EHD2 Interacts with the Insulin-Responsive Glucose Transporter (GLUT4) in Rat Adipocytes and May Participate in Insulin-Induced GLUT4 Recruitment
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- 作者：Seung Y. Park ; Byoung G. Ha ; Guem H. Choi ; Jiwon Ryu ; Beomsu Kim ; Chan Y. Jung ; and ; and Wan Lee
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 15, 2004
- 年：2004
- 卷：43
- 期：23
- 页码：7552 - 7562
- 全文大小：165K
- 年卷期：v.43,no.23(June 15, 2004)
- ISSN：1520-4995
文摘
Insulin-induced GLUT4 recruitment to the plasma membrane involves GLUT4 traffickingthrough multiple subcellular compartments regulated by multiple proteins, many of which are yet to beidentified. Here we describe a 65 kDa protein found in purified GLUT4 vesicles of rat adipocytes as apotential 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 EHdomain-containing proteins that have been implicated in vesicle trafficking. EHD2 in rat adipocytes was85% membrane-associated, including approximately 10% in immunopurified GLUT4 vesicles. Thisassociation of EHD2 with GLUT4 vesicles occurred in PM and three distinct endosomal fractions andwas 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 selectivelyenhanced this interaction in an endosomal fraction thought to contain GLUT4 exocytic vesicles. EHD2also interacted with the clathrin adaptor middle chain subunit 
1, 2, and rCALM in GST pull-downexperiments. Significantly, an affinity-purified EHD2 antibody and a peptide corresponding to the EHD2sequence Glu428-Glu535 drastically (by 75% and 35%, respectively) suppressed the insulin-induced increasein the plasma membrane GLUT4 contents in SLO-permeabilized rat adipocytes without affecting thebasal GLUT4 distribution. These findings strongly suggest that EHD2 interacts with GLUT4 in ratadipocytes and may play a key role in insulin-induced GLUT4 recruitment to the plasma membrane.
1, 2, and rCALM in GST pull-downexperiments. Significantly, an affinity-purified EHD2 antibody and a peptide corresponding to the EHD2sequence Glu428-Glu535 drastically (by 75% and 35%, respectively) suppressed the insulin-induced increasein the plasma membrane GLUT4 contents in SLO-permeabilized rat adipocytes without affecting thebasal GLUT4 distribution. These findings strongly suggest that EHD2 interacts with GLUT4 in ratadipocytes and may play a key role in insulin-induced GLUT4 recruitment to the plasma membrane.