Trail-induced Apoptosis Molecular Mechanism Study

DR4 (Death Receptor 4) belongs to the tumor necrosis factor receptor (TNFR) gene family, which is defined by similar, cysteine-rich extracellular domain and a homologous cytoplasmic sequence termed as "death domain". DR4 can transmit apoptosis signal initiated by Apo2L/TRAIL (TNF-related apoptosis inducing ligand). It can activate caspases within seconds of ligand binding and cause an apoptotic demise of the cell within hours. Despite several investigations, the mechanisms of apoptosis initiation by Apo2L/TRAIL remain unclear.To investigate the mechanism of apoptosis signaling mediated by DR4, the yeast two-hybrid system was used for cloning and identifying proteins binding with the death domain of death receptor 4 (DR4) for Apo2L/TRAIL (TNF-related apoptosis inducing ligand). In brief, the death-domain of DR4 (DR4DD) was used as the bait to screen the human leukocyte MATCHMAKER cDNA library. Sequence and functional domain analysis were carried out by using automatic sequencing, BLAST and ScanProsite softwares, respectively. Two positive clones, named as pADBl and pADB2, were obtained in this study. Sequencing analysis showed that the insert fragments in pADB 1 and pADB2 contained open reading frames fused with the GAL4AD sequence. PADBl contained an open reading frame of 504bp encoding 168 amino acids. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of human formyl peptide receptor-like 1 (FPRL1) was 97%. The insert of pADB2 shared no homologous with any known sequences in Genbank. The open reading frame of pADB2 was 141 bp encoding 47 amino acids. FPRL1 binds specifically with DR4CD (the cytoplasmic domain of DR4) in vitro and in vivo were confirmed by pull-down experiment and co-immunoprecipitation respectively.In order to study the function of FPRL1 in signal transduction of cell apoptosis...