Our studies are directed towards understanding the mechanism of action and biological role of the newly-discovered human growth factor, amphiregulin. To address these issues, we utilize techniques common to protein biochemistry, cell biology and molecular biology. Recent work has focused on the identification of the cell surface receptor for amphiregulin. We have determined that amphiregulin acts by binding to the extracellular domain of the epidermal growth factor receptor, resulting in auto- phosphorylation on tyrosine residues, activation of the receptor tyrosine kinase activity and cell division. In addition, we have shown that the interaction of amphiregulin and the epidermal growth factor receptor results in activation of the c-erbB-2 receptor tyrosine kinase, a kinase which is believed to be involved in the pathogenesis of a number of human malignancies. Other studies have focused on the structural characterization of the amphiregulin molecule and identification of protein domains critical to the bioactivity of amphiregulin. We have identified and purified multiple heparin-binding, glycosylated biologically active forms of the amphiregulin molecule. The predominant form of amphiregulin which is secreted by human epithelial cells is a 16.5 kDa molecule that contains complex N-linked oligosaccharide which is rich in sialic acid. The 16.5 kDa form of AR also contains disaccharides linked to serine/threonine residues. However, a smaller 9.5 kDa non-glycosylated biologically active form of amphiregulin has been isolated which contains the epidermal growth factor-like domain but possesses a truncated NH2-terminal extension. These results revealed that the NH2-terminal region of the amphiregulin molecule is not critical to the ability of amphiregulin to activate the epidermal growth factor receptor tyrosine kinase. Current studies are directed towards identifying the heparin-binding domain of amphiregulin, understanding the role of cell surface proteoglycan in the mechanism of amphiregulin action and determining the biological consequence of targeting of amphiregulin to the nucleus of human cells.
