The Intracellular Juxtamembrane Domain of the Epidermal Growth Factor (EGF) Receptor Is Responsible for the Allosteric Regulation of EGF Binding

We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238–19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain. We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238–19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain. The EGF 2The abbreviations used are: EGF, epidermal growth factor; CHO, Chinese hamster ovary. receptor is a tyrosine kinase composed of an ∼620-amino-acid extracellular domain that recognizes and binds EGF, a single pass α-helical transmembrane domain, and an intracellular tyrosine kinase domain, encompassing roughly residues 685–950 (1Ullrich 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 (1992) Google Scholar). In addition, the receptor contains an ∼230-amino-acid-long C-terminal tail that contains the bulk of the sites of receptor autophosphorylation (2Downward J. Parker P. Waterfield M.D. Nature. 1984; 311: 483-485Crossref PubMed Scopus (468) Google Scholar, 3Hsuan J.J. Totty N. Waterfield M.D. Biochem. J. 1989; 262: 659-663Crossref PubMed Scopus (49) Google Scholar, 4Margolis G. Li N. Koch A. Mohammadi M. Hurwitz D. Zilberstein A. Ullrich A. Pawson T. Schlessinger J. EMBO J. 1990; 9: 4375-4380Crossref PubMed Scopus (216) Google Scholar). An intracellular juxtamembrane domain of about 40 residues connects the transmembrane domain to the kinase domain and has been shown to be crucial in the allosteric activation of the EGF receptor kinase (5Thiel K.W. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19238-19243Crossref PubMed Scopus (118) Google Scholar, 6Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1172) Google Scholar). In the membrane, the EGF receptor exists as a monomer, but a wealth of data indicate that the binding of EGF induces the formation of EGF receptor dimers (7Clayton A.H.A. Walker F. Orchard S.G. Henderson C. Ruchs D. Rothacker J. Nice E.C. Burgess A.W. J. Biol. Chem. 2005; 280: 30392-30399Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Cochet C. Kashles O. Chambaz E.M. Borrello I. King C.R. Schlessinger J. J. Biol. Chem. 1988; 263: 3290-3295Abstract Full Text PDF PubMed Google Scholar, 9Liu P. Sudhaharan T. Koh R.M.L. Hwang L.C. Ahmed S. Maruyama I.N. Wohland T. Biophys. J. 2007; 93: 684-698Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Yarden Y. Schlessinger J. Biochemistry. 1987; 26: 1443-1451Crossref PubMed Scopus (443) Google Scholar). Dimerization appears to be mediated in large part by the extracellular domain of the receptor, which is comprised of four subdomains, designated I through IV. X-ray crystallography data suggest that in the absence of ligand, the extracellular domain is held in a closed configuration through the interaction of loops or arms that extend from the backs of subdomains II and IV (11Ferguson K.M. Berger M.B. Mendrola J.M. Cho H.-S. Leahy D.J. Lemmon M.A. Mol. Cell. 2003; 11: 507-517Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). Upon binding of EGF, this intramolecular tether is released, allowing the receptor to adopt an open conformation in which EGF is tightly bound between subdomains I and III. In this configuration, the “dimerization arm” that was previously involved in tethering the receptor closed mediates the formation of a back-to-back EGF receptor dimer (12Garrett T.P.J. McKern N.M. Lou M. Elleman T.C. Adams T.E. Lovrecz G.O. Zhu H.-J. Walker F. Frenkel M.J. Hoyne P.A. Jorissen R.N. Nice E.C. Burgess A.W. Ward C.W. Cell. 2002; 110: 763-773Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar, 13Ogiso H. Ishitani R. Nureki O. Fukai S. Yamanaka M. Kim J.-H. Saito K. Sakamoto A. Inoue M. Shirouzu M. Yokoyama S. Cell. 2002; 110: 775-787Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar). Analyses of the binding of 125I-EGF to its receptor have invariably resulted in concave up Scatchard plots that have been interpreted as indicating the presence of two classes of EGF binding sites. However, we have recently used global analysis of the binding of 125I-EGF to cells expressing increasing levels of EGF receptors to show that EGF binding is best described by a model involving negative cooperativity in an aggregating system (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar) (see Fig. 6). Ligand binding is negatively cooperative if the binding of ligand to the first site on a dimer reduces the affinity of the ligand for binding to the second site on the dimer. The concept of cooperativity only applies to existing dimers. It does not relate to the effect of ligand on the assembly or disassembly of those dimers. The effect of ligand on the formation of receptor dimers is captured in the concept of linkage (15Wong I. Lohman T.M. Methods Enzymol. 1995; 259: 95-127Crossref PubMed Scopus (41) Google Scholar, 16Wyman J. Gill S.J. Binding and Linkage: Functional Chemistry of Biological Macromolecules. University Science Books, Mill Valley, CA1990: 203-236Google Scholar). If ligand binding is positively linked to dimer formation, then ligand promotes the assembly of receptor dimers. In a monomer-dimer equilibrium, positive linkage arises when a ligand binds with higher affinity to the first site on the dimer than to the monomer. Under these circumstances, the ligand will preferentially bind to the dimer, shifting the equilibrium in favor of the dimeric species. In the case of the EGF receptor, biochemical data suggest that EGF induces receptor dimerization; however, evidence for positive linkage in binding studies has been lacking. By analyzing the binding of 125I-EGF to cells expressing various EGF receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is, in fact, positively linked to receptor dimerization. Autophosphorylation of the EGF receptor abolishes the positive linkage that is present during the initial phase of the ligand binding reaction. Negative cooperativity is present in both the phosphorylated and the non-phosphorylated states of the receptor. Structure-function analyses demonstrate that both cooperativity and linkage are lost when the EGF receptor is truncated immediately after the transmembrane domain. However, both forms of regulation are restored in receptors that include the additional 40 amino acids that correspond to the intracellular juxtamembrane domain. These data expand the role of the intracellular juxtamembrane domain to include the allosteric regulation of EGF binding by the extracellular domain and demonstrate the presence of inside-out signaling in the EGF receptor system. Construction of EGF Receptor Mutants and Plasmids—The V583D- and K721A-EGF receptor point mutants were constructed using the QuikChange mutagenesis kit (Stratagene). The c′698- and c′973-EGF receptors were generated using the QuikChange mutagenesis kit to introduce a stop codon immediately 3′ of the codon encoding Ala-698 or Phe-973 of the EGF receptor. The receptor J.L. Pike L.J. Biochemistry. PubMed Scopus Google Scholar) and the receptor J.L. Li Pike L.J. Biophys. 2006; PubMed Scopus Google Scholar) were generated as described mutants were and then the between the and sites as described (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar). These mutants are shown in Fig. were with cells using to the were by in were in and were and with of from to to 125I-EGF and was from 125I-EGF was using the of and J. PubMed Scopus Google Scholar). binding was in using and 125I-EGF and increasing of were on the of the cells were in and the were in The were to and for in a binding was by the data to a binding model and using the as binding were in and analysis were as described previously (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar). was used to the data from binding experiments to cells expressing increasing of EGF to the of EGF receptor (15Wong I. Lohman T.M. Methods Enzymol. 1995; 259: 95-127Crossref PubMed Scopus (41) Google Scholar). This can be from the of EGF receptors as by and Gill J. Gill S.J. Binding and Linkage: Functional Chemistry of Biological Macromolecules. University Science Books, Mill Valley, CA1990: 203-236Google Scholar). Receptor is as a of the receptor is to in the two of the for receptor were from of the number of receptors in a of in cells expressing a of S. Li Y. Pike L.J. Biophys. J. 2007; 93: Full Text Full Text PDF PubMed Scopus Google Scholar). the for are the of in the the for the for ligand binding in of on 125I-EGF these EGF receptor mutants were generated and a which not EGF receptors or were with these This to control the of EGF receptor by the of increasing of we have previously (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar) and as shown in Fig. the binding of 125I-EGF to cells expressing the EGF receptor a of binding that from to as the number of EGF of the data from binding the in the The are as the that were from the However, are as this is the used to The data are with (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar) and indicate that the affinity of EGF for binding to the receptor and the first site on the receptor dimer is and on the of is no binding of ligand to the dimer as with the monomer, no positive linkage. The binding of EGF to the second site on the dimer is of affinity of negative This is the the from to with increasing receptor higher receptor dimers are and are of the affinity sites present in the X-ray studies have shown that the EGF receptor exists in a configuration mediated by between the dimerization in II and the tethering in IV (11Ferguson K.M. Berger M.B. Mendrola J.M. Cho H.-S. Leahy D.J. Lemmon M.A. Mol. Cell. 2003; 11: 507-517Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). of in IV to was to the intramolecular tether and the binding of EGF F. Orchard S.G. Jorissen R.N. Hoyne P.A. Adams T.E. Ward C.W. Nice E.C. Burgess A.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this of the 125I-EGF binding we this in Fig. the binding for 125I-EGF binding to the receptor in cells expressing increasing levels of the receptor. the receptor, the binding for the receptor from to with increasing of can be from the the from the global of the data for the receptor are to those for the receptor. this does not to the ligand binding of the EGF receptor. of Receptor on Ligand binding was in cells expressing the K721A-EGF receptor In to the observed in cells expressing EGF receptor, the binding for the K721A-EGF receptor from to as the receptor number analysis of the data that were from those for EGF In the EGF receptor, the affinity of EGF for binding to the was the as its affinity for binding to the first site on the dimer. By in the K721A-EGF receptor, EGF bound to the with an affinity of but bound to the first site on the receptor dimer with an affinity of in the EGF receptor EGF binds with higher affinity to the first site on the dimer as with the monomer. is positive linkage between ligand binding and receptor dimerization. This is the from to with increasing receptor higher of receptor, dimers are and are of the affinity sites to which EGF can Negative cooperativity is present in the K721A-EGF receptor as the affinity of EGF for binding to the second site on the dimer was about than that for binding to the first site on the dimer. linkage between EGF binding and receptor dimerization was observed in an EGF receptor truncated the kinase domain. This receptor is but the C-terminal autophosphorylation in this Gill Cell. 1989; Full Text PDF PubMed Scopus Google Scholar, A. Gill 1990; PubMed Scopus Google Scholar). shown in Fig. the 125I-EGF binding of the c′973-EGF receptor were to those for the K721A-EGF from to with increasing of The were to those observed for the K721A-EGF receptor and indicate the presence of positive linkage with negative cooperativity in the truncated EGF receptor. of the in and that both positive linkage and negative cooperativity were present in the K721A-EGF receptor indicates that kinase is not required for these allosteric on EGF the domain was required for this an EGF receptor was generated in which the intracellular domain of the receptor been The 125I-EGF binding for this are shown in Fig. was a from to in the of the binding with increasing levels of receptor was as with was in the or c′973-EGF receptor analysis of the binding data that indicate a in both cooperativity and linkage in this EGF bound with only a higher affinity to the first site on the dimer as with the monomer. This is but than the in the and c′973-EGF receptors that the domain. In addition, the affinity of EGF for binding to the second site on the dimer is only than its affinity for binding to the first site on the dimer, a that is not These data indicate that both linkage and cooperativity are by of the intracellular domain. studies have the intracellular juxtamembrane domain of the EGF receptor in the allosteric activation of the kinase (5Thiel K.W. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19238-19243Crossref PubMed Scopus (118) Google Scholar, 6Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1172) Google Scholar). the role of this domain in the ligand binding of the receptor, a was generated in which the EGF receptor was truncated This a receptor that contains the extracellular and transmembrane the intracellular juxtamembrane domain but the of the kinase domain Methods Enzymol. PubMed Scopus Google Scholar) and the C-terminal shown in Fig. the binding of 125I-EGF to the receptor generated a of binding that from to as receptor levels with this the indicate that this positive the affinity of EGF for binding to the first site on the receptor dimer is higher than the affinity of EGF for binding to the receptor monomer. In addition, the negative cooperativity with EGF a affinity for binding to the second site on the receptor dimer as with the first site on the dimer These data suggest that the juxtamembrane domain is and to allosteric regulation of ligand binding on the EGF receptor. The of the juxtamembrane domain in cooperativity and linkage was by with an additional EGF receptor in the juxtamembrane domain. We have recently the signaling of an EGF receptor in which residues and the of the intracellular juxtamembrane domain were to allowing of the receptor these sites J.L. Pike L.J. Biochemistry. PubMed Scopus Google Scholar). This was in of only as receptor autophosphorylation as EGF receptor. this we generated 125I-EGF binding in cells expressing levels of receptors from to can be in Fig. binding were by a single to the binding of ligand to a single of sites with an affinity of the juxtamembrane domain both cooperativity and linkage in EGF It has been for that Scatchard analyses of the binding of 125I-EGF to its receptor concave up plots F. Orchard S.G. Jorissen R.N. Hoyne P.A. Adams T.E. Ward C.W. Nice E.C. Burgess A.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, D. P. Lemmon M.A. I. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. P. EMBO J. 1984; PubMed Scopus Google Scholar, M. Nature. PubMed Scopus Google Scholar). plots were interpreted as indicating the existence of two classes of EGF binding sites. However, using global analysis of 125I-EGF binding data from cells expressing levels of EGF receptors, we have recently that the in EGF binding is the of negative cooperativity in an aggregating system (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar). The experiments this to on the allosteric regulation of EGF binding and on the of the intracellular of the EGF receptor to the ligand binding of the extracellular domain. Our between the of linkage and in an as the EGF receptor, in which the binding of the ligand can the of a monomer-dimer equilibrium 6). If the affinity of a ligand for the dimeric of the receptor is higher than for the of the receptor ligand will preferentially bind to the and dimerization of the receptor. In this the system is to exhibit positive linkage between binding and dimerization. to a system in which the are system is negatively cooperative if binding of ligand to the first of the dimer the affinity with which ligand binds to the second in the dimer Both (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar) and analyses of the EGF receptor indicate the presence of negative cooperativity in EGF binding but show no linkage between ligand binding and receptor dimerization. This is as has been that binding of EGF to dimerization of its receptor (7Clayton A.H.A. Walker F. Orchard S.G. Henderson C. Ruchs D. Rothacker J. Nice E.C. Burgess A.W. J. Biol. Chem. 2005; 280: 30392-30399Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Cochet C. Kashles O. Chambaz E.M. Borrello I. King C.R. Schlessinger J. J. Biol. Chem. 1988; 263: 3290-3295Abstract Full Text PDF PubMed Google Scholar, 9Liu P. Sudhaharan T. Koh R.M.L. Hwang L.C. Ahmed S. Maruyama I.N. Wohland T. Biophys. J. 2007; 93: 684-698Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 10Yarden Y. Schlessinger J. Biochemistry. 1987; 26: 1443-1451Crossref PubMed Scopus (443) Google Scholar). The present an for the of linkage between EGF binding and receptor dimer Both the K721A-EGF receptor and the EGF receptor positive the binding of EGF to the receptor dimer was of higher affinity than the binding of EGF to the receptor monomer. We have previously a for the receptor (14Macdonald J.L. Pike L.J. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 112-117Crossref PubMed Scopus (138) Google Scholar) that contains a in the dimer that activation of the EGF receptor kinase X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1172) Google Scholar). The K721A-EGF receptor is The receptor is but the kinase domain be by The c′973-EGF receptor is but the C-terminal tail that contains the bulk of the sites for EGF receptor The of these receptors is that are not in to ligand This that in the unphosphorylated receptor, the binding of EGF is positively linked to receptor dimerization. In the receptor, dimerization to receptor activation and It is this that is captured in the binding as are to report on equilibrium binding of the receptor dimer to a in its affinity for By the appears to a on the binding of EGF to the as the affinity of EGF for receptor is higher than that for that be these to the affinity of EGF for receptor and dimers in the receptor. This the positive linkage that is present in the of ligand the mutants and c′973-EGF to exhibit positive linkage in the equilibrium binding the to the the of positive linkage between ligand binding and EGF receptor dimerization is to the EGF receptor positive linkage. However, the receptor the positive linkage is lost 6). autophosphorylation of the receptor to a in receptor affinity is not with the domain of the EGF receptor have that the C-terminal tail a conformation Gill J. Biol. Chem. Full Text PDF PubMed Google Scholar, Sci. 2006; PubMed Scopus Google Scholar, Sci. 2005; PubMed Scopus Google Scholar). In addition, we have recently a in the EGF receptor in cells using D. Pike L.J. J. Biol. Chem. 2008; Full Text Full Text PDF Scopus Google Scholar). the in ligand binding affinity in to an in the conformation of the domain of the receptor. This be to the the binding of and to the receptor, or the binding of that as a of receptor of the appears that the intracellular domain is to the extracellular domain to effect a in ligand binding This that is inside-out signaling in the EGF receptor system. the negative cooperativity observed the EGF receptor dimer is not by of the receptor. reduces the affinity of EGF for binding to both the first site and the second site on the dimer by about an of a the and receptors exhibit a of negative cooperativity appears to be of is that a that is distinct from that involved in positive linkage. both negative cooperativity and positive linkage to be on the presence of the juxtamembrane domain. In the absence of the domain of the receptor both positive linkage and negative cooperativity were the intracellular juxtamembrane domain restored both positive linkage and negative cooperativity to the presence of the juxtamembrane domain of the receptor the affinity of the receptor for ligand, is that to the presence of this intracellular domain is to the extracellular domain. These data evidence of inside-out signaling in the EGF receptor. It is that the of the juxtamembrane domain the or of the transmembrane that in the to the extracellular domain. The that the juxtamembrane domain to the both cooperativity and linkage is with the that the of the transmembrane and intracellular juxtamembrane is for the allosteric regulation of ligand binding to the EGF receptor. of the first residues of the intracellular juxtamembrane domain are or this is positively It has been that this with the negatively of the in the to the of this with the (1Ullrich 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 (1992) Google Scholar, S. M.J. D. J. 2005; PubMed Scopus Google Scholar). By S. J. G. I. O. 2005; PubMed Scopus Google Scholar, S. N. F. A. 2006; PubMed Scopus Google Scholar) have that a negatively of amino acids from residues in the C-terminal tail with the residues in the juxtamembrane domain of the EGF receptor. residues are in the c′698- and c′973-EGF receptors, both of which exhibit negative cooperativity and positive these negatively residues be involved in the allosteric regulation of EGF binding We favor the that the juxtamembrane domain negative cooperativity and positive linkage by with the or by in with the juxtamembrane domain of EGF receptor. will be to this and the conformation of the extracellular domain. In data demonstrate the existence of positive linkage between ligand binding and receptor dimerization only in the unphosphorylated EGF receptor. This an for the of equilibrium binding experiments on receptors to evidence for receptor dimer Negative cooperativity is in the receptor of its that is distinct from the phenomenon of linkage. Nonetheless, both linkage and cooperativity require the presence of the intracellular juxtamembrane domain. The fact that amino and the intracellular domain receptor binding affinity indicates that is inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (5Thiel K.W. Carpenter G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19238-19243Crossref PubMed Scopus (118) Google Scholar, 6Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1172) Google Scholar). Our experiments expand the role of this domain to include the allosteric control of ligand the of the juxtamembrane domain in the regulation of EGF receptor with