Structure-Function Studies of Ligand-Induced Epidermal Growth Factor Receptor Dimerization
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- 作者：Beena Neelam ; Audrey Richter ; Stephen G. Chamberlin ; Sarah M. Puddicombe ; Lynn Wood ; Mary Beth Murray ; Krishnadas Nandagopal ; Salil K. Niyogi ; and Donna E. Davies
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 7, 1998
- 年：1998
- 卷：37
- 期：14
- 页码：4884 - 4891
- 全文大小：128K
- 年卷期：v.37,no.14(April 7, 1998)
- ISSN：1520-4995
文摘
We present a novel 96-well assay which we have appliedto a structure-function study ofepidermal growth factor receptor dimerization. The basis of theassay lies in the increased probability ofEGFRs being captured as dimers by a bivalent antibody when they areimmobilized in the presence of acognate ligand. Once immobilized, the antibody acts as a tether,retaining the receptor in its dimericstate with a resultant 5-7-fold increase in binding of a radiolabeledligand probe. When the assay wasapplied to members of the EGF ligand family, murine EGF, transforminggrowth factor alpha, and heparin-binding EGF-like growth factor were comparable with human EGF(EC50 = 2nM); betacellulin, whichhas a broader receptor specificity, was slightly less effective.In contrast, amphiregulin(AR1-84), whichhas a truncated C-tail and lacks a conserved leucine residue, wasineffective unless used at >1 
f">M. Wefurther probed the involvement of the C-tail and the conserved leucineresidue in receptor dimerizationby comparing the activities of two genetically modified EGFs (thechimera mEGF/TGF
fchars/alpha.gif" BORDER=0>44-50 andtheEGF point mutant L47A) and a C-terminally extended form of AR(AR1-90) with those of twootherunrelated EGF mutants (I23T and L15A). The potency of theseligands was in the order EGF > I23T >mEGF/TGFfchars/alpha.gif" BORDER=0>44-50 > L47A = L15A
f"> AR1-90 >AR1-84. Although AR was muchworse than predictedfrom its affinity, this defect could be partially rectified byco-localization of the immobilizing antibodywith heparin. Thus, it seems likely that AR cannot dimerize theEGFR unless other accessory moleculesare present to stabilize its functional association with theEGFR.
f">M. Wefurther probed the involvement of the C-tail and the conserved leucineresidue in receptor dimerizationby comparing the activities of two genetically modified EGFs (thechimera mEGF/TGFfchars/alpha.gif" BORDER=0>44-50 andtheEGF point mutant L47A) and a C-terminally extended form of AR(AR1-90) with those of twootherunrelated EGF mutants (I23T and L15A). The potency of theseligands was in the order EGF > I23T >mEGF/TGFfchars/alpha.gif" BORDER=0>44-50 > L47A = L15Af"> AR1-90 >AR1-84. Although AR was muchworse than predictedfrom its affinity, this defect could be partially rectified byco-localization of the immobilizing antibodywith heparin. Thus, it seems likely that AR cannot dimerize theEGFR unless other accessory moleculesare present to stabilize its functional association with theEGFR.