Identification of hepatopoietin dimerization, its interacting regions and alternative splicing of its transcription
2002
Li, Yong | Wei, Kaihua | Lu, Chengrong | Li, Yingxian | Li, Ming | Xing, Guichun | Wei, Handong | Wang, Qingming | Chen, Jizhong | Wu, Chutse | Chen, Huipeng | Yang, Songcheng | He, Fuchu
Hepatopoietin (HPO) is a novel human hepatotrophic growth factor. Recently, we demonstrated that the extracellular ligand form of HPO can stimulate proliferation of hepatic cells via its specific receptor, which is on the surface of these cells. Also, the intracellular form of HPO potentiates the transcriptional factor APâ1. Intriguingly, a variety of HPO complexes with different molecular masses were detected in hepatocytes. In this study, we screened a human fetal liver cDNA library using the yeast two hybrid system with HPO as bait, hoping to find an HPOâbinding protein. Surprisingly, HPO, itself, and a previously unidentified alternatively spliced form of HPO (named HPO23) were identified as interacting with HPO, indicating that HPO may exist as a homodimer or heterodimer. These results were further confirmed in vitro and in vivo. First, mass spectrometry analysis demonstrated that recombinant human HPO exist as a homodimer and that disulfide bonds were involved in the formation of rhHPO dimer. Secondly, a pullâdown experiment confirmed that GST–HPO and HA–HPO, could bind together in vitro. Using HPO and various HPO deletion mutants, we identified the extreme 30 amino acids at both Nâ and Câtermini are essential for HPO dimerization. Finally, Western blotting analysis demonstrated that HPO exists as two isoforms at 15âkDa and 23âkDa (HPO23) in liver cells, the 15âkDa species is located in nucleus, and the 23âkDa species mainly in the cytoplasm. These results implicated that the capacity of HPO to form both homodimers and heterodimers with the alternatively spliced forms might contribute to the existence of various HPO complexes in hepatic cells.
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