Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

Abstract

A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-␣ in their recognition by the receptor. Cyclization of Gln 1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys 45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125ILabeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr 464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/ R45K is Lys 465 of the receptor.Epidermal growth factor (EGF) 1 and TGF␣ mediate their biological responses by binding to the EGF receptor in target cells (reviewed in Refs. 1-3). While the three-dimensional structures of mEGF (4, 5), hEGF (6), and TGF␣ (7) are known from high field NMR studies, no three-dimensional structure is yet available for the EGF receptor. Much structural information about the receptor has been gleaned, however, by mapping onto the derived primary sequence of the receptor (8) information from chemical, immunochemical, and molecular biological studies of the receptor (reviewed in Refs. 9 and 10). In the present study, we focus on the extracellular, ligand-binding domain of the receptor, identifying residues of the receptor adjacent to the carboxyl-terminal domain of bound EGF, a region shown to be important in the discrimination of EGF and TGF␣ by the EGF receptor (11).Murine EGF has no lysyl residues (12), so the ␣-amino terminus is the only primary amino group. We exploited this aspect of the chemistry of the hormone in a study in which the N terminus of wild-type mEGF was modified with SSFSB, a heterobifunctional cross-linking reagent, and affinity crosslinked to the receptor (13). The site of cross-linking was identified by Edman degradation of a purified fragment of the rece...

The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin

“…This bivalence model attributes formation of homo-and heterodimers to NDF isoform-speci®c selectivity of site 2 (Pinkas- Kramarski et al, 1996a). Two recent studies support relevance of a similar bivalence model to EGF: First, a nity labeling identi®ed two binding sites on ErbB-1, each located at the other side of the N-terminal cysteine-rich domain of this receptor, and interacts with one terminus of the ligand (Summer®eld et al, 1996). Second, all possible stoichiometries of EGF : ErbB-1 were measured in solution, but the predominant ratio determined was 2 : 2 (Lemmon et al, 1997).…”

Decorin Binds to a Narrow Region of the Epidermal Growth Factor (EGF) Receptor, Partially Overlapping but Distinct from the EGF-binding Epitope

“…According to the model structure (see Fig. 7A), His 394 -Ile 402 occur on the opposite side of the L2 domain putatively involved in EGF binding as evidenced by cross-linking experiments (55). This additional evidence suggests that His 394 -Ile 402 may not play a direct interacting role with EGF but are nevertheless likely to be important for the overall local conformation of L2.…”

“…In addition, this face contains Lys 465 (Fig. 6B), an amino acid that can be cross-linked to EGF (55). Although in the deletion mutant ⌬9 the top third of L2, which is closely associated with the hydrophobic patch of amino acids, was missing, there was still full EGF/EGFR and decorin/ EGFR interaction.…”

“…The various deleted regions of L2 are in purple. Lys 465 , which has been cross-linked to EGF (55), is shown in red. C, representative ␤-galactosidase assays for decorin/EGFR and EGF/EGFR interacting clones.…”

Disabling Receptor Ensembles with Rationally Designed Interface Peptidomimetics

“…The final monomeric structures were then obtained by running the CHARMm energy minimization in the Residue Topology File mode. To construct a dimeric erbB1⅐EGF model, the following assumptions were made based on the existing experimental evidence: erbB1⅐EGF complex has a 2:2 stoichiometry (32,33); the C-terminal part of subdomain IV is a dimeric interaction site (based on our results described below); the N terminus of bound EGF is close (within about 15 Å) to Tyr-101 (subdomain I) of erbB1 (34); the C-terminal Arg-45 of bound EGF is close (within about 15 Å) to Lys-465 (subdomain III) of erbB1 (35); the N terminus of EGF bound to erbB1 is about 67 Å (from 52 to 82 Å) away from the membrane surface (36); maximal dimensions of erbB1 are about 110 Å for the monomer and 120 Å for the dimer (32); EGF binds to the second face of subdomain III (37). Orientations of complex-forming two erbB1 and two EGF (Protein Data Bank code, 3EGF) molecules were adjusted manually to satisfy the criteria listed above based on two different models of the complex arrangement (see "Discussion").…”