Human CCL17/TARC (hCCL17) and CCL22/MDC (hCCL22) interact with their receptor CCR4, playing pivotal roles in various Th2 cell-dominant diseases. Rat Ccl17, Ccl22 and Ccr4 share 63.4%, 65.2% and 87.5% homology at the amino acid level, respectively, compared with their human homologues. Rat Ccl22 has been demonstrated to interact with rat Ccr4. However, it is not known whether rat Ccl17 is a functional ligand for rat Ccr4 and whether rat Ccr4 can interact with hCCL17 and hCCL22. In this study, we cloned rat Ccl17 and Ccr4 cDNA from rat thymus and expressed the recombinant rat Ccl17. The binding assay indicated rat Ccl17 could saturably bind rat Ccr4, and this binding could be competitively inhibited by unlabeled rat Cc17, hCCL17 or hCCL22. Both rat and human CCL17 or CCL22 could induce the chemotactic migration and calcium mobilization in rat Ccr4-transfected cells. They could also desensitize each other’s effect on chemotaxis and calcium mobilization. Furthermore, both rat Ccl17 and hCCL17 could induce significant receptor internalization in rat Ccr4-EGFP transfected cells. These results demonstrated the conserved CCL17/CCR4 interaction in rats and the further cross-species interaction of rat Ccr4 with hCCL17 and hCCL22. Likewise, they provided the molecular basis for the investigation of physiological and pharmacological roles of CCR4, its ligands, and their antagonists in rat disease models.
TIAN LinJie1,2, QI Hui1,2, XIE Yuan1,2, ZHANG YingMei1,2, ZHANG WenJuan1,2, SUN XiangYu1,2, WANG Ying1,2 & MA DaLong1,2 1 Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100191, China
Abstract CC chemokine receptor 4 （CCR4） is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-（2-（4-chloro-phenyl）-5-{[（naphthalen-1- ylmethyl）-carbamlyl]-methyl-4-oxo-thiazolidin-3-yl）-N-（3-morpholin-4-yl-propyi）-acetamide （S009） and the N-terminal extracellular tail （ML40） of CCR4 has been validated to be high affinity by capillary zone electrophoresis （CZE）.The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-I 1, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases.
Zhe SunLin-Jie TianQian LinXiao-Mei LingJun-Hai XiaoYing Wang
