Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function.
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Endothelial nitric-oxide synthase (eNOS) is regulated in part through specific protein interactions. Dynamin-2 is a large GTPase residing within similar membrane compartments as eNOS. Here we show that dynamin-2 binds directly with eNOS thereby augmenting eNOS activity. Double label confocal immunofluorescence demonstrates colocalization of eNOS and dynamin in both Clone 9 cells cotransfected with green fluorescent protein-dynamin and eNOS, as well as in bovine aortic endothelial cells (BAEC) expressing both proteins endogenously, predominantly in a Golgi membrane distribution. Immunoprecipitation of eNOS from BAEC lysate coprecipitates dynamin and, conversely, immunoprecipitation of dynamin coprecipitates eNOS. Additionally, the calcium ionophore, a reagent that promotes nitric oxide release, enhances coprecipitation of dynamin with eNOS in BAEC, suggesting the interaction between the proteins can be regulated by intracellular signals. In vitro studies demonstrate that glutathione S-transferase (GST)-dynamin-2 quantitatively precipitates both purified recombinant eNOS protein as well as in vitro transcribed (35)S-labeled eNOS from solution indicating a direct interaction between the proteins in vitro. Scatchard analysis of binding studies demonstrates an equilibrium dissociation constant (K(d)) of 27.6 nm. Incubation of purified recombinant eNOS protein with GST-dynamin-2 significantly increases eNOS activity as does overexpression of dynamin-2 in ECV 304 cells stably transfected with eNOS-green fluorescent protein. These studies demonstrate a direct protein-protein interaction between eNOS and dynamin-2, thereby identifying a new NOS-associated protein and providing a novel function for dynamin. These events may have relevance for eNOS regulation and trafficking within vascular endothelium. (+info)
Dynamin is involved in human epithelial cell vacuolation caused by the Helicobacter pylori-produced cytotoxin VacA.
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The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. To elucidate the molecular mechanism of vacuole formation by VacA, we examined the participation of dynamin, a GTPase functioning in intracellular vesicle formation, in human HeLa cells. Immunocytochemistry revealed that endogenous dynamin was localized to vacuoles induced by VacA. In cells transiently transfected with a GTPase-defective (dominant-negative) dynamin mutant, VacA failed to induce vacuolation. In contrast, VacA did induce vacuolation in cells transiently transfected with wild-type dynamin. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with the dominant-negative dynamin mutant. In contrast, uptake was markedly enhanced in cells stably transfected with wild-type dynamin. Moreover, VacA cytopathic effects on the viability of HeLa cells were inhibited in cells stably transfected with dominant-negative dynamin-1. Sequential immunocytochemical observation confirmed that expression of dominant-negative dynamin did not affect VacA attachment to or internalization into HeLa cells. We suggest that dynamin is involved in the intracellular vacuolation induced by VacA. (+info)
Beta-arrestin- and dynamin-dependent endocytosis of the AT1 angiotensin receptor.
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The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta-arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta-arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of beta-arrestin1(V53D) and beta-arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of beta-arrestin- and dynamin-dependent endocytosis of the AT1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations. (+info)
Phosphorylation of a synaptic vesicle-associated protein by an inositol hexakisphosphate-regulated protein kinase.
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Despite the fact that inositol hexakisphosphate (InsP(6)) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP(6). The substrate for the InsP(6)-regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP(6)-dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP(6)-dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse. (+info)
Dynamin and Rab5a-dependent trafficking and signaling of the neurokinin 1 receptor.
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Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca(2+) mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP. (+info)
Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor and bradykinin in HEK293 cells.
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In this study, we analysed the agonist-promoted trafficking of human B(2) (B(2)R) and B(1) (B(1)R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B(2)R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B(2)R using radioligand binding and by imaging of B(2)R-GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B(1)R was sequestered to a considerably lesser extent upon binding of des-Arg(10)-kallidin. B(2)R sequestration was rapid (half-life approximately 5 min) and reached a steady-state level that was significantly lower than that of BK sequestration. B(2)R sequestration was minimally inhibited by K44A dynamin (22.4+/-3.7%), and was insensitive to arrestin-(319-418), which are dominant-negative mutants of dynamin I and beta-arrestin respectively. Furthermore, the B(2)R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the beta(2)-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2+/-16.3%) and by arrestin-(319-418) (36.9+/-4.4%). Our results show that B(2)R is sequestered to a significantly greater extent than is B(1)R upon agonist treatment in HEK293 cells. Furthermore, B(2)R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and beta-arrestin-independent manner and, at least in part, involves caveolae. (+info)
A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis.
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Dynamin, a high-molecular-weight GTPase, plays a critical role in vesicle formation at the plasma membrane during endocytosis in animal cells. Here we report the identification of a new dynamin homolog in Arabidopsis named Arabidopsis dynamin-like 6 (ADL6). ADL6 is quite similar to dynamin I in its structural organization: a conserved GTPase domain at the N terminus, a pleckstrin homology domain at the center, and a Pro-rich motif at the C terminus. In the cell, a majority of ADL6 is associated with membranes. Immunohistochemistry and in vivo targeting experiments revealed that ADL6 is localized to the Golgi apparatus. Expression of the dominant negative mutant ADL6[K51E] in Arabidopsis protoplasts inhibited trafficking of cargo proteins destined for the lytic vacuole and caused them to accumulate at the trans-Golgi network. In contrast, expression of ADL6[K51E] did not affect trafficking of a cargo protein, H(+)-ATPase:green fluorescent protein, destined for the plasma membrane. These results suggest that ADL6 is involved in vesicle formation for vacuolar trafficking at the trans-Golgi network but not for trafficking to the plasma membrane in plant cells. (+info)
Agonist-induced internalization and mitogen-activated protein kinase activation of the human prostaglandin EP4 receptor.
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We examined the pathway of prostaglandin E(2) (PGE(2))-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative beta-arrestin (319-418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for beta-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE(2) stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and co-expression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization. (+info)