Affinity between TBC1D4 (AS160) phosphotyrosine-binding domain and insulin-regulated aminopeptidase cytoplasmic domain measured by isothermal titration calorimetry

Abstract

Uptake of circulating glucose into the cells happens via the insulin-mediated signalling pathway, which translocates the glucosetransporter 4 (GLUT4) vesicles from the intracellular compartmentto the plasma membrane. RabㆍGTPases are involvedin this vesicle trafficking, where RabㆍGTPase-activatingproteins (RabGAP) enhance the GTP to GDP hydrolysis.TBC1D4 (AS160) and TBC1D1 are functional RabGAPs in theadipocytes and the skeletonal myocytes, respectively. Theseproteins contain two phosphotyrosine-binding domains (PTBs)at the amino-terminus of the catalytic RabGAP domain. Thesecond PTB has been shown to interact with the cytoplasmicregion of the insulin-regulated aminopeptidase (IRAP) of theGLUT4 vesicle. In this study, we quantitatively measured the∼μM affinity (KD) between TBC1D4 PTB and IRAP using isothermaltitration calorimetry, and further showed that IRAP residues1-49 are the major region mediating this interaction. Wealso demonstrated that the IRAP residues 1-15 are necessarybut not sufficient for the PTB interaction.

Keywords

• IRAP
• Isothermal titration calorimetry
• Protein-protein interaction
• PTB domain
• TBC1D4 (AS160)