The Study Of The Molecular Mechanism Of H4R Activating The Pathway Of ERK And The Assay Of Quantitively Analysis Of The GPCR Internalization

The Histamine H4 receptor (H4R) is a newly identified histamine receptor, and has been found to play an important role the immune system. The H4R acts via Gαi-protein to inhibit adenylate cyclase activity and modulate MAPK activity. We aim to elucidate the molecular mechanisms for H4R-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), using CHO-K1 cells expressing human H4R. Our data demonstrated that the H4R-mediated activation of ERK1/2 is blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H4R agonist histamine, H4R is shown to induce ERK1/2 phosphorylation via Ca2+-independent PKC, extracellular calcium and platelet-derived growth factor receptor (PDGFR) transcativation-dependent pathways. Furthermore, it is indicated that the βγ-subunits play a key role in H4R-activated ERK1/2 phosphorylation. These results could be the foundation for the further investigation of the physiological functions of H4R. Meanwhile, we have developed the DnaE-intein based assay for the quantitative analysis of GPCR internalization. Based on the interaction of internalized GPCR with Rab5 which is localized in the cell surface of endosomes, we have designed a new assay to quantitatively analyze GPCR internalization. This new assay can be applied to many kinds of GPCRs, and is very sensitive. It would be an efficient tool to research the GPCRs.