HER4 RECEPTOR ACTIVATION AND PHOSPHORYLATION OF SHC PROTEINS BY RECOMBINANT HEREGULIN-FC FUSION PROTEINS

Heregulins (HRGs) are mosaic glycoproteins that bind to and induce the tyrosine phosphorylation of the HER4/p180(erbB4) receptor. This work was aimed at studying the biological effects induced by recombinant epidermal growth factor (EGF)-like domains of KRGs as well as identifying intracellular molecules involved in HERA signaling. To this end, we cloned the EGF-like domains of HRG-alpha, -beta 2, and -beta 3 into a eukaryotic expression vector in frame with sequences encoding a thrombin cleavage site followed by the Fc portion of a human IgG1. These chimeric genes directed the expression of recombinant fusion proteins, rHRGs-T-Fc, which specifically stimulated the phosphorylation of HER4/p180(erbB4). We also show that rHRG-alpha-T-Fc bound to human breast cancer cells that express HER4 receptors and induced the expression of intercellular adhesion molecule-1. After thrombin protease cleavage of rHRGs-T-Fc, their EGF-like domains were purified and shown to stimulate protein phosphorylation in HER4-expressing cells. Moreover, the rHRG-beta 2 EGF-like domain markedly induced the phosphorylation of She proteins on tyrosine, suggesting a role for these adaptor molecules in HRG-mediated signaling.