EHD2 interacts with the insulin-responsive glucose transporter (GLUT4) in rat adipocytes and may participate in insulin-induced GLUT4 recruitment

Insulin-induced GLUT4 recruitment to the plasma membrane involves GLUT4 trafficking through multiple subcellular compartments regulated by multiple proteins, many of which are yet to be identified. Here we describe a 65 kDa protein found in purified GLUT4 vesicles of rat adipocytes as a potential GLUT4 traffic regulatory protein. On the basis of MALDI-TOF MS, RT-PCR, gene cloning, protein sequencing, and immunoreactivity data, we identified this protein as EHD2, a member of the EH domain-containing proteins that have been implicated in vesicle trafficking. EHD2 in rat adipocytes was 85% membrane-associated, including approximately 10% in immunopurified GLUT4 vesicles. This association of EHD2 with GLUT4 vesicles occurred in PM and three distinct endosomal fractions and was not significantly affected by cellular insulin treatment. In co-immunoprecipitation experiments, however, EHD2 physically interacted with GLUT4 in each of these fractions, and cellular insulin treatment selectively enhanced this interaction in an endosomal fraction thought to contain GLUT4 exocytic vesicles. EHD2 also interacted with the clathrin adaptor middle chain subunit mu(1), mu(2), and rCALM in GST pull-down experiments. Significantly, an affinity-purified EHD2 antibody and a peptide corresponding to the EHD2 sequence Glu(428)-Glu(535) drastically (by 75% and 35%, respectively) suppressed the insulin-induced increase in the plasma membrane GLUT4 contents in SLO-permeabilized rat adipocytes without affecting the basal GLUT4 distribution. These findings strongly suggest that EHD2 interacts with GLUT4 in rat adipocytes and may play a key role in insulin-induced GLUT4 recruitment to the plasma membrane.