Screening Of Proteins Interacted With Intracellular Domain Of Mouse Fibroblast Growth Factor Receptor-3 (FGFR3)

Fibroblast growth factor receptor 3 (FGFR3) plays a particularly important role in skeletal development. Disruption of murine FGFR3 produces severe and progressive bone dysplasia with enhanced endochondral bone growth, suggesting that FGFR3 mediates the negative regulation of bone growth. Besides PLC-γand FRS2, little is known about the substrates of FGFR3. To identify the proteins that interact with FGFR3 and mediate FGFR3-dependent signaling, the following experiments were performed in this study.1. Construction of pGBKT7 DNA-BD/R3-ID and identification of its expression in the yeast.By means of PCR, the cDNA fragment of the intracellular domain of FGFR3, was inserted into pGBKT7 DNA-BD vector to make pGBKT7 DNA-BD/R3-ID. The sequence of the intracellular domain of FGFR3 and its open reading frame were comfirmed correct by sequencing.2. Amplification of GAL4 AD/mouse 17.5 day embryo cDNA library, identifcation of autonomous activation, measurement of cell toxicity and His3 leaky expression in the yeast.(1) cDNA library plasmids were amplified according to the operating manual, and extracted by means of alkaline lysis, and purified using polyethylene glycol precipitation.(2) The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformant in the selected medium SD/-Trp was observed. The results showed that colony size of transformant was the same as that of the controls.β-galactosidase activity of positive clones was tested by using both liquid assay and plate assay. The experimental results suggested that the intracellular domain of FGFR3 has neither the ability to autonomously activate the reporter gene nor the toxicity to yeast cell. The recombinant plasmid transformant could not grow in the SD/-His medium, and did not have His leaky expression.