A specific binding site for a fragment of the B-loop of epidermal growth factor and related peptides Dmitry S. Gembitsky, Zsolt Bozso ´, Martina O’Flaharty, Ferenc O ¨ tvo ¨s, Richard F. Murphy, Sa ´ndor Lovas* Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, Nebraska 68178-0405, USA Received 11 May 2001; accepted 13 August 2001 Abstract Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125 I-EGF and [3'4'- 3 H-Tyr 22,29 , Abu 20,31 ]EGF(20 –31)-NH 2 . Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125 I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity. © 2002 Elsevier Science Inc. All rights reserved. Keywords: B-loop fragment; Epidermal growth factor; Specific binding; Transforming growth factor alpha 1. Introduction Epidermal growth factor (EGF) is a 53 residues polypep- tide which is present in many tissues and body fluids of mammals [4]. Transforming growth factor- (TGF-) has 35% sequence similarity (Fig. 1) with EGF and exhibits a spectrum of biological actions similar to those of EGF [10,11,29]. Other structurally and functionally related polypeptides, including vaccinia growth factor (VGF), am- phiregulin (ARG), epiregulin (ERG), heregulin- (HRG-), betacellulin (BTC) and heparin binding EGF-like growth factor (HB-EGF) have been described. The EGF receptor family is represented by four receptor tyrosine kinases erbB-1 (EGF receptor), erbB-2, erbB-3 and erbB-4 [21]. Upon binding of the ligand, homo- and/or heterodimeriza- tion of the erbB receptors results in activation of the intrin- sic kinase activity [5,16,21]. This triggers several signal transduction reactions which are crucial for the regulation of cell proliferation [20,24]. EGF, TGF-, VGF, ARG, ERG, BTC and HB-EGF bind to and activate erbB-1 [2,25,26]. HRG- and other heregulins bind to and activate erbB-3 and erbB-4 [5]. No ligand for erbB-2 is known but it can be activated through HRG- or EGF-induced heterodimeriza- tion with other members of the erbB receptor family [19, 28]. The structure of the EGF molecule in all species is highly conserved and consists of three loops which are held in place by three disulfide bonds [18,33]. In human (h), mouse (m), guinea pig (gp) and rat (r) EGFs, these bonds occur between Cys 6 and Cys 20 to form the A-loop, Cys 14 and Cys 31 to form the B-loop, and Cys 33 and Cys 42 to form the C-loop. This pattern of six Cys residues, the so-called EGF motif, is present in all other members of the EGF/TGF- peptide family (Fig. 1). The structure of EGF has been well characterized [3,8,18,22,33] and contains two domains. The “N domain” includes a helical segment in the A-loop and a triple-stranded -sheet incorporating the N-terminal tail and * Corresponding author. Tel.: +1-402-280-5753; fax: +1-402-280- 2690. E-mail address: slovas@bif1.creighton.edu (S. Lovas). Abbreviations used for amino acids and peptides follow the recom- mendations of the IUPAC-IUB Commission of Biochemical Nomencla- ture, Eur. J. Biochem. (1984) 138, 9 –37. Other abbreviations: Abu, -ami- nobutyric acid; DIEA, N, N' -diisopropylethylamine; DMF, N, N' - dimethylformamide; DMSO, dimethyl sulfoxide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(benzotriazol-1-yl)-N, N, N' , N' - tetramethyluronium hexafluorophosphate; HEPES, 4(-2-hydroxyethyl)-1- piperazineethanesulfonic acid; HOBt, 1-hydroxybenzotriazole; NMP, N- methylpyrrolidinone; OtBu, O-tret-butyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol. Peptides 23 (2002) 97–102 0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0196-9781(01)00584-8