Structure-Function Studies of Ligand-Induced Epidermal Growth Factor Receptor Dimerization † Beena Neelam, ‡ Audrey Richter, ‡ Stephen G. Chamberlin, ‡ Sarah M. Puddicombe, ‡ Lynn Wood, Mary Beth Murray, § Krishnadas Nandagopal, § Salil K. Niyogi, § and Donna E. Davies* ,‡ CRC Wessex Medical Oncology Unit, Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, U.K., and Life Sciences DiVision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-8080 ReceiVed October 15, 1997; ReVised Manuscript ReceiVed December 29, 1997 ABSTRACT: We present a novel 96-well assay which we have applied to a structure-function study of epidermal growth factor receptor dimerization. The basis of the assay lies in the increased probability of EGFRs being captured as dimers by a bivalent antibody when they are immobilized in the presence of a cognate ligand. Once immobilized, the antibody acts as a tether, retaining the receptor in its dimeric state with a resultant 5-7-fold increase in binding of a radiolabeled ligand probe. When the assay was applied to members of the EGF ligand family, murine EGF, transforming growth factor alpha, and heparin- binding EGF-like growth factor were comparable with human EGF (EC 50 ) 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR 1-84 ), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 μM. We further probed the involvement of the C-tail and the conserved leucine residue in receptor dimerization by comparing the activities of two genetically modified EGFs (the chimera mEGF/TGFR 44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR 1-90 ) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFR 44-50 > L47A ) L15A . AR 1-90 > AR 1-84 . Although AR was much worse than predicted from its affinity, this defect could be partially rectified by co-localization of the immobilizing antibody with heparin. Thus, it seems likely that AR cannot dimerize the EGFR unless other accessory molecules are present to stabilize its functional association with the EGFR. The epidermal growth factor receptor (EGFR) 1 is a transmembrane receptor tyrosine kinase which plays a pivotal role in the regulation of normal cell growth and differentia- tion. It can be activated extracellularly by one of several ligands that share a common EGF structural motif character- ized by three loops imposed by intramolecular disulfide bonds (1). Six ligands have been well characterized to date: epidermal growth factor (EGF) (2), transforming growth factor alpha (TGFR)(3), amphiregulin (AR) (4), heparin-binding EGF-like growth factor (HB-EGF) (5), betacellulin (BTC) (6), and epiregulin (7). AR, HB-EGF, and BTC possess extended N-terminal tails that, in the case of AR and HB-EGF, include heparin binding motifs. Although it was originally believed that these ligands bind exclusively to the EGFR, BTC and HB-EGF have recently been shown to have a broader specificity binding to c-erbB4 as well as EGFR (8-10). Ligand binding to the EGFR promotes receptor dimerization which in turn results in elevation of its intrinsic tyrosine kinase activity, initiation of intracellular signal transduction, and ultimately a cellular response (11). Clinical interest in the EGFR stems from the observation that it is frequently overexpressed in a variety of common carcinomas where its expression is associated with disease recurrence, reduced survival, and the presence of metastases (12). Given that this receptor controls many cellular processes which can be subverted by the tumor cells to promote not only their growth, but also their survival and dissemination (13), considerable effort has been directed toward development of novel chemotherapeutic agents capable of blocking EGFR-mediated cellular activation. Although several candidate compounds have been developed which inhibit intracellular signal transduction from the EGFR (14), no effective agents have been developed which are capable of blocking ligand binding or receptor dimerization. However, by working extracellularly, such reagents would offer considerable advantages; they would bypass the requirement to cross the plasma membrane in order to reach a specific intracellular compartment, they would not be exposed to drug resistance mechanisms such as the multidrug † This work was supported by the Cancer Research Campaign, U.K. and by USDOE Contract DE-AC05-96OR22464 to Lockheed Martin Energy Research Corporation. M.B.M. was supported by NCI Post- doctoral Training Grant CA 09336, and K.N. by NSF Grant BES 9421774. * Author to whom correspondence should be addressed. ‡ CRC Wessex Medical Oncology Unit. § Oak Ridge National Laboratory. 1 Abbreviations: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor (h or m prefix refers specifically to the human or murine sequence, respectively); TGFR, transforming growth factor R; BTC, betacellulin; HB-EGF, heparin-binding EGF-like growth factor; AR, amphiregulin; aFGF, acidic fibroblast growth factor; HEPES, N-2- hydroxyethyl piperazine-N′-2-ethanesulfonic acid; ELISA; enzyme- linked immunosorbent assay; MoAb, monoclonal antibody; BSA, bovine serum albumin; TMB, 3, 3′, 5, 5′-tetramethylbenzidine dihy- drochloride; HSPG, heparan sulfate proteoglycan; ATCC, American Type Culture Collection; SAXS, small-angle X-ray scattering. 4884 Biochemistry 1998, 37, 4884-4891 S0006-2960(97)02548-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/20/1998