The functional analysis of a novel adaptor protein binding to GLUT4

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PurposeIt has been supposed that a protein binding to GLUT4 could play an important role during the recycling of GLUT4 vesicle and insulin-induced GLUT4 translocation. We assumed the existence of GLUT4 "adaptor protein" in adipocytes, and we have identified a novel protein "p61" that interacts with GLUT4, and analyzed its function.MethodUsing the yeast two-hybrid system, we screened from rat adipocyte cDNA library, and have cloned p61 as an interesting protein that interacts with GLUT4. The intracellular localization of p61 in L6-GLUT4myc myotubes, stably expressing myc-tagged GLUT4, was examined by the immunohistochemistry using the myc tag antibody. The interaction of GLUT4 and p61 was confirmed by the yeast two-hybrid system, and also the immunoprecipitation. After overexpression of p61 in 3T3-L1 adipocytes, the GLUT4 translocation and the insulin-induced glucose uptake were examined.ResultsThe expression of p61 in adipocytes was confirmed at the mRNA level using Northern blotting. The p61 consists of 540 amino acids with the ANK (ankyrin) structure thought to be a protein-protein interaction motif. The p61 existed in perinuclear region in the L6-GLUT4myc myotubes as a result of the immunohistochemistry. Moreover, it was confirmed that the interaction of p61 and GLUT4 by yeast two-hybrid system and the immunoprecipitation. In addition, overexpression of p61 in 3T3-L1 adipocytes using adenoviral system, increased the amount of GLUT4 in membrane fraction after insulin stimulation, and also significantly facilitated insulin-induced glucose uptake.ConclusionIt is suggested that a novel ANK structure protein p61 facilitates GLUT4 translocation, and then activates insulin-induced glucose uptake.