The Study On Structure And Function Of KGF-2

Keratinocyte growth factor(KGF)-2, a member of the fibroblast growth factor(FGF) family, is a specific mitogen for epithelial cells. During development, KGF-2 regulates the morphogenesis of various tissues and organs. Studies on animal models showed that KGF-2 had significant effects on accelerating cure of wounds, defending against damages caused by radiation, and promoting healing of the ulceration and enteritis. While inappropriately expressed, KGF-2 would contribute to pathogenesis of cancer. By binding to and activating its main receptor, FGFR2IIIb on the epithelial cell membrane, KGF-2 exerts its important biological functions. It is important to identify the crucial receptor binding sites on KGF-2, which will provide valuable clues for controlling the signal transduction of KGF-2 and reasonably designing medicines or antagonists of KGF-2 used to treat human diseases. Many researches have proven that extraneous KGF-2 can be translocated into nucleus, but the mechanism and function of this process are still unclear.Based on the results of computer molecular modeling and analysis system, combined with the homologous sequences' comparison, five KGF-2 mutants: AGBH, T114A, T114R, G160H and LY, were designed aiming at the critical amino acid that played important role in KGF-2 binding to FGFR2IIIb. KGF-2 cDNA was obtained from human fetal liver cDNA library by PCR, then the DNA fragments encoding the KGF-2 mutants were obtained using overlap extension polymerase chain reaction, and then inserted into the plasmid pET-17b. Their expression levels were studied in E.coli BL21, which were 13% ~ 30%, and mainly expressed as soluble form. All recombinant proteins were purified by cation exchange chromatography. The purity of recombinant proteins was over 90%. The affinity to receptor FGFR2IIIb and the effect to rat trachea epithelium(RTE) cell of these recombinant proteins were tested using competitiveELISA and MTT, respectively. The results showed that the standard, KGF-2 and mutants had mitogenic effect to RTE, and their effects depended on their concentrations. KGF-2 had the same receptor affinity and mitogenic effect to RTE as the standard. Compared with KGF-2, the receptor affinity and mitogenic effect of LY rose dramatically, although AGBH increased a little in receptor affinity but had notable enhanced mitogenic effect. T114A had the same receptor affinity as KGF-2 but greatly depressed mitogenic effect to RTE. The receptor affinity and mitogenic effect of T114R and G160H were dramatically lower than KGF-2. Above results suggested that Thr114, A, B, Gly 160 and L were all important amino acids in KGF-2 binding to receptor, and their mutations would greatly affect the receptor affinity and function of KGF-2. Meanwhile, the receptor affinities of AGBH and T114A had a little or no change, but the mitogenic effect of both had greatly changed. It suggested that Thr114, A and B were crucial amino acids for receptor activation by KGF-2. The structure of each KGF-2 mutant was modeled using SGI Origin 300 graphic workstation and BIOSYM/MSI molecular modeling system. Mainly aimed at the mutated amino acids, the interactions with FGFR2IIIb were analyzed and compared with that pre-mutation. The result showed the receptor affinity was determined by the offset and balance between all changed interactions, but not any one of them.Two proteins, PRO2605 and RPL22, interacting with KGF-2 were found using the yeast two-hybrid system. Further study was done to investigate the interaction between KGF-2 and PRO2605 or RPL22 using mammalian two-hybrid system.The results suggested that only the interaction between RPL22 and KGF-2 was confirmed in COS7. RPL22 has a classical nuclear localization signal at its N terminal, so this protein is very likely involved in the nuclear import of KGF-2.