Nrg-1 Induced Erbb Heterodimer Trans Phosphorylation And Its Downstream Signaling Pathway Research

ErbB Receptors belong to subclass I of the superfamily of receptor tyrosine kinases (RTKs), also termed EGFR family. There are four members of the EGFR family, ErbB1(EGFR), ErbB2 (neu/p185), ErbB3 and ErbB4. ErbB2 does not directly bind to ligands, but serves as a heterodimer partner for ErbB3 or ErbB4. Neuregulin-1 (NRG-1) is a ligand for ErbB3 or ErbB4. When NRG-1 binds to ErbB3 or ErbB4, ErbB3 or ErbB4 forms heterodimer with ErbB2. Both ErbB2 and ErbB4 contain high level of kinase activity, while the kinase activity of ErbB3 is impaired. NRG-1 has lower affinities (l-100nM) with ErbB3 or ErbB4, and higher affinities (<1nM) with ErbB2/ErbB3 or ErbB2/ErbB4 heterodimers. The analysis of genetically modified mice had proved the importance of ErbB receptors and their ligands in development. Furthermore, ErbB3 or ErbB4 gene-inactivated mice have similar or overlapping phenotypes to NRG-1 or ErbB2 knockout mice. The data strongly suggests that ErbB2 participates in NRG-1-activated ErbB3 or ErbB4 signaling pathways in vivo.We studied the phosphorylation of heterodimers, ErbB2/ErbB3 and ErbB2/ErbB4, in a transient gene expression system. The ErbB2 was phosphorylated by ligand-indepndent homodimers in the cells transfected ErbB2 alone. In the cells co-transfected ErbB2 & ErbB3, phosphorylation of ErbB3 was up-regulated after stimulated by NRG-1, and more ErbB2/ErbB3 heterodimers formed. We constructed a truncated ErbB3 mutant, ErbB3-676, which contains the Extracellular domain and transmembrane domain. In the co-transfected cells, the 310KD heterodimers of ErbB2/ErbB3-676 increased after stimulated by NRG-1, while the phosphorylation of ErbB2 was decreased, which indicated that the up-regulated phosphorylation was due to the full length of ErbB3 in the heterodimers. The immunoblot of the ErbB2 and ErbB3 separately had showed that the phosphorylation of ErbB2/ErbB3 heterodimers was up-regulated through the tyrosine residues of ErbB3 only. And the up-regulated phosphorylation of ErbB3 was significantly higher (according to three independent examinations). The data showed that only trans-phosphorylation had occurred in theheterodimers after the stimulated by NRG-1. In other words, the intrinsic tyrosine kinase domain of ErbB2 had phosphorylated the specific tyrosine residues of ErbB3 only. NRG-1 could up-regulated the phosphorylation of ErbB2 in the cells co-transfected ErbB2 & ErbB4, which was due to more ErbB2/ErbB4 heterodimers formed after stimulated by NRG-1. The data indicated that the phosphorylation of ErbB2 in the ErbB2/ErbB4 heteodimers was up-regulated by NRG-1. In contrast with the ErbB2/ErbB3 heterodimer, the phosphorylation of ErbB2 was due to the ErbB4, which has normal tyrosine kinase activation. In a word, the phosphorylation between the ErbB heterodimers was not up-regulated by cis-phosphorylation, but only trans-phosphorylation.We found that rhNRG had therapeutic effect on the MI rat model by injection 10ug/kg rhNRG through tail vein (10 days' continuous injection). Followed studies showed that rhNRG activated the ErbB4 of left ventricle, then three important signal proteins, Erkl/2&Akt, were significantly activated too. The phosphorylation of Erk1/2 kept continuous up-regulation, and was significantly higher than that of negative control at 4.5 hours. The phosphorylation of Akt was decreased and even was significantly repressed. After injection U0126 (the MEK1/2 inhibitor) the phosphorylation of Erk 1/2 activated by the rhNRG was very significantly repressed. The EF values of MI rats showed that U0126 didn't have significant effect on the therapeutic effect. Wortmannin (the inhibitor of PI3K) could significantly repress the phosphorylation of Akt up-regulation by rhNRG DMSO could significantly up-regulated the phosphorylation of Akt. The MI rat trial showed that Wortmannin had very significantly improved the therapeutic effect. We also found that wortmannin could repress the phosphorylation of Erkl/2 too, while U0126 had no significant effect on the phosphorylation of Akt. Therefore, the reasonable explain was that PI3Kcould regulated the phosphorylation of Erk1/2, in other words MAPK and PI3K had cross-talk in the rat heart. According to data above, we concluded that the inhibition of PI3K plays the key role on the therapeutic effect of rhNRG on MI rat.