Molecular Characterization Of The Melatonin Receptor MT1Sigual Transduction And Establishing An Intein Based Model For GPCR Actiyation Detection

G protein-coupled receptors (GPCRs) are the most abundance cell membrane receptors, and are involved in regulation of multiple important physiological and pathological progress. In this paper, we investigated the melatonin receptor MT1signaling pathway and constructed a DnaE-intein based model real-time monitoring chemokine receptor CCR5activation. In the part of MT1signaling research, we found MT1could couple to Gs protein and activate the cAMP/PKA/CREB patheay besides the reported Gi coupling pathway. We found that the intracellular loop2and the NSXXNAXXY motif in TM7of MT1were important interaction region of the MT1-Gs coupling, and we also found the lipid rafts may be right place for the Gs protein dependent MT1signaling. We further investigated the detail pathway of the MT1’s endocytosis and mediated MAPK/ERK phosphorylation. We found that MT1-Gi coupling is the necessary premise of MT1’s clathrin dependent endocytosis. We also found that both the Gi/PI3K/PDK1/PKC pathway and Gs/cAMP/PKA pathway involved in the MAPK/ERK phosphorylation, in a time and concentration different manner. In the part of DnaE intein based modeling of CCR5activation, we construct a reporter system which is based on the findings of a natural split DnaE intein from Synechococcus elongatus PCC7942. Using the reporter system, we construct a real-time activated-CCR5dependent cell-cell fusion assay, and this assay is suitable for the screening of CCR5antagonist. The achieved assay provides a new approach for the anti-HIV infection drug preliminary screening.