title: Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes.
abstract: Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
authors.forename: Adilson
authors.initials: A
authors.lastname: Guilherme
authors.forename: Neil A
authors.initials: NA
authors.lastname: Soriano
authors.forename: Paul S
authors.initials: PS
authors.lastname: Furcinitti
authors.forename: Michael P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D015870:Gene Expression; D051275:Glucose Transporter Type 4; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; C120700:EHD1 protein, human; C484942:EHD2-binding protein 1, mouse; C485640:Ehd1 protein, mouse; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; D033921:Vesicular Transport Proteins