Assessment of biological activity of synthetic fragments of transforming growth factor‐alpha

Abstract

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.

Interaction of Transforming Growth Factor .alpha. with the Epidermal Growth Factor Receptor: Binding Kinetics and Differential Mobility within the Bound TGF-.alpha.

“…Over the past decade, various attempts have been made to define the functionally important domains and residues within EGF and TGF-a. These include recombinant DNA techniques to identify functional residues (Defeo-Jones et al, 1988; Lazar et al, 1988Lazar et al, , 1989Engler et al, 1988Engler et al, , 1991Matsunanmi et al, 1991), monoclonal antibodies studies to identify important peptide segments (Katsuura & Tanaka, 1989;Hoeprich et al, 1989;Richter et al, 1992), and the mitogenic activity of synthetic peptides derived from EGF and TGF-a (Heath & Merrifield, 1986; Darlak et al, 1988;Eppstein et al, 1989;Tam et al, 1991). These studies have implicated certain residues in TGF-a such as R42 and L48 (or R41 and L47 in EGF) as critical for function (Engler et al, 1990;Matsunanmi et al, 1991) and have suggested that large portions of more than one loop are required for mitogenic activity (Tam et al, 1991;Richter et al, 1992).…”

Multidomain binding of transforming growth factor .alpha. to the epidermal growth factor receptor

“…Measurements of the mitogenic activity of synthetic peptides have also been used to locate receptor-binding regions in TGFa and EGF, with an almost uniform lack of success. Hence, a large series of peptides, which included sequences cognate to the A-, B-, and C-loops of TGFa, failed to induce DNA synthesis (Defeo-Jones et al, 1988;Darlak et al, 1988, Tam et al, 1991. Several groups (Darlak et al, 1988;Eppstein et al, 1989) have also failed to reproduce an earlier claim (Nestor et al, 1985) that the linear peptide corresponding to residues 34-43 of TGFa blocks the mitogenic action of EGF.…”

Structure-function analysis of human transforming growth factor-α by site-directed mutagenesis

“…Structural analysis of TGF-has revealed that the six cysteine residues in the molecule define thr looped regions required for biological activity. Any disruption of disulphide bridges renders the protein inactive, and attempts to evoke a response using individual synthetic loops or linear peptides have been unsuccessful (Komoriya et al, 1984;Darlak et al, 1988;DeFeo-Jones et al, 1988). The structures of EGF and TGF-a are well documented (Montelione et al, 1986;Campbell et al, 1990) and we have found that the segment from Phe-15 to Asp-47 is conformationally well defined (Kline et al, 1990).…”