Insertion of Argos Sequences into the B-Loop of Epidermal Growth Factor Results in a Low-Affinity Ligand with Strong Agonistic Activity1'

Epidermal growth factor (EGF) 1 belongs to a family of structurally related growth factors which all exert their action by binding to the epidermal growth factor receptor (Carpenter & Wahl, 1991). Many tumor cells express this receptor, and also secrete members of this family of EGF-like molecules, thus creating the possibility of an autocrine growth factor cycle. In particular, the role of transforming growth factor a (TGF a) in the outgrowth of various tumors has been well established (Lee et al., 1995). Many studies have been performed on the structure—function relationship of EGFlike molecules, with the final aim of developing EGFreceptor antagonists, which can be used clinically to interfere with such autocrine processes (Carpenter & Wahl, 1991; Groenen et al., 1994; Prigent & Lemoine, 1992). 2D NMR studies have provided evidence that amino acids surrounding the second and sixth cysteine residues are in close contact with each other, a nonlinear receptor binding pocket (Hommel et al., 1992). Among these are Y13, L15, HI 6 , R41, E43, and L47 in hEGF. In addition, our previous data, using exchange mutants of hEGF and hTGFa, have shown that also R45 in hEGF belongs to the receptor binding domain (Van de Poll et al., 1995). There is still discrepancy in the literature as to whether the B-loop of hEGF, the main /9-sheet structure, located f This work was supported by Grant K U N 93-493 from the Dutch Cancer Society. * Corresponding author. Phone: ( 3 1 )24-3652707. Fax: (31 )243652999. E-mail: mvdpolI@ sci.kun.nl. ® Abstract published in Advance ACS Abstracts, June 1, 1997. 1 Abbreviations: h(m)EGF, human (murine) epidermal growth factor; hTGFa, human transforming growth factor a ; M APK, mitogenactivated protein kinase. between the third and fourth cysteine, is directly involved binding or only of structural importance for in ■o r V i determining the correct conformation required for binding. 123 and A30 cause a significant reduction in binding affinity [see for a recent review Groenen et al. (1994)], but EGF molecules with truncated forms of the B-loop have been claimed to be biologically active (Taggart et al., 1993). Domainstudies between EGF and TG Fa have also not given definite conclusions in this respect (Kramer et al., al., 1995). In recent years, various studies have been performed on the characterization of EGF-like molecules in Drosophila. Spitz, a 26 kDa trans