Identification Of The Interaction Site Of ErbB2 And Its Autoinhibitor And The Mechanism Of Cell Growth Inhibition By Herstatin

ErbB2, a 185 kDa receptor, is encoded by ErbB2 oncogene. It is one of the members of epidermal growth factor receptor (EGFR) family, which consists of four receptors including epidermal growth factor receptor (EGFR, also called HER-1 or ErbB-1), ErbB2 (HER-2), ErbB3 (HER-3) and ErbB4 (HER-4). ErbB2 overexpression is associated with poor progrnosis and short time to relapse. ErbB2 also enhances the metastatic potential of cancer cells. The most important intracellular signal transduction pathways activated by ErbB2 are Ras-MAPK and PI3K-Akt which play an important role in proliferation, differentiatio, even resistanse to TNF- α ,γ -radiation and chemotherapy. ErbB2 is frequently overexpressed in many human tumors. Therefore, ErbB2 is a good candidate target for immunotherapy and also a research hotspot. Recently, herstatin, an autoinhibitor of ErbB2, has been reported. It consists of subdomains L1 and S1 of ErbB2 and a novel C-terminal sequence encoded by intron 8. It has been demonstrated that Herstatin can bind to ErbB2 with high affinity. It disrupts the formation of heterodimers with EGFR or HER3 and homodimer with itself, reduces tyrosine phosphorlation of EGFR family and inhibits proliferation of cancer cells that overexpress ErbB2. However, the mechanism of the interaction between Herstatin and ErbB2 has not been fully understood and the binding sites are not clear now. The binding region of Herstatin on ErbB2 ectodomain might be a potential target region for the drug design.In the present study, the full-length sequence of Herstatin was amplified by over-lapping PCR. A plasmid containing Herstatin was constructed. The cells were transfected with the plasmid pcDNA3.1/Hst and Herstatin was expressed in the transfected cells. Herstatin mRNA and an about 60 KDa protein were detected by RT-PCR and West-blotting respectively. The results showed that Herstatin can inhibit cell proliferation and induce apoptosis by using Hoechst staining and flowcytometry.To investigate the interaction site on ErbB2 with Herstatin, the 3-D structure of Herstatin and ErbB2 complex was constructed by using computer-aided homology method. Based on the predicted secondary structure of C-terminal region of 79 aa by using GOR IV method, the 3-D model of the sequence of aa 341-419 representing C-terminal region of 79 aa was constructed by ab initio modeling method. The 3-D sequence of Herstatin from aa 1-340 were derived from the 3-D crystal structure of ErbB2 ECD. According to the 3-D crystal structure of ErbB2 ECD, the theoretical spatial structure of ErbB2 ECD was modeled with computer-aided homology method. Subsequently AUTODOCK3.0 program used to dock Herstatin- and ErbB2, the final optimized 3-D complex structure of ErbB2 ECD and Herstatin was modeled. The interaction region of ErbB2 ECD was at the C-terminal portion of S1, while the binding domain of Herstatin to ErbB2 ECD was at the 79 aa region encoded by intron 8.