Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.     

Increased gene expression and production of murine endothelin receptors after birth

We developed the real-time PCR quantification of endothelin-A (ET-A) and endothelin-B (ET-B) receptor genes and present their relative expression levels in various adult tissues and during development in mouse using the 2(-Delta Delta C(T)) method. ET-A and ET-B receptors were detected in all tissues examined. Gene expression of ET-A and ET-B receptors increases during the later stages of embryonic development in lung, heart, liver, kidney, and skin and reaches a maximum on the first one or two days after birth. The results, in agreement with our data on endothelin (ET) ligands, suggest that the ET system may be involved in the emergence and maintenance of functions vital after birth in these organs. These findings were corroborated through observation of the correlation between the gene expression and (poly)peptide production of the ET system in normal skin before and after parturition.     

Intersectin regulates fission and internalization of caveolae in endothelial cells

Intersectin, a multiple Eps15 homology and Src homology 3 (SH3) domain-containing protein, is a component of the endocytic machinery in neurons and nonneuronal cells. However, its role in endocytosis via caveolae in endothelial cells (ECs) is unclear. We demonstrate herein by coimmunoprecipitation, velocity sedimentation on glycerol gradients, and cross-linking that intersectin is present in ECs in a membrane-associated protein complex containing dynamin and SNAP-23. Electron microscopy (EM) immunogold labeling studies indicated that intersectin associated preferentially with the caveolar necks, and it remained associated with caveolae after their fission from the plasmalemma. A cell-free system depleted of intersectin failed to support caveolae fission from the plasma membrane. A biotin assay used to quantify caveolae internalization and extensive EM morphological analysis of ECs overexpressing wt-intersectin indicated a wide range of morphological changes (i.e., large caveolae clusters marginated at cell periphery and pleiomorphic caveolar necks) as well as impaired caveolae internalization. Biochemical evaluation of caveolae-mediated uptake by ELISA showed a 68.4% inhibition by reference to control. We also showed that intersectin interaction with dynamin was important in regulating the fission and internalization of caveolae. Taken together, the results indicate the crucial role of intersectin in the mechanism of caveolae fission in endothelial cells.     

Endothelin-3 induced mesenteric vasoconstriction and PMN infiltration in the rat small intestine: role of endothelin receptors

The objectives of this study were to characterize endothelin (ET)-3-induced alterations in intestinal hemodynamics and to evaluate whether ET-3 administration alters the tissue levels of polymorphonuclear leukocytes (PMNs) and modulates the epithelial barrier function of the small intestine. ET-3 (100 pmol/kg/min) was infused into the superior mesenteric artery (SMA) for 10 min, and tissue samples were obtained 30 min after terminating the infusion. SMA blood flow was significantly decreased throughout the experiment following ET-3 infusion. Pretreatment with bosentan (ET-A and ET-B receptor antagonist), ET-B receptor antagonist BQ-788 or ET-A receptor antagonist BQ-485 completely inhibited the ET-3-induced decrease in the SMA blood flow. Similar results were obtained from the resistance data, in which ET-3-induced increases in SMA resistance were significantly reduced by all ET receptor antagonists. ET-3 administration significantly elevated tissue MPO activity, blood-to-lumen clearance of (51)Cr-EDTA and caused a marked microscopic damage in the intestinal mucosa. ET-3-induced elevations in tissue PMN infiltration and mucosal damage were significantly inhibited by pretreatments with ET-A or ET-B receptor antagonists. Overall, our data indicate that ET-3 causes microscopic damage, PMN infiltration and mucosal dysfunction in the rat small intestine. In addition, ET-3-induced hemodynamic alterations as well as tissue PMN infiltration and mucosal damage are mediated by both ET-A and ET-B receptors.     

Dynamin mediates caveolar sequestration of muscarinic cholinergic receptors and alteration in NO signaling

In cardiac myocytes, agonist binding to muscarinic acetylcholine receptors (mAchRs) leads to the targeting of stimulated receptors to plasmalemmal microdomains termed caveolae. Here, we examined whether this translocation leads to mAchR internalization and alteration in downstream NO signaling. Differential binding of membrane-permeant and -impermeant mAchR radioligands on caveolae-enriched membranes revealed that carbachol stimulation of cardiac myocytes induces sequestration of mAchRs through caveolae fission. GTP but not its non-hydrolyzable analog GTP gamma S drove the further detachment of caveolae from myocyte sarcolemma. Also, incubation of extracts of carbachol-stimulated myocytes with recombinant GTPase dynamin induced mAchR sequestration in budded caveolae, while dominant-negative K44A dynamin inhibited it. These data were confirmed by immunofluorescence microscopy on m2 mAchR-expressing COS cells. Finally, repeated carbachol stimulations of mAchRs co-expressed in COS cells with endothelial nitric oxide synthase (eNOS) and wild-type, but not mutant, dynamin led to a progressive increase in mAchR sequestration and a concurrent stabilization of the inhibitory eNOS-caveolin complex. These findings emphasize the role of caveolae in mAchR trafficking and NO signaling, and suggest that caveolae fission may contribute to G-protein-coupled receptor desensitization.     

Endothelin enhances adenosine and isoprenaline elevated cyclic AMP levels in rat cerebellar slices.

In this study, we present evidence showing that endothelin (ET) potentiates the responses to adenosine, to 5'-N-ethylcarboxamidoad, a nonhydrolyzable adenosine agonist, and to isoprenaline. These responses seem to occur through ET-B receptors, as all three endothelin isopeptides have the same potency, sarafotoxin 6c has the same effect as ET-1, BQ-123, an ET-A receptor antagonist has no effect, and BQ-788, an ET-B receptor antagonist that totally suppresses the responses analyzed.     

Regulation and intracellular trafficking pathways of the endothelin receptors

The effects of endothelin (ET) are mediated via the G protein-coupled receptors ET(A) and ET(B). However, the mechanisms of ET receptor desensitization, internalization, and intracellular trafficking are poorly understood. The aim of the present study was to investigate the molecular mechanisms of ET receptor regulation and to characterize the intracellular pathways of ET-stimulated ET(A) and ET(B) receptors. By analysis of ET(A) and ET(B) receptor internalization in transfected Chinese hamster ovary cells in the presence of overexpressed betaARK, beta-arrestin-1, beta-arrestin-2, or dynamin as well as dominant negative mutants of these regulators, we have demonstrated that both ET receptor subtypes follow an arrestin- and dynamin/clathrin-dependent mechanism of internalization. Fluorescence microscopy of Chinese hamster ovary and COS cells expressing green fluorescent protein (GFP)-tagged ET receptors revealed that the ET(A) and ET(B) subtypes were targeted to different intracellular routes after ET stimulation. While ET(A)-GFP followed a recycling pathway and colocalized with transferrin in the pericentriolar recycling compartment, ET(B)-GFP was targeted to lysosomes after ET-induced internalization. Both receptor subtypes colocalized with Rab5 in classical early endosomes, indicating that this compartment is a common early intermediate for the two ET receptors during intracellular transport. The distinct intracellular routes of ET-stimulated ET(A) and ET(B) receptors may explain the persistent signal response through the ET(A) receptor and the transient response through the ET(B) receptor. Furthermore, lysosomal targeting of the ET(B) receptor could serve as a biochemical mechanism for clearance of plasma endothelin via this subtype.     

Three-dimensional reconstruction of dynamin in the constricted state

Members of the dynamin family of GTPases have unique structural properties that might reveal a general mechanochemical basis for membrane constriction. Receptor-mediated endocytosis, caveolae internalization and certain trafficking events in the Golgi all require dynamin for vesiculation. The dynamin-related protein Drp1 (Dlp1) has been implicated in mitochondria fission and a plant dynamin-like protein phragmoplastin is involved in the vesicular events leading to cell wall formation. A common theme among these proteins is their ability to self-assemble into spirals and their localization to areas of membrane fission. Here we present the first three-dimensional structure of dynamin at a resolution of approximately 20 A, determined from cryo-electron micrographs of tubular crystals in the constricted state. The map reveals a T-shaped dimer consisting of three prominent densities: leg, stalk and head. The structure suggests that the dense stalk and head regions rearrange when GTP is added, a rearrangement that generates a force on the underlying lipid bilayer and thereby leads to membrane constriction. These results indicate that dynamin is a force-generating 'contrictase'.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

Tobacco-associated pulmonary vascular dysfunction in smokers: role of the ET-1 pathway

Pulmonary vascular remodeling and dysfunction associated to tobacco smoking might pave the way for the subsequent development of pulmonary hypertension. Its prognosis is dreadful and its underlying mechanisms are so far largely unknown in humans. To assess the potential role of endothelin-1 and its receptors in smokers' pulmonary artery vasoactive properties. Endothelium-dependent vasodilation to ACh was assessed in pulmonary vascular rings from 34 smokers and compared with that of 10 nonsmokers. The effects of ET-A (BQ 123) or ET-B (BQ 788) blockers and that of an ET-B activator (sarafotoxin) were evaluated. Endothelin-1 was quantitated by ELISA. Expression of its receptors was quantitated by Western blotting. Smokers exhibited an impaired pulmonary endothelium-dependent vasodilation compared with nonsmokers (P < 0.01). In the former group, 8 of 34 subjects exhibited a marked endothelial dysfunction (ED(+)) whereas 26 (ED(-)) (P < 10(-4)) displayed a vasorelaxation to ACh that was comparable to that of nonsmokers. In ED(+) subjects, ET-A was overexpressed (P < 0.05) and inversely correlated (P < 10(-2)) with the response to ACh. Sarafotoxin significantly improved vasodilation in all subjects (P < 10(-2)). In conclusion, tobacco smoking is associated to an impaired pulmonary vasorelaxation at least partly mediated by an ET-1/ET-A-dependent dysfunction.     

Regulation of the rat brain endothelin system by endogenous beta-endorphin

Several lines of evidence indicate that the central endogenous opioid and endothelin (ET) system regulate each other. To explore this idea further, we determined the effect of intracerebroventricular (i.c.v.) administration of anti-beta-endorphin IgG (rabbit) on the expression level of the opioid, corticotropin-releasing hormone and endothelin receptors, and tissue concentration of ET-1. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-beta-endorphin IgG (2.5 microg/microl) on days 1, 3 and 5. On day 6, animals were euthanized and caudate, cortex and hippocampus collected for Western blot analysis. Anti-beta-endorphin IgG down-regulated ET-A receptor protein expression in the caudate (51%), but had no effect on the expression of mu, delta, kappa opioid, ET-B, CRH-1 and CRH-2 receptors in any brain region. Anti-beta-endorphin IgG increased tissue ET-1 levels in the caudate by 30.3%. [35S]GTP-gamma-S binding assays demonstrated that anti-beta-endorphin IgG increased the efficacy of [D-Ala2-MePhe4, Gly-ol5]enkephalin without altering its potency in caudate. Control experiments showed that there was no detectable rabbit IgG in caudate, cortex and hippocampus samples. These results suggest that beta-endorphin in the CSF coordinately regulates ET-1 levels and the ET-A receptor in rat caudate. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between opioid and ET system.     

Type-specific sorting of G protein-coupled receptors after endocytosis

The beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are structurally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed that these receptors differ substantially in their membrane trafficking after endocytosis. B2AR expressed in stably transfected HEK293 cells exhibits negligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functional receptors) and immunoblotting (to detect total receptor protein). In contrast, DOR exhibits substantial (>/=50%) agonist-induced down-regulation when examined by similar means. Degradation of internalized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytometric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non-recycling pathways (respectively) can be detected within approximately 10 min after endocytosis, significantly before the onset of detectable proteolytic degradation of receptors ( approximately 60 min after endocytosis). Studies using pulsatile application of agonist suggest that after this sorting event occurs, later steps of membrane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observations establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substantial down-regulation of receptors.     

From membranes to organelles: Emerging roles for dynamin-like proteins in diverse cellular processes

Dynamin is a GTPase mechanoenzyme most noted for its role in vesicle scission during endocytosis, and belongs to the dynamin family proteins. The dynamin family consists of classical dynamins and dynamin-like proteins (DLPs). Due to structural and functional similarities DLPs are thought to carry out membrane tubulation and scission in a similar manner to dynamin. Here, we discuss the newly emerging roles for DLPs, which include vacuole fission and fusion, peroxisome maintenance, endocytosis and intracellular trafficking. Specific focus is given to the role of DLPs in the budding yeast Saccharomyces cerevisiae because the diverse function of DLPs has been well characterized in this organism. Recent insights into DLPs may provide a better understanding of mammalian dynamin and its associated diseases.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

What is the role of endothelin system?

Endothelins are a family of three peptides of 21 amino acids with strong vasoconstrictor effects. The three peptides are encoded by three different genes and derived from precursors (" big endothelins") which are cleaved by metalloproteases, named endothelin-converting enzyme. Two receptors have been cloned, ET-A and ET-B which bind the three endothelins with various affinities. The diverse expression pattern of the endothelin system (ET) components is associated with a complex pharmacology and its counteracting physiological actions. New modulators of the ET system have been described : retinoic acid, leptin, prostaglandins, hypoxia. Endothelins can be considered as regulators working in paracrine and autocrine fashion in a variety of organs in different cellular types. The ET system has beneficial and detrimental roles in mammals. The different components have been shown to be essential for a normal embryonic and neonatal development, for renal homeostasis and maintenance of basal vascular tone. They are involved in physiological and tumoral angiogenesis. They affect the physiology and pathophysiology of the liver, muscle, skin, adipose tissue and reproductive tract. The endothelin system participates in the development of atherosclerosis as well as pulmonary hypertension, and mediates cardiac remodeling in heart failure. Elaboration of new animal models (knock-out, pathophysiological models em leader ) will allow the clear genetic dissection of physiological and pathophysiological roles of the endothelin system.     

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.     

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.     

Presence of dynamin--syntaxin complexes associated with secretory granules in adrenal chromaffin cells

Dynamin proteins have been implicated in many aspects of endocytosis, including clathrin-mediated endocytosis, internalization of caveolae, synaptic vesicle recycling, and, more recently, vesicular trafficking to and from the Golgi complex. To provide further insight into the function(s) of dynamin in neuroendocrine cells, we have examined its intracellular distribution in cultured chromaffin cells by subcellular fractionation, immunoreplica analysis, and confocal immunofluorescence. We found that dynamin, presumably the dynamin-2 isoform, is associated specifically with the membrane of purified secretory chromaffin granules. Oligomerization state analysis by sucrose density velocity gradients indicated that the granule-associated dynamin is in a monomeric form. Immunoprecipitation experiments coupled to double-labeling immunofluorescence cytochemistry revealed that the granular dynamin is associated with a syntaxin component that is not involved in the granule-bound SNARE complex. The possibility that dynamin participates in the coupling of the exocytotic and endocytotic reaction through the building of a granular membrane subset of proteins is discussed.     

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.