissociation constant for the soluble, recombinant extracellular domain lacking the membrane-spanning segment, as judged by cross-linking [125I]iodinated EGF to its binding site, were both 500 nm suggests that comparisons of these two proteins should be meaningful. It is also reassuring that these values for the apparent dissociation constants compare favorably with values of 350 nm (21Greenfield C. Hils I. Waterfield M.D. Federwisch M.L. Wollmer A. Blundell T.L. McDonald N. EMBO J. 1989; 8: 4115-4123Crossref PubMed Scopus (140) Google Scholar), 250 nm (7Lax I. Mitra A.K. Ravera C. Hurwitz D.R. Rubinstein M. Ullrich A. Stroud R.M. Schlessinger J. J. Biol. Chem. 1991; 266: 13828-13833Abstract Full Text PDF PubMed Google Scholar), and 150 nm(22Hurwitz D.R. Emanuel S.L. Nathan M.H. Sarver N. Ullrich A. Felder S. Lax I. Schlessinger J. J. Biol. Chem. 1991; 266: 22035-22043Abstract Full Text PDF PubMed Google Scholar) for other preparations of soluble, recombinant EGF receptor. Nevertheless, it must be kept in mind that the conditions under which the dimerization of the soluble, recombinant extracellular domain and the dimerization of the extracellular domain containing the membrane-spanning segment were assessed in the present experiments were not identical because these two proteins were prepared by different protocols. The soluble, recombinant extracellular domain was provided to us as a protein that had been purified from the culture medium surrounding the Chinese hamster ovary cells that had excreted it, and the extracellular domain containing the membrane-spanning segment was expressed in murine B82 cells and tested for dimerization in the context of a crude lysate of these cells. We did examine these two proteins under identical conditions of temperature, pH, and detergent concentration. In fact, even at a 20-fold dilution, the condition most important for its comparison with the soluble, recombinant extracellular domain, the extracellular domain containing the membrane-spanning segment showed no decrease in the amount of dimerization (Fig. 3). Under these conditions, the remaining cytoplasm of the lysed cells had been diluted more than 200-fold over its concentration in the cell, and the solution should be even more similar to that in which the purified soluble, recombinant extracellular domain was dissolved. For all of these reasons, we believe that valid comparisons can be made between the behavior of these two proteins. Therefore, dimers of both intact, native EGF receptor and of the extracellular domain containing the membrane-spanning segment have dissociation constants at least 10,000-fold smaller than that of the soluble, recombinant extracellular domain. It follows that addition of the segment of the 23 amino acids GMVGALLLLLVVALGIGLFMRRR, which contains the sequence spanning the membrane with an additional three to six amino acids that extend into the cytoplasm, converts a protein that dimerizes reluctantly into a protein that dimerizes avidly in response to the addition of EGF. This observation suggests that rather than playing a passive role in the dimerization of EGF receptor produced by EGF, the two membrane-spanning domains from the two monomers of EGF receptor that dimerize upon the binding of EGF provide molecular surfaces that associate to yield a significant portion of the standard free energy of dimerization. This possibility has always been an implication of the fact that the mutation of valine 664 to a glutamate (17Bargmann C.I. Hung M. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (812) Google Scholar) in the membrane-spanning segment of the rat Neu protein turns an inactive, monomeric protein tyrosine kinase into a constitutively active (18Bargmann C.I. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5394-5398Crossref PubMed Scopus (178) Google Scholar), constitutively dimeric (19Weiner D.B. Liu J. Cohen J.A. Williams W.V. Greene M.I. Nature. 1989; 339: 230-231Crossref PubMed Scopus (358) Google Scholar) protein tyrosine kinase. It has been argued that the high concentrations of soluble, recombinant extracellular domain that are necessary to observe dimerization result from the fact that the protein is in free solution rather than in the membrane (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar). While it is true that intact EGF receptor in a cell membrane is restricted to diffusion in two, rather than three, dimensions, native intact EGF receptor or its extracellular domain containing the membrane-spanning segment when they are dissolved in solutions of Triton X-100 have the same degrees of diffusional freedom as a soluble, recombinant extracellular domain of EGF receptor, yet high concentrations of these proteins are not required to observe the dimerization produced by EGF. Because a 1% solution of Triton X-100 is 0.1 mm in micelles (24Yedgar S. Barenholz Y. Cooper V.G. Biochim. Biophys. Acta. 1974; 363: 98-111Crossref PubMed Scopus (100) Google Scholar, 25Kushner L.M. Hubbard W.D. J. Phys. Chem. 1954; 58: 1163-1167Crossref Scopus (134) Google Scholar), a concentration over 1,000-fold greater than the concentration of native EGF receptor in even undiluted detergent extracts of A431 cells, it can be concluded that the small minority of the micelles that are occupied by molecules of native EGF receptor or the extracytoplasmic domain containing the membrane-spanning segment will contain only one molecule of the respective monomeric protein. In fact, if a significant fraction of the molecules of unactivated monomeric intact EGF receptor or monomeric unactivated extracellular domain containing the membrane-spanning segment shared the same micelle with any protein, let alone one of its twins, then cross-linking of it with these other molecules of protein would be unavoidable because of their close proximity, and clear bands of immunostained monomeric protein would not be observed on the respective Western blots, but they are (1Canals F. Biochemistry. 1992; 31: 4493-4501Crossref PubMed Scopus (72) Google Scholar). If each molecule of monomeric native EGF receptor in these solutions is alone in its micelle, there is no fundamental difference between its diffusion through the solution and the diffusion of the soluble, recombinant extracellular domain through the solution, and direct comparisons between the two in terms of dissociation constants are valid. Nor is it proper to consider the micelles of Triton X-100 as a separate phase (5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar) when evaluating the dissociation constants of the intact native EGF receptor or its extracellular domain containing the membrane-spanning segment. The proteins are equal to or greater in mass (molar masses of 71,300 g mol−1 and 132,000 g mol−1) than a micelle (24Yedgar S. Barenholz Y. Cooper V.G. Biochim. Biophys. Acta. 1974; 363: 98-111Crossref PubMed Scopus (100) Google Scholar, 25Kushner L.M. Hubbard W.D. J. Phys. Chem. 1954; 58: 1163-1167Crossref Scopus (134) Google Scholar) of Triton X-100 alone (molar mass of 90,000 g mol−1). Furthermore, the size of a micelle of Triton X-100 does not dramatically increase as phospholipid dissolves in it (24Yedgar S. Barenholz Y. Cooper V.G. Biochim. Biophys. Acta. 1974; 363: 98-111Crossref PubMed Scopus (100) Google Scholar, 26Robson R.J. Dennis E.A. Biochim. Biophys. Acta. 1979; 573: 489-500Crossref PubMed Scopus (27) Google Scholar); in the relevant range of mole fraction, as molecules of phospholipid are incorporated into a micelle, the number of molecules of Triton X-100 present increases at most by 50% (24Yedgar S. Barenholz Y. Cooper V.G. Biochim. Biophys. Acta. 1974; 363: 98-111Crossref PubMed Scopus (100) Google Scholar). Therefore, the mixed micelles of Triton X-100 and endogenous phospholipid surrounding the dissolved transmembrane segments of the proteins in the detergent extracts used in these experiments are simply accretions upon the proteins themselves (27Kyte J. Structure in Protein Chemistry. Garland, NY1995: 521-523Google Scholar). Consequently, the molar concentrations relevant to quantification of the equilibria are those of the complexes between the proteins and the detergent expressed in moles of complex for each liter of aqueous solution, not in moles for each liter of Triton X-100. Finally, it is unlikely that the simple fusion of the two micelles, one from each monomer of the native enzyme or of the extracellular domain containing the membrane-spanning segment, contributes any favorable free energy of association to the formation of a dimer. This conclusion follows from the fact that above the critical micelle concentration, increasing the concentration of Triton X-100 has little effect on the mass of either micelles of pure Triton X-100 or mixed micelles of phospholipid and Triton X-100 (24Yedgar S. Barenholz Y. Cooper V.G. Biochim. Biophys. Acta. 1974; 363: 98-111Crossref PubMed Scopus (100) Google Scholar, 25Kushner L.M. Hubbard W.D. J. Phys. Chem. 1954; 58: 1163-1167Crossref Scopus (134) Google Scholar). This observation indicates that a micelle represents the most stable oligomer of the detergent at the given pH, ionic strength, and temperature (16Tanford C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes. 2nd Ed. John Wiley & Sons, NY1980: 42-89Google Scholar), and doubling its size by fusion should, if anything, be unfavorable. When both free EGF and EGF bound to the extracellular domain of EGF receptor containing the membrane-spanning segment were removed from the solution by passage over an immunoadsorbent for EGF (15Tanner K. Biochemistry. 1997; 36: 14889-14896Crossref PubMed Scopus (5) Google Scholar), the dimeric protein reverted to monomer within 5 min of the removal of the EGF (Fig. 5). This behavior is distinct from the behavior of dimeric, enzymatically active intact EGF receptor from which the EGF has been removed by passage over the same immunoadsorbent. Unliganded dimer of intact EGF receptor remains dimerized over several hours (15Tanner K. Biochemistry. 1997; 36: 14889-14896Crossref PubMed Scopus (5) Google Scholar). If it is assumed that the half time for dissociation of the dimer of the extracellular domain containing the membrane-spanning segment is less that 4 min, then the rate constant for its dissociation must be greater than 0.2 min−1. The rate of dissociation of the unliganded native enzyme has been estimated to be 0.26 h−1. Therefore, the dissociation of a dimer of the extracellular domain containing the membrane-spanning segment is at least 40-fold faster than the dissociation of a dimer of the native enzyme. This result suggests that regions of the cytoplasmic domain of EGF receptor must form strong associations in the native dimer of the intact enzyme. The facts that the soluble, recombinant extracellular domain of EGF receptor does not avidly form dimers in response to addition of EGF, that adding EGF to the extracellular domain of EGF receptor containing the membrane-spanning segment results in its rapid dimerization in even the most dilute solutions of the protein, that this dimer rapidly dissociates upon removal of the EGF, and that intact EGF receptor remains dimerized even in the absence of EGF lead to a new proposal that is an alternative to that presented by Lemmon et al.(5Lemmon M.A. Bu Z. Ladbury J.E. Zhou M. Pinchasi D. Lax I. Engelman D.M. Schlessinger J. EMBO J. 1997; 16: 281-294Crossref PubMed Scopus (303) Google Scholar) for the mechanism of dimerization of EGF receptor. The unoccupied extracellular, ligand-binding domain of intact, native EGF receptor, rather than being the source of its dimerization, sterically inhibits its dimerization. The binding of EGF produces a conformational change in the extracellular domain relieving the steric inhibition. It is this relief of the steric effect that permits the protein to undergo rapid homodimerization. Some of the contacts that are necessary to realize this dimerization in the presence of EGF, must be in the segment of amino acids that spans the membrane because only feeble dimerization is observed with the extracellular domain alone. These contacts between membrane-spanning segments are sufficient to stabilize the dimer but only in the presence of EGF because, when EGF is removed from the dimer of the extracellular domain containing the membrane-spanning segment, it rapidly reverts to monomer. This suggests that the conformational change relieving the steric inhibition is rapidly reversible upon dissociation of EGF. When the cytoplasmic domain of EGF receptor is present, however, additional surfaces from these domains in the dimer of EGF receptor must interact favorably to provide a significant portion of the stabilization of the homodimer because dimerization of the intact native EGF receptor persists for hours following the removal of EGF by passage over the immunoadsorbent. We thank Dr. Gordon Gill, Department of Medicine, University of California at San Diego for providing us with the B-82 cells used in these experiments and for his consistent interest and encouragement. Samples of the purified soluble, recombinant extracellular domain of human EGF receptor were kindly provided by Dr. Deborah Cadena, Department of Medicine, University of California at San Diego.