Study On GIPC2 Interaction Protein

Objective:GIPC2 is a natural, small molecular protein. We have found expression level of GIPC2 changes in a variety of tumors. For example, GIPC2 is upregulated in gastric cancer, while downregulted in kidney cancer, acute lymphoblastic leukemia and adrenal cortical carcinoma. However, GIPC2 function in the tumorigenesis is still unclear, and interaction proteins of GIPC2 are rarely reported. In this study, we want to find new protein-prtein interactions of GIPC2,which can play important roles in the study of GIPC2 biological function.Methods:First, we use two methods, yeast-two-hybrid and immunoprecipitation-massspectrometry to study GIPC2 interaction protein.We have three points:GIPC2 PDZ domain screening PDZ ligand library, GIPC2 screening cDNA library and GIPC2 screening 293T cells expressing protein three to validate protein-protein interaction.Then,we screen the initial results by biological functions to obtain candidate proteins. Thirdly, we considerate the tissue specificity and species specificity of protein expression, We confirm the protein-protein interactions in PC 12 and 293T cells by fluorescence resonance energy transfer and co-immunoprecipitation.Results:In the yeast-two-hybrid system, we find 14 candidate ligands by GIPC2 PDZ domain screening the PDZ ligand library, and four candidate ligands are obtained by GIPC2 screening cDNA library. In 293T cells, we gain hundreds of possible ligand proteins by immunoprecipitation-mass spectrometry. Then, we screen the initial results by biological function and subcellular localization to receive 19 candidate proteins. In PC 12 cells, we confirm 19 candidate proteind showed positive results with GIPC2 by FRET. In 293 t cells, we identify NONO as interaction protein for GIPC2 by CO-IP.Conclusion:Using yeast-two-hybrid, immunoprecipitation-mass spectrometry, fluorescence resonance energy transfer and co-immunoprecipitation, we successfully screen and validate new interaction proteins for GIPC2, which will provide important clues for studying the biological functions of GIPC2 related tumors.