The small molecular weight G-protein RAB7 is localized to both early and late endosomes and has been shown to be critical for trafficking through the endocytic pathway. The role of RAB7 in the endocytic pathway has been controversial, with some groups reporting that it regulates trafficking from early to late endosomes and others ascribing its role to trafficking between late endosomes and lysosomes. In this study, we use RNA interference to identify the exact step RAB7 regulates in the movement of the epidermal growth factor receptor (EGFR) from the cell surface to the lysosome. In the absence of RAB7, trafficking of the EGF⅐EGFR complex through the early endosome to the late endosome/multivesicular body (LE/MVB) does not change, but exiting from the LE/MVB is blocked. Ultrastructural analysis reveals that RAB7 is not required for formation of intraluminal vesicles of the LE/MVB, since RAB7-deficient cells have an increased number of enlarged LE/MVBs densely packed with intraluminal vesicles. Biochemical data indicate that the EGFR complex is sequestered in these intraluminal vesicles. Together, these data provide evidence that RAB7 is required for the transfer of cargo from the LE/MVB to the lysosome and for endocytic organelle maintenance.The endocytic pathway regulates a number of fundamental cellular processes. These include the uptake of nutrients, immune response, intracellular transport, and regulation of cell surface receptor signaling (1). Disruption of normal endocytic trafficking can affect cellular homeostasis and lead to changes in cell physiology that range from hyperproliferation to cell death. Understanding the molecular regulation of endocytic trafficking will provide a better understanding of basic cell biology as well as identify potential molecular targets for diseases characterized by defects in endocytic trafficking.By following the postinternalization events of cell surface receptors, considerable work has been done to elucidate the molecular details of the endocytic pathway (2). Many cell surface receptors, either constitutively or in response to ligand, use this degradative pathway to regulate receptor and/or ligand levels. Following clathrin-mediated internalization, the endocytic pathway is composed of a series of dynamic stages that progressively shuttle cargo from clathrin-coated vesicles to early endosomes, to late endosomes/multivesicular bodies (LE/MVBs), 2 and finally to lysosomes for degradation. Each of these endocytic stages is defined by the morphology and protein composition of the organelle.Endocytic trafficking is coordinated by a variety of proteins that regulate endosome maturation, movement, fission, and fusion. Primary among these are the small molecular weight G-proteins called RABs (3). Rab proteins are members of the Ras superfamily of GTPases that cycle between GTP-bound active and GDPbound inactive states. The nucleotide bound state of the RAB determines whether it can interact with downstream effectors. Individual RAB proteins have been shown to act as hubs that...

Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2-and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.The binding of insulin to its heterotetrameric integral-membrane receptor activates its intracellular tyrosine kinase domain and thereby triggers a signaling cascade resulting in the translocation and fusion of intracellular GLUT4 1 isoform-containing vesicles to the plasma membrane (1-3). Although most cell types also constitutively express the GLUT1 isoform at the cell surface, the insulin-stimulated increase in plasma membrane-associated GLUT4 protein accounts for the majority of post-prandial glucose disposal in both muscle and adipose tissue (4).The insulin-stimulated translocation of these GLUT4-containing vesicles has several features in common with the regulated exocytosis pathway of synaptic vesicle trafficking in neurotransmitter release (5). The machinery involved in the regulation of synaptic vesicle priming/docking/fusion entails the pairing of protein complexes in the vesicle compartment (v-SNAREs, for vesicle SNAP receptors) with their cognate receptor complexes at the target membrane (t-SNAREs, for target membrane SNAP receptors). Recently, several of the vand t-SNARE proteins have been identified that specifically participate in the insulin-regulated docking and fusion of GLUT4 vesicles with the adipocyte plasma membrane. GLUT4 vesicles co-purify with both the VAMP2 and VAMP3 v-SNARE isoforms and specific proteolytic cleavage of VAMP2, expression of a dominant-interfering VAMP2 mutant or inhibitory peptides impairs insulin-stimulated GLUT4 translocation (6 -11). In addition, transferrin-horseradish peroxidase ablation of recycling endosomes resulted in a selective loss ...

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We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both A␤40 and A␤42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of ␤-cleaved amyloid precursor protein (APP) carboxylterminal fragments (␤CTF), the rate-limiting proteolytic intermediate in A␤ generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in ␤-cleaved soluble APP relative to ␣-cleaved soluble APP was also observed following Rab5 overexpression. ␤CTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, ␤CTF generation, and A␤ production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a ␤-amyloidogenic pathway.In the brain parenchyma, the deposition of the small peptide A␤, a proteolyic fragment of the amyloid precursor protein (APP), 1 is an invariant feature of Alzheimer's disease (AD) (1). Generation of A␤ requires two sequential proteolytic steps:␤-cleavage in the lumenal domain of APP followed by intramembranous ␥-cleavage (reviewed in Ref.2). ␤-Cleavage appears to be mediated by the BACE family of aspartyl-proteases (3, 4), whereas ␥-cleavage requires the presenilin protein complex (5, 6). The intracellular site(s) of A␤ generation has been the subject of intense investigation for a number of years, and both the secretory and endocytic pathways have been implicated (7-13). Confounding the clear delineation of A␤ generation sites is the broad intracellular distribution of the key proteins involved. APP is predominantly localized to the trans-Golgi network (TGN), but significant amounts of the protein are found at the cell surface and within endosomes and other transport vesicles (2). BACE has been placed within early endosomes and/or throughout the endosomal-lysosomal system (3, 14, 15), although a predominant TGN localization has also been reported (16,17). The presenilin proteins were initially thought to be located primarily within the endoplasmic reticulum and Golgi apparatus (18 -23), but more recent evidence suggests a distribution also within the plasma membrane and endosomes (18, 24 -27), and studies of nicastrin also strongly suggest that the mature presenilin complex is found in compartments distal to the endoplasmic reticulum (28).Our previous studies have shown that abnormalities of the neuronal endocytic pathwa...

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.Receptor-mediated endocytosis is an essential mechanism for several important physiological processes. These include down regulation of cell surface receptors, degradation of the receptor and/or ligand, absorption and retention of essential nutrients, transcellular transport of specific ligands, and the activation of intracellular signal transduction cascades (42,49,52). It is well established that the insulin receptor undergoes endocytosis upon insulin stimulation (3,28,35,37); however, the exact physiological significance of insulin receptor internalization is poorly understood. Several studies have demonstrated that following insulin stimulation, the endosome-localized insulin receptor exhibits increased autophosphorylating and exogenous substrate tyrosine kinase activity compared to plasma membrane-associated insulin receptors (3,31,32). Based on the time course of endosome association, kinase activation, and compartmentalization of effector substrates, it has been hypothesized that the internalized endosome-associated insulin receptor population is responsible for the activation of intracellular signaling pathways (12,18,31). However, other studies have reported that low-temperature (4°C) blockade of insulin receptor internalization does not impair either insulin receptor autophosphorylation or tyrosine phosphorylation of the major insulin receptor substrate, IRS1 (9, 26). Thus, in contrast, these data suggest that activa...