Role of clathrin-mediated endocytosis in CXCR2 sequestration, resensitization, and signal transduction.
(9/999)
CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines interleukin-8, melanoma growth-stimulatory activity/growth-regulatory protein, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we explored the mechanism responsible for ligand-induced CXCR2 endocytosis. Here, we demonstrate that dynamin, a component of clathrin-mediated endocytosis, is essential for CXCR2 endocytosis and resensitization. In HEK293 cells, dynamin I K44A, a dominant-negative mutant of dynamin that inhibits the clathrin-mediated endocytosis, blocks the ligand-stimulated CXCR2 sequestration. Furthermore, co-expression of dynamin I K44A significantly delays dephosphorylation of CXCR2 after ligand stimulation, suggesting that clathrin-mediated endocytosis plays an important role in receptor dephosphorylation and resensitization. In addition, ligand-mediated receptor down-regulation is attenuated when receptor internalization is inhibited by dynamin I K44A. Interestingly, inhibition of receptor endocytosis by dynamin I K44A does not affect the CXCR2-mediated stimulation of mitogen-activated protein kinase. Most significantly, our data indicate that the ligand-stimulated receptor endocytosis is required for CXCR2-mediated chemotaxis in HEK293 cells. Taken together, our findings suggest that clathrin-mediated CXCR2 internalization is crucial for receptor endocytosis, resensitization, and chemotaxis. (+info)
Contribution of active zone subpopulation of vesicles to evoked and spontaneous release.
(10/999)
Our previous work on Drosophila synapses has suggested that two vesicle populations possessing different recycling pathways, a fast pathway emanating from the active zone and a slower pathway emanating from sites away from the active zone, exist in the terminal. The difference in recycling time between these two pathways has allowed us to create a synapse that possesses the small, active zone subpopulation without the larger, nonactive zone population. Synapses were depleted using the temperature-sensitive endocytosis mutant, shibire, which reversibly blocks vesicle recycling at the restrictive temperature. In the depleted state, both the excitatory junction potential (EJP) and spontaneous release are abolished. After shibire-induced depletion, the active zone population begins to reform within 30 s at the permissive temperature, whereas the nonactive zone population does not begin to reform until approximately 10-15 min later. Evoked release recovered at approximately the same time as the active zone population. During the time when the active zone population existed in the terminal without the nonactive zone population, enough transmitter release was available to sustain a normal evoked response for many minutes at frequencies above those produced during normal activity (flight) by this motor neuron. When only the active zone population existed in the terminal, the frequency of spontaneous release was greatly attenuated and possessed abnormal release characteristics. Spontaneous release recovered its predepletion frequency and release characteristics only after the nonactive zone population was reformed. (+info)
U50,488H-induced internalization of the human kappa opioid receptor involves a beta-arrestin- and dynamin-dependent mechanism. Kappa receptor internalization is not required for mitogen-activated protein kinase activation.
(11/999)
Agonist-promoted internalization of some G protein-coupled receptors has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether opioids induced internalization of the human and rat kappa opioid receptors stably expressed in Chinese hamster ovary cells, the potential mechanisms involved in this process and its possible role in activation of mitogen-activated protein (MAP) kinase. Exposure of the human kappa receptor to the agonists U50,488H, U69,593, ethylketocyclazocine, or tifluadom, but not etorphine, promoted receptor internalization. However, none of these agonists induced significant internalization of the rat kappa opioid receptor. U50, 488H-induced human kappa receptor internalization was time- and concentration-dependent, with 30-40% of the receptors internalized following a 30-min exposure to 1 microM U50,488H. Agonist removal resulted in the receptors gradually returning to the cell surface over a 60-min period. The antagonist naloxone blocked U50, 488H-induced internalization without affecting internalization itself, while pretreatment with pertussis toxin had no effect on U50, 488H-induced internalization. In contrast, incubation with sucrose (0.4-0.8 M) significantly reduced U50,488H-induced internalization of the kappa receptor. While co-expression of the wild type GRK2, beta-arrestin, or dynamin I had no effect on kappa receptor internalization, co-expression of the dominant negative mutants GRK2-K220R, beta-arrestin (319-418), or dynamin I-K44A significantly inhibited receptor internalization. Whether receptor internalization is critical for MAP kinase activation was next investigated. Co-expression of dominant negative mutants of beta-arrestin or dynamin I, which greatly reduced U50,488H-induced internalization, did not affect MAP kinase activation by the agonist. In addition, etorphine, which did not promote human kappa receptor internalization, was able to fully activate MAP kinase. Moreover, U50,488H or etorphine stimulation of the rat kappa receptor, which did not undergo internalization, also effectively activated MAP kinase. Thus, U50,488H-induced internalization of the human kappa opioid receptor in Chinese hamster ovary cells occurs via a GRK-, beta-arrestin-, and dynamin I-dependent process that likely involves clathrin-coated pits. In addition, internalization of the kappa receptor is not required for activation of MAP kinase. (+info)
Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth.
(12/999)
Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C. psittaci GPIC and C. trachomatis LGV/L2 into HeLa cells and demonstrated that it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When clathrin-dependent endocytosis was inhibited by overexpression of the dynK44A mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in the proliferation of Chlamydia and the growth of the chlamydia vacuole (inclusion). When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia was unaltered. These results indicate that the effect on the growth of Chlamydia in the dynK44A cells was not simply due to a deprivation of nutrients taken up by endocytosis. Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion and intracellular membrane compartments. In addition, cytochalasin D inhibited Chlamydia entry by more than 90%, indicating that chlamydiae enter epithelial cells by an actin-dependent mechanism resembling phagocytosis. Finally, dynamin is apparently not involved in the formation of phagocytic vesicles containing Chlamydia. (+info)
Genetic evidence for an equilibrium between docked and undocked vesicles.
(13/999)
By using the shibire mutation to block endocytosis in a temperature-dependent fashion, we have manipulated the number of synaptic vesicles in a nerve terminal and have observed a remarkable proportionality of the number of quanta released to the size of the total vesicle pool. In the experiments described below we determine that approximately 0.3% of the vesicle pool is released per stimulus. The data suggest that the pool of readily releasable docked vesicles does not represent the saturation of a limiting number of release sites, but instead represents a subset of vesicles that is in equilibrium with the larger pool of vesicles. Before presenting this data and the significance of the finding for the regulation of neurotransmission, we will briefly review the use of Drosophila genetics as a tool for dissecting synaptic transmission. (+info)
Endocytosis: How dynamin sets vesicles PHree!
(14/999)
The dynamin GTPase is required for clathrin-dependent, receptor-mediated endocytosis. Exciting new studies have shown that dynamin's pleckstrin homology domain binds to phosphatidylinositol 4, 5-bisphosphate in vivo, thus localising dynamin directly at the plasma membrane and ultimately enabling vesiculation. (+info)
Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination. (+info)
A model for dynamin self-assembly based on binding between three different protein domains.
(16/999)
Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral. (+info)