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PubMed1998

High-affinity binding of epidermal growth factor (EGF) to EGF receptor is disrupted by overexpression of mutant dynamin (K44A).

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 degreesC EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degreesC. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.

Dynamins↗
PubMed1999

EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?

In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling.

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.

CSK Tyrosine-Protein Kinase↗
PubMed2005

Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease.

Synaptic dysfunction is one of the earliest events in the pathogenesis of Alzheimer disease (AD). However, the molecular mechanisms underlying synaptic defects in AD are largely unknown. We report here that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that is essential for synaptic vesicle recycling and, hence, for memory formation and information processing. The Abeta-induced dynamin 1 decrease occurred in the absence of overt synaptic loss and was also observed in the Tg2576 mouse model of AD. In addition, our results provided evidence that the Abeta-induced decrease in dynamin 1 was likely the result of a calpain-mediated cleavage of dynamin 1 protein and possibly the down-regulation of dynamin 1 gene expression. These data suggest a mechanism to explain the early cognitive loss without a major decline in synapse number observed in AD and propose a novel therapeutic target for AD intervention.

Alzheimer Disease↗
PubMed2005

Opposite roles of FAP-1 and dynamin in the regulation of Fas (CD95) translocation to the cell surface and susceptibility to Fas ligand-mediated apoptosis.

Human melanoma is the most aggressive form of skin cancer and is extremely resistant to radiation and chemotherapy. One of the critical parameters of this resistance is down-regulation of Fas (CD95) cell-surface expression. Using TIG3 normal human fibroblasts and human melanoma cell lines, we investigated transcriptional regulation of FAP-1, a regulator of Fas translocation in the cell. Protein-tyrosine phosphatase FAP-1 (PTPN13, PTP-BAS) interacts with human Fas protein and prevents its export from the cytoplasm to the cell surface. In contrast, dynamin-2 facilitates Fas protein translocation from the Golgi apparatus via the trans-Golgi network to the cell surface. Suppression of dynamin functions by dominant negative dynamin K44A blocks Fas export, whereas the down-regulation of FAP-1 expression by specific RNA interference restores Fas export (a phenomenon that could still be down-regulated in the presence of dominant-negative dynamin). Based on the FAP-1- and dynamin-dependent regulation of Fas translocation, we have created human melanoma lines with different levels of surface expression of Fas. Treatment of these melanoma lines with soluble Fas ligand resulted in programmed cell death that was proportional to the pre-existing levels of surface Fas. Taking into consideration the well known observations that FAP-1 expression is often up-regulated in metastatic tumors, we have established a causal connection between high basal NF-kappaB transcription factor activity (which is a hallmark of many types of metastatic tumors) and NF-kappaB-dependent transcriptional regulation of FAP-1 gene expression that finally restricts Fas protein trafficking, thereby, facilitating the survival of cancer cells.

Antigens, CD↗
PubMed1993

Mutations in human dynamin block an intermediate stage in coated vesicle formation.

The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.

Alternative Splicing↗
PubMed1998

Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus.

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69-80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915-934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949-966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.

Biological Transport↗
PubMed2001

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

3T3 Cells↗
PubMed1999

The dynamin-like protein DLP1 is essential for normal distribution and morphology of the endoplasmic reticulum and mitochondria in mammalian cells.

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

Animals↗
PubMed2001

Mammalian dynamin-like protein DLP1 tubulates membranes.

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) but not GTP. EM of the GTPgammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPgammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

Amino Acid Substitution↗
PubMed2003

Modulation of Rac localization and function by dynamin.

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin(-2) inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin(-2) depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.

Animals↗
PubMed2002

The dynamin A ring complex: molecular organization and nucleotide-dependent conformational changes.

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self-assembles into rings and helices in a nucleotide-dependent manner, similar to human dynamin-1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three-dimensional map, which shows that the nucleotide-free complex consists of two layers with 11-fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide-dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission.

Animals↗
PubMed2000

A novel Arabidopsis thaliana dynamin-like protein containing the pleckstrin homology domain.

A full-length cDNA encoding a novel type of plant dynamin-like protein, ADL3, was isolated from Arabidopsis thaliana. ADL3 is a high molecular weight GTPase whose GTP-binding domain shows a low homology to those of other plant dynamin-like proteins. ADL3 contains the pleckstrin homology domain as is in mammalian dynamins, although other plant dynamin-like proteins reported lack this domain. The ADL3 gene was expressed weakly in various tissues, except for siliques with high level expression, which is distinct from the case for other plant dynamin-like protein genes. Taken together, it is predicted that the mode of activation of ADL3 is different from those of other plant homologues.

Amino Acid Sequence↗
PubMed2003

The Arabidopsis dynamin-like proteins ADL1C and ADL1E play a critical role in mitochondrial morphogenesis.

Dynamin-related proteins are high molecular weight GTP binding proteins and have been implicated in various biological processes. Here, we report the functional characterization of two dynamin homologs in Arabidopsis, Arabidopsis dynamin-like 1C (ADL1C) and Arabidopsis dynamin-like 1E (ADL1E). ADL1C and ADL1E show a high degree of amino acid sequence similarity with members of the dynamin family. However, both proteins lack the C-terminal Pro-rich domain and the pleckstrin homology domain. Expression of the dominant-negative mutant ADL1C[K48E] in protoplasts obtained from leaf cells caused abnormal mitochondrial elongation. Also, a T-DNA insertion mutation at the ADL1E gene caused abnormal mitochondrial elongation that was rescued by the transient expression of ADL1C and ADL1E in protoplasts. In immunohistochemistry and in vivo targeting experiments in Arabidopsis protoplasts, ADL1C and ADL1E appeared as numerous speckles and the two proteins colocalized. These speckles were partially colocalized with F1-ATPase-gamma:RFP, a mitochondrial marker, and ADL2b localized at the tip of mitochondria. These results suggest that ADL1C and ADL1E may play a critical role in mitochondrial fission in plant cells.

Arabidopsis↗
PubMed2004

Regulating actin dynamics at membranes: a focus on dynamin.

Dynamin, the large guanosine triphosphatase, is generally considered to have a key role in deforming membranes to create tubules or vesicles. Dynamin, particularly dynamin2 isoforms, also are localized with actin filaments, often at locations where cellular membranes undergo remodeling. Perturbing dynamin function interferes with endocytic traffic and actin function. Thus, dynamin may regulate actin filaments coordinately with its activities that remodel membranes. This review will highlight recent observations that provide clues to mechanisms whereby dynamin might coordinate membrane remodeling and actin filament dynamics during endocytic traffic, cell morphogenesis and cell migration.

Actins↗
PubMed2004

The granzyme B-serglycin complex from cytotoxic granules requires dynamin for endocytosis.

Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake.

Apoptosis↗
PubMed2005

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid beta protein.

BACKGROUND: The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by alpha-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of beta- and gamma-secretases generates the amyloid beta protein (Abeta). In this study, we investigated the effects of modulators of endocytosis on APP processing. RESULTS: Human embryonic kidney cells were transfected with a dominant negative mutant of dynamin I, an important mediator of clathrin-dependent endocytosis, and APP proteolysis was analyzed. Overexpression of the mutant dynamin (dyn I K44A) resulted in increased shedding of the APP ectodomain (sAPPalpha), accumulation of the C-terminal alpha-secretase product C83, and a reduction in the release of Abeta. Levels of mature APP on the cell surface were increased in cells expressing dyn I K44A, and internalization of surface-immunolabeled APP, assessed by fluorescence microscopy, was inhibited. Dynamin is a substrate for protein kinase C (PKC), and it was hypothesized that activators of PKC, which are known to stimulate alpha-secretase-mediated cleavage of APP, might exert their effects by inhibiting dynamin-dependent endocytosis. However, the internalization of surface-biotinylated APP was unaffected by treatment of cells with phorbol 12-myristate 13-acetate in the presence of the alpha-secretase inhibitor TAPI-1. CONCLUSION: The results indicate that APP is internalized by a dynamin-dependent process, and suggest that alterations in the activity of proteins that mediate endocytosis might lead to significant changes in Abeta production.

Amyloid Precursor Protein Secretases↗
PubMed2006

Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization.

BACKGROUND: The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. METHODS: Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319-418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. RESULTS: In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319-418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. CONCLUSION: From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.

Animals↗