POSTER NO: 206

Cloning and identifying of a novel protein binding with death domain of the death receptor 4

Xiaoling Li, Yanxin Liu, Shilian Liu, Dexian Zheng
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, PUMC and CAMS, Room 227, 5 Dong Dan San Tiao, Beijing 100005, China

Objective: To clone and identify novel proteins binding with the death domain of death receptor 4 (DR4) for Apo2L/TRAIL (TNF-related apoptosis inducing ligand) and investigate the mechanism of apoptosis signaling mediated by DR4.

Methods: The yeast two-hybrid system was used for this study. In brief, the death-domain of DR4 (DR4DD) was amplified by PCR and cloned into the GAL4 DNA-BD vector to generate the DR4DD expression plasmids. And the plasmid was then co-transformed into the yeast strain Y190 with the human leukocyte MATCHMAKER cDNA library constructed in the GAL4 AD vector. The transformants were plated on minimal medium lacking Leu, Trp and His to select for the clones (His+) containing the two plasmids. Primary His+ transformants were tested for the expression of the LacZ reporter gene using a sensitive and rapid filter assay of beta-galactosidase activity. The same and false positive clones were eliminated by PCR and re-cotransformation respectively. The real positive clones must be the ones in which the reporter gene was expressed only when the two plasmids above were introduced into the yeast reporter strain (Y190) simultaneously. Automatic sequencing was carried out for DNA sequence determination. Sequence and functional domain analysis was carried out by BLAST and ScanProsite software, respectively.

Result: By using yeast two-hybrid system, two positive clones, named as pADB1 and pADB2, were obtained. Sequencing analysis showed that the insert fragments in pADB1 and pADB2 contained open reading frames fused with the GAL4 AD sequence. pADB1 contained an open reading frame of 522 bp encoding 174 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 in pADB2 was 141 bp encoding 47 amino acids. Functional domain analysis by ScanProsite showed that DR4DD contained 2 N-glycosylation sites, 3 protein kinase C phosphorylation sites, 4 casein kinase II phosphorylation sites and 4 N-myristoylation sites. Similarly, FPRL1 contains 2 N-glycosylation sites, 2 protein kinase C phosphorylation sites, 2 casein kinase II phosphorylation sites, 3 N-myristoylation sites and 1 G-protein coupled receptors family 1 signature, suggesting that there might well be a possible interaction between DR4DD and FPRL1.

Discussion: It is reported that the binding of FPRL1 with its agonists results in a G-protein signaling cascade that leads to cell adhesion, migration, release of oxygen intermediates, and activation of mitogen-activated protein kinase. Activation FPRL1 also attenuates the expression and function of several chemokine receptors such as CXCR2 and CCR5 through PKC-mediated receptor phosphorylation and heterologous desensitization. Our results showed that DR4DD contain 3 protein kinase C phosphorylation sites, therefore, one could deduce that activation of FPRL1 might be able to result in DR4DD phosphorylation and attenuate the function of DR4DD in signaling pathway triggered by TRAIL. In addition, it is known that N-glycosylation of FPRL1 is essential for recognition of its ligand LXA4 and domain analysis showed that there are 2 N-glycosylation sites in the molecules of DR4DD and FPRL1, these results future argued for the possible interaction between FPRL1 and DR4DD. We therefore postulated that activation of FPRL1 might induce DR4 phosphorylation, heterologous desensitization, and contribute to the cell sensitivity to Apo2L/TRAIL. The functional studies of FPRL1 in signaling pathway mediated by TRAIL are in active progress in our laboratory.