Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.     

Endosome fusion induced by diphtheria toxin translocation domain

Endosomal cargo travels through a dynamic vesicle network en route to degradation by lysosomes or recycling through the Golgi apparatus back to the cell surface. Rab5 is a key determinant of the early endosomes by organizing effector proteins in specific subdomains and mediating early endosome fusion. We find that early endosome morphogenesis and maturation is disrupted by diphtheria toxin (DT). Rab5 bound endosomes increase in size and in Rab5 content due to luminal toxin exposure, whereas Rab7 positive endosomes are not detectably altered. These changes appear to be caused by an activity of the toxin entry domain (T domain) as mutations inactivating either the receptor binding (CRM107) or ADP-ribosyl transferase (CRM197) activities do not inhibit the effect of DT on endosome morphogenesis. In contrast, mutations in the T domain or diminishing the endosomal pH gradient, which prevents T domain membrane insertion, inhibit these endosome changes. The change in size appears to be due to changing the early endosome fission-fusion equilibrium. The Rab5 membrane exchange rate, assessed with photoactivatable GFP-Rab5, decreases in the presence of DT. These changes to endosomes may reflect activities of the T domain that mediate toxin entry to the cytosol. The nontoxic mutant DT, CRM197, yields a new tool to manipulate endosome dynamics in living cells.     

Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified NPC1 gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.     

Copper incorporation into ceruloplasmin is regulated by Niemann–Pick C1 protein

Aim: Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans‐Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann–Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes. Methods: We induced the NPC phenotype using U18666A to modulate the vesicle traffic from the late endosome to TGN. Then, we examined the effect of NPC1 overexpression on the localization of ATP7B and secretion of holo‐Cp, a copper‐binding mature form of Cp. Results: Overexpression of NPC1 increased holo‐Cp secretion to culture medium of U18666A‐treated cells, but did not affect the secretion of albumin. Manipulation of NPC1 function affected the localization of ATP7B and late endosome markers, but did not change the localization of a TGN marker. ATP7B co‐localized with the late endosome markers, but not with the TGN marker. Conclusion: These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via an NPC1‐dependent pathway and incorporated into Cp.     

Rab5a is a common component of the apical and basolateral endocytic machinery in polarized epithelial cells.

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.     

Angiotensin (1-7) induces MAS receptor internalization

Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(1-7). For this purpose, we generated a chimera between the MasR and the yellow fluorescent protein (YFP; MasR-YFP). MasR-YFP-transfected HEK 293T cells were incubated with Ang-(1-7), and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 minutes. Following the time course of [(125)I]Ang-(1-7) endocytosis, we observed that half of MasR-YFP underwent endocytosis after 10 minutes, and this was blocked by a MasR antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1, and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin 1, suggesting that at some point MasR-YFP traverses caveolin 1-positive compartments. In conclusion, MasR undergoes endocytosis on stimulation with Ang-(1-7), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell.     

Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.

During receptor mediated endocytosis, at least a fraction of recycling cargo typically accumulates in a pericentriolar cluster of tubules and vesicles. However, it is not clear if these endosomal structures are biochemically distinct from the early endosomes from which they are derived. To better characterize this pericentriolar endosome population, we determined the distribution of two endogenous proteins known to be functionally involved in receptor recycling [Rab4, cellubrevin (Cbvn)] relative to the distribution of a recycling ligand [transferrin (Tfn)] as it traversed the endocytic pathway. Shortly after internalization, Tfn entered a population of early endosomes that contained both Rab4 and Cbvn, demonstrated by triple label immunofluorescence confocal microscopy. Tfn then accumulated in the pericentriolar cluster of recycling vesicles (RVs). However, although these pericentriolar endosomes contained Cbvn, they were strikingly depleted of Rab4. The ability of internalized Tfn to reach the Rab4-negative population was not blocked by nocodazole, although the characteristic pericentriolar location of the population was not maintained in the absence of microtubules. Similarly, Rab4-positive and -negative populations remained distinct in cells treated with brefeldin A, with only Rab4-positive elements exhibiting the extended tubular morphology induced by the drug. Thus, at least with respect to Rab4 distribution, the pathway of Tfn receptor recycling consists of at least two biochemically and functionally distinct populations of endosomes, a Rab4-positive population of early endosomes to which incoming Tfn is initially delivered and a Rab4-negative population of recycling vesicles that transiently accumulates Tfn on its route back to the plasma membrane.     

Differential regulation of CXCR2 trafficking by Rab GTPases

Intracellular trafficking of chemokine receptors plays an important role in fine-tuning the functional responses of neutrophils and lymphocytes in the inflammatory process and HIV infection. Although many chemokine receptors internalize through clathrin-coated pits, regulation of the receptor trafficking is not fully understood. The present study demonstrated that CXCR2 was colocalized with transferrin and low-density lipoprotein (LDL) after agonist treatment for different periods of time, suggesting 2 intracellular trafficking pathways for this receptor. CXCR2 was colocalized with Rab5 and Rab11a, which are localized in early and recycling endosomes, respectively, in response to agonist stimulation for a short period of time, suggesting a recycling pathway for the receptor trafficking. However, overexpression of a dominant-negative Rab5-S34N mutant significantly attenuated CXCR2 sequestration. The internalized CXCR2 was recycled back to the cell surface after removal of the agonist and recovery of the cells, but receptor recycling was inhibited by overexpression of a dominant-negative Rab11a-S25N mutant. After prolonged (4-hour) agonist treatment, CXCR2 exhibited significantly increased colocalization with Rab7, which is localized in late endosomes. The colocalization of CXCR2 with LDL and LAMP-1 suggests that CXCR2 is targeted to lysosomes for degradation after prolonged ligand treatment. However, the colocalization of CXCR2 with Lamp1 was blocked by the overexpression of a dominant-negative Rab7-T22N mutant. In cells overexpressing Rab7-T22N, CXCR2 was retained in the Rab5- and Rab11a-positive endosomes after prolonged (4-hour) agonist treatment. Our data suggest that the intracellular trafficking of CXCR2 is differentially regulated by Rab proteins.     

Aquaporin-2 is retrieved to the apical storage compartment via early endosomes and phosphatidylinositol 3-kinase-dependent pathway

Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.     

Visualizing microtubule-dependent vasopressin type 2 receptor trafficking using a new high-affinity fluorescent vasopressin ligand

The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)(2)-Suc-TMR(8)]VP or (VP(TMR)). This ligand is fully functional as revealed by its high binding affinity toward V2R [(K(d)) =157 ± 52 nM] and ability to increase intracellular cAMP 32-fold. VP(TMR) induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VP(TMR) and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VP(TMR) and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VP(TMR)-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VP(TMR), and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VP(TMR) ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism.     

TBC1D16 is a Rab4A GTPase activating protein that regulates receptor recycling and EGF receptor signaling

Rab4A is a master regulator of receptor recycling from endocytic compartments to the plasma membrane. The protein TBC1D16 is up-regulated in melanoma, and TBC1D16-overexpressing melanoma cells are dependent on TBC1D16. We show here that TBC1D16 enhances the intrinsic rate of GTP hydrolysis by Rab4A. TBC1D16 is both cytosolic and membrane associated; the membrane-associated pool colocalizes with transferrin and EGF receptors (EGFRs) and early endosome antigen 1, but not with LAMP1 protein. Expression of two TBC1D16 isoforms, but not the inactive R494A mutant, reduces transferrin receptor recycling but has no effect on transferrin receptor internalization. Expression of TBC1D16 alters GFP-Rab4A membrane localization. In HeLa cells, overexpression of TBC1D16 enhances EGF-stimulated EGFR degradation, concomitant with decreased EGFR levels and signaling. Thus, TBC1D16 is a GTPase activating protein for Rab4A that regulates transferrin receptor recycling and EGFR trafficking and signaling.     

Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes

CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.     

Agonist-induced endocytosis of rat somatostatin receptor 1

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.     

The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.     

Cell membrane GM1 ganglioside is a functional coreceptor for fibroblast growth factor 2

Free gangliosides bind fibroblast growth factor 2 (FGF2), thus preventing cell interaction and biological activity of the growth factor in endothelial cells. Here we investigated the role of cell-associated gangliosides in mediating the biological activity of FGF2. Treatment of endothelial cells of different origin with the ganglioside biosynthesis inhibitors fumonisin B1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or D-1-threo-1-phenyl-2-hexa-decanoylamino-3-pyrrolidino-1-propanol-HCl, impairs their capacity to proliferate when exposed to FGF2. Also, the mitogenic activity of FGF2 is inhibited by the GM1-binding cholera toxin B subunit (CTB). Conversely, overloading of endothelial GM 7373 cell membranes with exogenous GM1 causes a 10-fold increase of the mitogenic potency of FGF2. 125I-FGF2 binds to cell membrane GM1 (K(d) = 3 nM) in complex ganglioside/heparan sulfate-deficient Chinese hamster ovary (CHO)-K1-pgsA745 cell mutants that were overloaded with exogenous GM1. Moreover, FGF2 competes with FITC-CTB for the binding to cell membrane GM1 in different CHO cell lines independently of their capacity to express heparan sulfate proteoglycans. Conversely, CTB inhibits cell proliferation triggered by FGF2 in CHO cells overexpressing the tyrosine kinase FGF receptor 1. Finally, GM1-overloading confers to FGF receptor 1-transfected, complex ganglioside-deficient CHO-K1 cell mutants the capacity to proliferate when stimulated by FGF2. This proliferation is inhibited by CTB. Cell proliferation triggered by serum or by phorbol 12-myristate 13-acetate is instead independent of the cell membrane ganglioside milieu. In conclusion, cell membrane GM1 binds FGF2 and is required for the mitogenic activity of the growth factor. Our data indicate that cell-associated gangliosides may act as functional FGF2 co-receptors in different cell types.     

Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:WT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (<50%) compared with control Leishmania. Addition of exogenous hemin recovers the growth of Rab7:T21N mutant cells almost to the control level, suggesting that intracellular heme generated by Rab7-mediated Hb degradation is required for optimal growth of the parasites. Thus, our results identify a potential target which might be exploited to suppress the growth of Leishmania.     

Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells

Rab3 is a subfamily of the small GTP-binding protein Rab family and plays an important role in exocytosis. Several potential effectors of Rab3, including rabphilin3 and Rims (Rim1 and Rim2), have been isolated and characterized. Noc2 was identified originally in endocrine pancreas as a molecule homologous to rabphilin3, but its role in exocytosis is unclear. To clarify the physiological function of Noc2 directly, we have generated Noc2 knockout (Noc2(-/-)) mice. Glucose intolerance with impaired insulin secretion was induced in vivo by acute stress in Noc2(-/-) mice, but not in wild-type (Noc2(+/+)) mice. Ca(2+)-triggered insulin secretion from pancreatic isles of Noc2(-/-) mice was markedly impaired, but was completely restored by treatment with pertussis toxin, which inhibits inhibitory G protein Gi/o signaling. In addition, the inhibitory effect of clonidine, an alpha(2)-adrenoreceptor agonist, on insulin secretion was significantly greater in Noc2(-/-) islets than in Noc2(+/+) islets. Impaired Ca(2+)-triggered insulin secretion was rescued by adenovirus gene transfer of wild-type Noc2 but not by that of mutant Noc2, which does not bind to Rab3. Accordingly, Noc2 positively regulates insulin secretion from endocrine pancreas by inhibiting Gi/o signaling, and the interaction of Noc2 and Rab3 is required for the effect. Interestingly, we also found a marked accumulation of secretory granules in various exocrine cells of Noc2(-/-) mice, especially in exocrine pancreas with no amylase response to stimuli. Thus, Noc2, a critical effector of Rab3, is essential in normal regulation of exocytosis in both endocrine and exocrine cells.     

Prolactin secretion sites contain syntaxin-1 and differ from ganglioside monosialic acid rafts in rat lactotrophs

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.     

Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and up-regulate it rapidly to the cell surface upon activation

Activation and subsequent differentiation of naive CD8+ T cells lead to the development of memory subsets with distinct homing and effector capacities. On nonlymphoid homing subsets, expression of "inflammatory" chemokine receptors (such as CXCR3, CCR5, CX3CR1, and CXCR1) is believed to promote migration into sites of infection/inflammation. Here we show that CXCR1 can be up-regulated to the cell surface within minutes of activating human CD8+ T cells. No concurrent up-regulation of other inflammatory chemokine receptors was observed. Up-regulation of CXCR1 preferentially occurred on central memory CD8+ T cells-that is, cells with a lymph node homing phenotype-and was functionally relevant. Immunofluorescence microscopy showed CXCR1 to be present in intracellular vesicles that do not significantly colocalize with perforin, RANTES (regulated upon activation normal T cell expressed and secreted), or the lysosomal marker CD63. By contrast, partial colocalization with the Golgi marker GM130, the constitutive secretory pathway marker beta2-microglobulin, and the early endosome marker EEA1 was observed. Up-regulation of CXCR1 did not occur after T-cell receptor cross-linking. By contrast, supernatants from activated neutrophils, but not from monocytes or dendritic cells, induced its up-regulation. These results suggest that CD8+ T cells can rapidly adapt their homing properties by mobilizing CXCR1 from a distinct intracellular compartment.     

Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles thereby specifying signal output

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A(165)a and VEGF-A(165)b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1-binding VEGF-A(165)a led to sequential NRP-1-mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles. Recycling was accompanied by dephosphorylation of VEGFR-2 between Rab4 and Rab11 vesicles and quantitatively and qualitatively altered signal output. In cells stimulated with VEGF-A(165)b, an isoform unable to bind NRP-1, VEGFR-2 bypassed Rab11 vesicles and was routed to the degradative pathway specified by Rab7 vesicles. Deletion of the GIPC (synectin) binding motif of NRP-1 prevented transition of VEGFR-2 through Rab11 vesicles and attenuated signaling. Coreceptor engagement was specific for VEGFR-2 because EGFR recycled through Rab11 vesicles in the absence of known coreceptors. Our data establish a distinct role of NRP-1 in VEGFR-2 signaling and reveal a general mechanism for the function of coreceptors in modulating RTK signal output.