Systemic lupus erythematosus (SLE) is characterized by the dysfunction of T cells, B cells, and dendritic cells, the release of pro-inflammatory nuclear materials from necrotic cells, and the formation of antinuclear antibodies (ANA) and immune complexes of ANA with DNA, RNA, and nuclear proteins. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a key factor in abnormal activation of T and B cells in SLE. In T cells, increased production of nitric oxide and mitochondrial hyperpolarization (MHP) were identified as metabolic checkpoints upstream of mTOR activation. mTOR controls the expression T-cell receptor-associated signaling proteins CD4 and CD3zeta through increased expression of the endosome recycling regulator Rab5 and HRES-1/Rab4 genes, enhances Ca2+ fluxing and skews the expression of tyrosine kinases both in T and B cells, and blocks the expression of Foxp3 and the generation of regulatory T cells. MHP, increased activity of mTOR, Rab GTPases, and Syk kinases, and enhanced Ca2+ flux have emerged as common T and B cell biomarkers and targets for treatment in SLE.

ExoS (453 amino acids) is a bi-functional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96-219 include the Rho GTPase-activating protein (RhoGAP) domain, and residues 234-453 include the 14-3-3-dependent ADP-ribosyltransferase domain. Earlier studies also identified an N-terminal domain (termed the membrane localization domain) that comprises residues 51-77 and includes a novel leucine-rich motif that targets ExoS to the perinuclear region of cultured cells. There is limited information on how ExoS or other type III cytotoxins enter and target intracellular host proteins. Type III-delivered ExoS localized to both plasma membrane and perinuclear region, whereas ExoS(DeltaMLD) was localized to the cytosol. Plasma membrane localization of ExoS was transient and had a half-life of approximately 20 min. Type III-delivered ExoS co-immunoprecipitated 14-3-3 proteins and Rab9, Rab6, and Rab5. Immunofluorescence experiments showed that ExoS colocalized with Rab9, Rab6, and Rab5. Fluorescent energy transfer was detected between ExoS and 14-3-3 proteins but not between ExoS and Rabs proteins. Together, these results indicate that type III-delivered ExoS localizes on the host endosomes and utilizes multiple pathways to traffic from the plasma membrane to the perinuclear region of intoxicated host cells.

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.

NADPH oxidase 2 (Nox2)-generated reactive oxygen species (ROS) are critical for neutrophil (polymorphonuclear leukocyte (PMN)) microbicidal function. Nox2 also plays a role in intracellular signaling, but the site of oxidase assembly is unknown. It has been proposed to occur on secondary granules. We previously demonstrated that intracellular NADPH oxidase-derived ROS production is required for endotoxin priming. We hypothesized that endotoxin drives Nox2 assembly on endosomes. Endotoxin induced ROS generation within an endosomal compartment as quantified by flow cytometry (dihydrorhodamine 123 and Oxyburst Green). Inhibition of endocytosis by the dynamin-II inhibitor Dynasore blocked endocytosis of dextran, intracellular generation of ROS, and priming of PMN by endotoxin. Confocal microscopy demonstrated a ROS-containing endosomal compartment that co-labeled with gp91(phox), p40(phox), p67(phox), and Rab5, but not with the secondary granule marker CD66b. To further characterize this compartment, PMNs were fractionated by nitrogen cavitation and differential centrifugation, followed by free flow electrophoresis. Specific subfractions made superoxide in the presence of NADPH by cell-free assay (cytochrome c). Subfraction content of membrane and cytosolic subunits of Nox2 correlated with ROS production. Following priming, there was a shift in the light membrane subfractions where ROS production was highest. CD66b was not mobilized from the secondary granule compartment. These data demonstrate a novel, nonphagosomal intracellular site for Nox2 assembly. This compartment is endocytic in origin and is required for PMN priming by endotoxin.

Dysregulation of EGFR expression and signaling is well documented to contribute to disease progression and metastasis in many types of cancer including breast cancer. EGF-stimulated EGFR activation leads to receptor internalization and endocytic degradation to control EGFR-mediated signaling. This process is frequently deregulated in cancer cells, leading to increased EGFR expression and mitogenic signaling. Here, we demonstrate that Bif-1, a tumor suppressor, plays a role in EGFR endocytic degradation and chemotactic migration in MDA-MB-231 breast cancer cells. Our data reveal that suppression of Bif-1 expression delays EGFR degradation and sustains Erk1/2 activation in response to EGF stimulation. Mechanistically, loss of Bif-1 sequesters internalized EGF in Rab5-positive endosomes and delays EGFR trafficking to lysosomes. Recruitment of Rab7 to EGF-positive vesicles and the activation of Rab7 are impaired in Bif-1 knockdown cells. Additionally, intracellular pH and the localization of acidic vesicles are altered by suppression of Bif-1. Furthermore, inhibition of Bif-1 increases chemotactic cell migration in response to EGF or serum, which correlates with prolonged cytoskeletal reorganization. Importantly, the effect of Bif-1 on EGF-induced cell migration is abolished by gefitinib, an EGFR-specific inhibitor. Taken together, these data suggest a novel function for Bif-1 as a suppressor of breast cancer cell migration by promoting EGFR degradation through the regulation of endosome maturation.

African American men in the United States have higher mortality due to prostate cancer (PCa) compared to other races. One reason for this disparity is the lack of in-depth understanding of the PCa biology in African Americans. For example, hypoxia in prostate tumor microenvironment is associated with adverse prognosis; still, no hypoxia-related studies have been reported in African Americans. Here, we compared African-American and Caucasian PCa cells for exosome secretion under normoxic (21% O2) and hypoxic (1% O2) conditions. All cell lines showed higher exosome secretion under hypoxia but it was clearly more prominent in African-American PCa cells. Further, under hypoxia, Rab5 (a biomarker for early endosome) was clustered in perinuclear region; and CD63 (a biomarker for exosomes and multivesicular endosomes) showed greater co-localization with actin cytoskeleton especially in African American PCa cells. Importantly, exosome biogenesis inhibitors GW4869 (10-20 µM) or DMA (10-20 µg/ml) significantly decreased cell viability and clonogenicity in PCa cells. Interestingly, we also observed higher level of lactic acid loaded in exosomes secreted under hypoxia. Overall, under chronic hypoxia, PCa cells secrete more exosomes as a survival mechanism to remove metabolic waste.

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

To identify genes which are differentially transcribed in colorectal tumor cells, we compared the two human tumor cell lines, SW480 and HCT116, with the cell line, NCM460, from normal colon epithelium as a control. Using the methods of differential display reverse transcription PCR and Northern blot hybridization, we detected the differential transcription of seven genes: cholecystokinin, reticulocalbin, Rab5 guanine nucleotide exchange factor Rabex5, caldesmon, differentiation related gene 1 (drg1), taxol resistant associated gene 3 (Trag-3) and the gene for the placental protein, diff33. The yet unidentified cDNA of the human Rabex5 gene and the 3' untranslated region of the human caldesmon gene were cloned.

Drosophila endocytosis-defective cells develop tumour overgrowths in the imaginal discs. We have analysed the tumorigenic potential of cells mutant for Rab5, a gene involved in endocytosis. We found that while a compartment entirely made by Rab5 mutant cells can grow indefinitely, clones of Rab5 cells surrounded by normal cells are eliminated by cell competition. However, when a group of about 400 cells are simultaneously made mutant for Rab5, they form an overgrowing tumour: mutant cells in the periphery are eliminated, but those inside survive and continue proliferating because they are beyond the range of cell competition. These results identify group protection as a mechanism to evade the tumour-suppressing function of cell competition in Drosophila. Furthermore, we find that the growth of the tumour depends to a large extent on the presence of apoptosis inside the tumour: cells doubly mutant for Rab5 and the proapoptotic gene dronc do not form overgrowing tumours. These results suggest that the apoptosis caused by cell competition acts as a tumour-stimulating factor, bringing about high levels of Jun N-terminal kinase and subsequently Wg/Dpp signalling and high proliferation levels in the growing tumour. We conclude that under these circumstances cell competition facilitates the progression of the tumour, thus reversing its normal antitumour role.

We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.

Metastatic disease is dependent on tumor cell migration through the venous and lymphatic systems and requires dynamic rearrangement of adherens junctions. Endocytosis of cadherins is a key mechanism to dynamically arrange adherens junctions, signaling, and motility in tumor cells; however, the role of shear in regulating this process in metastatic cells is unknown. In this study, the role of shear in regulating cell surface expression of E-cadherin was investigated. We found that exposure to venous shear (shear rate, 200/s) induced internalization of E-cadherin in adherent metastatic oesophageal tumor cells (OC-1 tumor cell line). Internalized E-cadherin was found localized to Rab5-positive endosomes and was not present in lysosomes. As the Src family of tyrosine kinase have been implicated in regulating cadherin expression, we investigated the role of shear in regulating E-cadherin through Src activity. Pretreatment of OC-1 cells with the specific Src kinase inhibitor 4-amino-5- (4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) prevented shear-induced internalization of E-cadherin. Direct measurement of Src activity (phosphorylation on Y416) showed that Src is activated in sheared OC-1 cells and that the shear-induced increase in phospho-Src is inhibited by the presence of PP1. Moreover, we show that shear stress significantly increased the invasive capacity of OC-1 cells (P < 0.001), a process inhibited by the presence of PP1. These results indicate a novel role for shear in regulating the endocytosis of E-cadherin and invasiveness in metastatic cells.

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.

Classical swine fever virus (CSFV), a member of the genus Pestivirus within the family Flaviviridae, is a small, enveloped, positive-strand RNA virus. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. However, the mechanisms of CSFV entry into cells are not well characterized. In this study, we used systematic approaches to dissect CSFV cell entry. We first observed that CSFV infection was inhibited by chloroquine and NH4Cl, suggesting that viral entry required a low-pH environment. By using the specific inhibitor dynasore, or by expressing the dominant negative (DN) K44A mutant, we verified that dynamin is required for CSFV entry. CSFV particles were observed to colocalize with clathrin at 5 min postinternalization, and CSFV infection was significantly reduced by chlorpromazine treatment, overexpression of a dominant negative form of the EPS15 protein, or knockdown of the clathrin heavy chain by RNA interference. These results suggested that CSFV entry depends on clathrin. Additionally, we found that endocytosis of CSFV was dependent on membrane cholesterol, while neither the overexpression of a dominant negative caveolin mutant nor the knockdown of caveolin had an effect. These results further suggested that CSFV entry required cholesterol and not caveolae. Importantly, the effect of DN mutants of three Rab proteins that regulate endosomal traffic on CSFV infection was examined. Expression of DN Rab5 and Rab7 mutants, but not the DN Rab11 mutant, significantly inhibited CSFV replication. These results were confirmed by silencing of Rab5 and Rab7. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab7 during the early phase of infection within 45 min after virus entry. These results indicated that after internalization, CSFV moved to early and late endosomes before releasing its RNA. Taken together, our findings demonstrate for the first time that CSFV enters cells through the endocytic pathway, providing new insights into the life cycle of pestiviruses.

Bovine viral diarrhea virus (BVDV), a single-stranded, positive-sense pestivirus within the family Flaviviridae, is internalized by clathrin-dependent receptor-mediated endocytosis. However, the detailed mechanism of cell entry is unknown for other pestiviruses, such as classical swine fever (CSF) virus (CSFV). CSFV is the etiological agent of CSF, a highly contagious disease of swine that causes numerous deaths in pigs and enormous economic losses in China. Understanding the entry pathway of CSFV will not only advance our knowledge of CSFV infection and pathogenesis but also provide novel drug targets for antiviral intervention. Based on this objective, we used systematic approaches to dissect the pathway of entry of CSFV into PK-15 cells. This is the first report to show that the entry of CSFV into PK-15 cells requires a low-pH environment and involves dynamin- and cholesterol-dependent, clathrin-mediated endocytosis that requires Rab5 and Rab7.

Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca(2+)/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.

Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C. jejuni lipooligosaccharide (LOS) structures, generating human nerve ganglioside mimics, is important for intestinal epithelial translocation. We here show that C. jejuni isolates expressing ganglioside-like LOS bound in larger numbers to the Caco-2 intestinal epithelial cells than C. jejuni isolates lacking such structures. Next, we found that ganglioside-like LOS facilitated endocytosis of bacteria into Caco-2 cells, as visualized by quantitative microscopy using the early and late endosomal markers early endosome-associated protein 1 (EEA1), Rab5, and lysosome-associated membrane protein 1 (LAMP-1). This increased endocytosis was associated with larger numbers of surviving and translocating bacteria. Next, we found that two different intestinal epithelial cell lines (Caco-2 and T84) responded with an elevated secretion of the T-cell attractant CXCL10 to infection by ganglioside-like LOS-expressing C. jejuni isolates. We conclude that C. jejuni translocation across Caco-2 cells is facilitated by ganglioside-like LOS, which is of clinical relevance since C. jejuni ganglioside-like LOS-expressing isolates are linked with severe gastroenteritis and bloody stools in C. jejuni-infected patients.

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.

In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogenesis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Eps15 mutants or depleting K(+) trapped AdipoR1 at the plasma membrane, and K(+) depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoR1 and adiponectin is clathrin-dependent. Depletion of K(+) and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might downregulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoR1 is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.

G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

Dysfunction of alsin, particularly its putative Rab5 guanine-nucleotide-exchange factor activity, has been linked to one form of juvenile onset recessive familial amyotrophic lateral sclerosis (ALS2). Multiple lines of alsin knockout (ALS2(-/-)) mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2(-/-) neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2(-/-) neurons, we introduced green fluorescent protein (GFP)-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes. Here we report that Rab5-mediated endocytosis was severely altered in ALS2(-/-) neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2(-/-) neurons, which correlated with a significant reduction in endosomal motility and augmentation in endosomal conversion to lysosomes. Consequently, a significant increase in endosome/lysosome-dependent degradation of internalized glutamate receptors was observed in ALS2(-/-) neurons. These phenotypes closely resembled the endosomal trafficking abnormalities induced by a constitutively active form of Rab5 in wild-type neurons. Therefore, our findings reveal a negatively regulatory mechanism of alsin in Rab5-mediated endosomal trafficking, suggesting that enhanced endosomal degradation in ALS2(-/-) neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.

Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (mu2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the mu-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4-acetamido-4'-isothiocyabatostilbene-2',2-disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation.