Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.

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 (⌬EH-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.Insulin stimulates glucose transport in skeletal muscle and adipose tissues by promoting the translocation of the glucose transporter GLUT4 from intracellular pools to the plasma membrane (1-6). Although this phenomenon was documented for the first time Ͼ20 years ago (7,8), the mechanisms by which insulin regulates GLUT4 recycling and the cellular processes that retain GLUT4 within intracellular storage membranes in the basal state are still largely unknown. In the absence of insulin, GLUT4 cycles slowly between the plasma membrane and some intracellular compartments distinct from the constitutive recycling pathway, with the vast majority of the transporter residing within the cell interior near the juxtanuclear region. Activation of the insulin receptor tyrosine kinase triggers a cascade of signaling events that impact the GLUT4 recycling system, leading ultimately to the recruitment of intracellular GLUT4-containing vesicles to the cell surface (1, 2). This process appears to involve both motor-driven movements of these vesicles on microtubule-and actin-based cytoskeletal tracks (9 -11) as well as fusion of the GLUT4-containing vesicles with the plasma membrane (12). Substantial data indicate that activat...

Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3␣ (GSK-3␣) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes.Insulin is the primary hormone that regulates glucose homeostasis, and impairment of insulin action and secretion plays a critical role in the pathogenesis of diabetes mellitus (1, 2). One of the primary metabolic responses mediated by insulin is the stimulation of glucose transport and glycogen synthesis in muscle and adipose tissue (3, 4). It is now established that translocation of glucose transporter 4 (GLUT4) 1 from intracellular compartments to the plasma membrane is mainly responsible for the insulin-stimulated increase in glucose uptake in these tissues (5-8). This process is initiated when insulin binds and activates its receptor tyrosine kinase at the cell surface. The activated insulin receptor phosphorylates the insulin receptor substrate (IRS) family of proteins on tyrosine residues. IRS proteins propagate insulin signaling to the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase, which activates the p110 catalytic subunit (3, 4). A growing number of experiments indicate that insulin-induced PI 3-kinase activation is critical for insulin-induced metabolic actions, including glucose uptake and glycogen synthesis (9 -11). Thus, phosphorylation of phosphatidylinositol 4,5-bisphosphate by activated PI 3-kinase increases levels of PI(3,4,5)P 3 at cellular membranes. This phosphorylated lipid is a potent modulator of the protein kinases PDK1, atypical protein kinase C /, and Akt/Protein kinase B (12, 13). Blockade of the PI 3-kinase signaling pathway through the use of specific inhibitors or by the expression of a dominant negative p85 regulatory sub...

Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and transGolgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNAbased gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.Blood glucose homeostasis is maintained by insulin in part through glucose disposal into muscle and fat tissues. This is achieved by means of a redistribution of the facilitative glucose transporter GLUT4 from an internal sequestration compartment to the cell surface where it facilitates glucose uptake into these cells. In the absence of insulin GLUT4 recycles very slowly from internal membranes to the plasma membrane, whereas insulin markedly increases the exocytic rate of these GLUT4-containing vesicles. This results in a net increase of GLUT4 at the plasma membrane. This regulated trafficking pathway for GLUT4 appears to be distinct from that of many other membrane proteins such as the transferrin receptor or the GLUT1 glucose transporter protein.Insulin stimulation of adipocytes increases the abundance of these latter proteins at the cell surface by ϳ2-fold, whereas the amount of GLUT4 is increased between 10 -20-fold (1, 2). Other insulin-regulated exocytic pathways likely operate in adipocytes as well, such as a secretory mechanism for peptides like Acrp30/adiponectin. Like transferrin receptor recycling to the cell surface, Acrp30 secretion is only modestly stimulated by insulin (3). This large difference in insulin sensitivity indicates that the molecular mechanisms of GLUT4 sequestration and trafficking have unique regulatory elements. To understand insulin action on glucose transport it will be necessary to elucidate the nature of GLUT4 sequestration in intracellular membranes, the transit of GLUT4-containing intracellular membranes toward the cell surface, and the fusion of these membranes with the plasma membrane.Although the trafficking of the transferrin receptor and GLUT4 differ in their response to insulin stimulation, it appears that ϳ50% of the ...