Different behavior of l-afadin and neurabin-II during the formation and destruction of cell-cell adherens junction.
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We have recently isolated two novel actin filament-binding proteins, l-afadin and neurabin-II and shown that they are localized at cell-cell adherens junction (AJ) in epithelial cells. We found here that l-afadin, neurabin-II, ZO-1, and E-cadherin showed similar and different behavior during the formation and destruction of cell-cell AJ in MDCK cells. In MDCK cells, the accumulation of both l-afadin and E-cadherin, but not that of ZO-1, changed in parallel depending on Rac small G protein activity. Dissociation of MDCK cells by culturing the cells at 2 microM Ca2+ caused rapid endocytosis of E-cadherin, but not that of l-afadin or ZO-1. Addition of phorbol 12-myristate 13-acetate to these dissociated cells formed a tight junction-like structure where ZO-1 and l-afadin, but not neurabin-II or E-cadherin, accumulated. We furthermore found that, in non-epithelial EL cells, which expressed E-cadherin and attached to each other, l-afadin, neurabin-II, ZO-1 and E-cadherin were all localized at AJ. In cadherin-deficient L cells, I-afadin was mainly localized at cell-cell contact sites, but ZO-1 was mainly localized at the tip area of cell processes. Neurabin-II did not accumulate at the plasma membrane area. Neither l-afadin nor neurabin-II significantly interacted with alpha-, beta-catenin, E-cadherin, ZO-1 or occludin. (+info)
Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization.
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We have previously reported the identification of four autophosphorylation sites on the KDR VEGF receptor. Two of these sites (tyrosines 951 and 996) are located in the receptor's kinase insert domain, and two (tyrosines 1054 and 1059) are located in the catalytic domain. In order to clarify the functional significance of these sites, we made DNA constructs in which tyrosine codons were replaced with those for phenylalanine, and expressed the DNA constructs in 293 cells. VEGF binding to cells expressing the native receptor led to a rapid increase in receptor and PLC-gamma phosphorylation, and a slower increase in the phosphorylation of p125FAK and paxillin. VEGF binding to KDR(Y951F) and KDR(Y996F) expressing cells resulted in phosphorylation of all cellular substrates tested, although the level of PLCgamma phosphorylation was decreased for KDR(Y996F). The decreased level of PLCgamma phosphorylation was not because PLCgamma-containing SH2 domains bind to the Y996 autophosphorylation site. We conclude that there exists receptor autophosphorylation sites not previously identified which allow for signaling via PLCgamma, as well as p125FAK and paxillin. VEGF binding to cells expressing KDR mutated at both tyrosine's 1054 and 1059 activated receptor autophosphorylation but at a level which was only 10% of that seen for cells expressing native receptor. Tyrosine phosphorylation of cell signaling proteins was not observed in KDR(Y1054,1059) expressing cells. Utilizing an in vitro assay which directly measures receptor catalytic activity allowed us to determine that the tyrosine kinase activity of the native receptor was significantly greater than that for the double mutant. We conclude from this result that VEGF-induced autophosphorylation at tyrosines 1054 and 1059 is a required step for allowing maximal KDR kinase activity. Maximal rates of receptor kinase activity is required for VEGF-induced receptor internalization, as internalization was delayed in the KDR(Y1054,1059F) expressing cells when compared to cells expressing native receptor. (+info)
Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling.
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The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis. (+info)
Multiple distinct coiled-coils are involved in dynamin self-assembly.
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Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association. (+info)
The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions.
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Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes. (+info)
Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein.
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The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10(5) Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 degrees C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose. (+info)
Inhibition of clathrin-coated pit assembly by an Eps15 mutant.
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Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits. (+info)
New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites.
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We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin. These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization. Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A. Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site. We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177. The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin. The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket. Actin purified from one of these mutants has a reduced affinity for tolytoxin. In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin. We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket. (+info)