Structure−Function Studies of Ligand-Induced Epidermal Growth Factor Receptor Dimerization

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 (EC50 = 2nM); betacellulin, which has a broader receptor specificity, was slightly less effective. In contrast, amphiregulin (AR1-84), which has a truncated C-tail and lacks a conserved leucine residue, was ineffective unless used at >1 microM. 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/TGFalpha44-50 and the EGF point mutant L47A) and a C-terminally extended form of AR (AR1-90) with those of two other unrelated EGF mutants (I23T and L15A). The potency of these ligands was in the order EGF > I23T > mEGF/TGFalpha44-50 > L47A = L15A >> AR1-90 > AR1-84. 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.

Proteasome Inhibition Blocks Ligand-Induced Dynamic Processing and Internalization of Epidermal Growth Factor Receptor via Altered Receptor Ubiquitination and Phosphorylation

“…1D) for the EGFR-FLuc reporter degradation. This compared favorably to the known EC 50 of 16 ng/mL for EGF-induced EGFR dimerization documented in the literature (26). Consequently, a saturating concentration of EGF (100 ng/mL) was used in all subsequent experiments.…”

Successful Piglet Production in a Chemically Defined System for In-vitro Production of Porcine Embryos: Dibutyryl Cyclic AMP and Epidermal Growth Factor-family Peptides Support In-vitro Maturation of Oocytes in the Absence of Gonadotropins

“…In the present study, EGF and betacellulin worked well at 10 ng/ml, whereas amphiregulin was not effective at 10 ng/ml, but was effective at 1000 ng/ml. This difference in the effective concentration may be due to the difference in binding affinity, since the affinity of amphiregulin is several orders of magnitude less than that of EGF [26,27].…”

“…In the present study, EGF and betacellulin worked well at 10 ng/ml, whereas amphiregulin was not effective at 10 ng/ml, but was effective at 1000 ng/ml. This difference in the effective concentration may be due to the difference in binding affinity, since the affinity of amphiregulin is several orders of magnitude less than that of EGF [26,27].Furthermore, it is very interesting that a combination of EGF with another EGF-family member (amphiregulin or betacellulin), as compared with EGF alone, improved the percentage of oocytes developing to the metaphase-II stage. A similar synergistic effect on oocyte maturation has been observed in buffalo COCs exposed to EGF plus IGF-I [28].…”

Induction of amphiregulin by p53 promotes apoptosis via control of microRNA biogenesis in response to DNA damage

“…Soluble AREG directly binds to EGFR as a ligand to transmit growth signal (3). However, AREG is not able to activate EGFR signaling effectively compared with other EGFR ligands (4). It is well established that AREG translocates to the plasma membrane; however, few reports showed that AREG localizes in the nucleus (5,6).…”