Direct identification of residues of the epidermal growth factor receptor in close proximity to the amino terminus of bound epidermal growth factor.

Abstract

We have recently developed a kinetically controlled, step-wise affinity cross-linking technique for specific, high-yield, covalent linkage of murine epidermal growth factor (mEGF) via its N terminus to the EGF receptor. EGF receptor from A431 cells was cross-linked to radiolabeled mEGF (MImEGF) by this technique and the 12'I-mEGF-receptor complex was purified and denatured. (19)(20)(21)(22)(23), and functional analysis ofchicken/human receptor chimera (23, 24) and receptor deletion mutants (25). Covalent cross-linking of 1251-labeled EGF (125I-EGF) to the receptor has been used in previous work, in which identification of the portion of the receptor to which 125I-EGF was linked was deduced from the electrophoretic mobility and immunochemical reactivity of the labeled products of proteolytic and glycosylytic digests (26,27). Most recently, EGF bound to the soluble, extracytoplasmic portion of the receptor has been visualized in electron microscopic images (28) and crystallized for x-ray diffraction studies (29).It is convenient to define the extracellular portion of the human EGF receptor in terms of the following domains (26): a cysteine-poor N-terminal region (domain I, residues 1-146), and a second cysteine-poor region (domain III, residues 333-460) that separates two cysteine-rich regions (domain II, residues 147-332; domain IV, residues 461-621). The bulk of existing evidence, including the results of all cross-linking studies reported to date, points to domain III as containing determinants for receptor binding to EGF (20,23,24,26,27); however, some studies have also implicated domain I (23,25).We have recently developed a kinetically controlled, stepwise affinity cross-linking technique to achieve high yields of the EGF receptor covalently linked to the N terminus of murine EGF (mEGF) (30). Here we report purification of cross-linked species and conditions for denaturation and tryptic digestion of 125I-mEGF-linked receptor. A radiolabeled receptor fragment that did not comigrate in SDS/ Tricine gels with products resulting from tryptic digestion of 125I-mEGF was further purified; direct protein microsequencing revealed the uniquely migrating peptide to be derived from domain I of the extracellular portion of the EGF receptor. 11

Isolation and characterization of four alternate c-erbB3 transcripts expressed in ovarian carcinoma-derived cell lines and normal human tissues

“…Similarly, the proteins encoded by clones R1F and R2F contain subdomains I, II, and the N-terminal part of subdomain III. Hence, if ligand-binding by ErbB-3 is directed by subdomains I and/or III, as is the case for ErbB-1 (Woltjer et al, 1992), these truncated ErbB-3 products may also be able to bind to neuregulin. Since ErbB-3 can initiate signal transduction following neuregulin binding through heterodimerization with other members of the ErbB family, the expression of various truncated ErbB-3 receptors with varying anities for the neuregulins and/or with the potential to heterodimerize with membrane-bound ErbB receptors may add a new level of regulation to the growth control pathways mediated by this growth factor/receptor family.…”

Disabling Receptor Ensembles with Rationally Designed Interface Peptidomimetics

“…8 shows molecular models for possible arrangement of the erbB1⅐EGF complex. Based on the existing experimental evidence, this complex is likely to have a 2:2 receptor-ligand stoichiometry with EGF bound to both subdomains I and II (32)(33)(34)(35). There are two possible ways in which EGF can interact with subdomains I and III within the complex.…”

Expression of herstatin, an autoinhibitor of HER-2/neu, inhibits transactivation of HER-3 by HER-2 and blocks EGF activation of the EGF receptor

“…Subdomains I and II appear to be a repeating unit of III and IV that may have arose by a gene duplication event (Ullrich et al, 1984). Subdomain III contains the high a nity ligand binding site (Lax et al, 1989(Lax et al, , 1991Woltjer et al, 1992), and subdomain I may serve as a low a nity, promiscuous ligand binding site (Lax et al, 1989(Lax et al, , 1991Tzahar et al, 1997). Dimerization can be achieved in the absence of subdomains I and II, since their deletion from the EGF receptor results in constitutive dimerization and oncogenic transformation in a ligandindependent fashion (Carter and Kung, 1994;Moscatello et al, 1996;Qian et al, 1994).…”