Construction and expression of different functional forms of mouse discoidin domain receptor 2 / 解放军医学杂志

ABSTRACT

Objective Discoidin domain receptor 2 is a kind of receptor tyrosine kinases, which was found to be over-expressed in synovial fibroblasts in rheumatoid arthritis (RA). The present study was to construct and express wild type (FLDDR2), Fc chimera (FcDDR2) and truncated form of mouse discoidin domain receptor 2 (ttDDR2) for further study. Methods Full-length, truncated form of mouse DDR2 were amplified by RT-PCR. A chimera form of mouse DDR2 was constructed by replacing part of the extracellular domain with Fc fragment of human immunoglobulin G1 (IgG1). Eukaryotic expression vectors of the different forms of mouse DDR2 were constructed by subcloning the PCR products into pcDNA3.1(+) or pMKIT-Neo. Then COS-7 cells were transient transfected with the eukaryotic expression vectors of full-length (FLDDR2), truncated (ttDDR2) and chimeric form of mouse DDR2 (FcDDR2). Successful transfection and expression was confirmed by Western blot(WB) and Immunoprecipitation (IP)/WB. With or without stimulation with soluble type I collagen for 3h, phosphorylation level of transfected cells were detected by IP/WB. Eukaryotic expression vectors of full-length, truncated form and chimeric forms of mouse DDR2 were successfully constructed and confirmed by sequencing. After transient transfection, the expression of these three forms in the respectively transfected cells was observed by Western blot. Results The result of IP/WB suggested that the chimeric form of mouse DDR2(FcDDR2) could be properly expressed in the COS-7 cells. Under the condition of collagen, decreased tyrosine phosphorylation of FLDDR2 was detected in the COS-7 cells that were cotransfected with ttDDR2 and FLDDR2, comparing with that in COS-7 cells transected with only FLDDR2. There was no obvious difference in phosphorylation level between FcDDR2 without collagen stimulation and FLDDR2 with the collagen stimulation. Conclusion Three different forms of mouse DDR2 were successfully constructed. FcDDR2 could be an activator for its auto-phosphorylation. And ttDDR2 could be a partially negative competitor.