RESUMO
GPCR-G alpha fusion protein has been used to investig ate receptors in recent years. The expression of GPCR-G alpha fusion protein en sured defined 1∶1 stoichiometry, the physical proximity of GPCR to G alpha subu nit, and was suitable for high-throughput ligand screening. The fusion protein technology provided a novel strategy to study orphan GPCRs. Obviously, it will p lay important roles in ligand screening of the orphan GPCRs and exert its signif icances in the R&D of novel drugs targeted at oGPCRs.
RESUMO
AIM: To investigate the influence and mechanisms of unsaturated fatty acid on PAI-1 expression in HepG-2 cells. METHODS: PAI-1 activity and mRNA expression were determined by colorimetric assay and RT-PCR, respectively. Two types of CAT reporter gene plasmid containing different deletions in PAI-1 promoter were constructed and transfected into HepG-2 cells, respectively. The transcriptional activity of PAI-1 was demonstrated by the CAT's expression. RESULTS: The mRNA and activity of PAI-1 significantly increased in the groups of oleic acid and linoleic acid compared with the control. When co-transfected with PPAR?-pSG5, the level of PAI-1 transcription was significantly increased. In the HepG-2 cells transfected with NF-?B-like sequence-deletion-pCAT construct linoleic acid, the PAI-1 transcriptional activity increased, and no significant change was observed when transfected with VLDL/fatty acid response element-deletion-pCAT construct. CONCLUSIONS: Unsaturated fatty acids induce PAI-1 activity and mRNA expression in HepG-2 cells. PPAR? may be one of transcription factors playing a role in the regulation of PAI-1 gene expression. The VLDL/fatty acid response element in the PAI-1 promoter may play an important role in the regulation, but not the NF-?B-like sequence.