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Biomedical subjects

Mitsunori Fukuda

Publications and source records attributed to Mitsunori Fukuda.

At least 37 records · Page 2Linked to original sources
PubMed2005

Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells.

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.

Amiloride↗
PubMed2005

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells.

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.

Animals↗
PubMed2005

AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

Adipocytes↗
PubMed2005

Adenovirus-mediated silencing of synaptotagmin 9 inhibits Ca2+-dependent insulin secretion in islets.

Synaptotagmins (Syts) are involved in Ca(2+)-dependent insulin release. However, which Syt isoform is functional in primary beta-cells remains unknown. We demonstrate by electron microscopy of pancreatic islets, the association of Syt 9 with insulin granules. Silencing of Syt 9 by RNA interference adenovirus in islet cells had no effect on the expression of Syt 5, Syt 7 and Syt 3 isoforms. The latter was localized at the plasma membrane of pancreatic polypeptide cells. Insulin release in response to glucose or tolbutamide was strongly inhibited in Syt 9 deficient islets, whereas exocytosis potentiated by raising cAMP levels, was unaltered. Thus, Syt 9 may act as Ca(2+) sensor for beta-cell secretion.

Adenoviridae↗
PubMed2005

Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner.

Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic beta-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a.syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane.

Amylases↗
PubMed2005

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis.

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.

Acrosome↗
PubMed2005

The short apical membrane half-life of rescued {Delta}F508-cystic fibrosis transmembrane conductance regulator (CFTR) results from accelerated endocytosis of {Delta}F508-CFTR in polarized human airway epithelial cells.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the apical plasma membrane. Rescue of DeltaF508-CFTR by reduced temperature or chemical means reveals that the DeltaF508 mutation reduces the half-life of DeltaF508-CFTR in the apical plasma membrane. Because DeltaF508-CFTR retains some Cl(-) channel activity, increased expression of DeltaF508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued DeltaF508-CFTR that lead to the decreased apical membrane half-life of DeltaF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the DeltaF508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Functional analysis of Slac2-c/MyRIP as a linker protein between melanosomes and myosin VIIa.

Slac2-c/MyRIP, an in vitro Rab27A- and myosin Va/VIIa-binding protein, has recently been proposed to regulate retinal melanosome transport in retinal pigment epithelium cells by directly linking melanosome-bound Rab27A and myosin VIIa; however, the exact function of Slac2-c in melanosome transport has never been clarified. In this study, we used melanosome transport in skin melanocytes as a model for retinal melanosome transport and analyzed the in vivo function of Slac2-c in melanosome transport by the ectopic expression of Slac2-c, together with myosin VIIa, in Slac2-a-depleted melanocytes. In vitro binding experiments revealed that myosin VIIa had a greater affinity for Slac2-c, compared with the binding affinity of myosin Va, and that the myosin VIIa-binding domain of Slac2-c is different from the previously characterized myosin Va-binding domain that is conserved between Slac2-a/melanophilin and Slac2-c. Consistent with this result, cyan fluorescent protein-tagged Slac2-c expressed in melanocytes was localized on melanosomes via the specific interaction with Rab27A and recruited co-expressed yellow fluorescent protein-tagged myosin VIIa to the melanosomes without interfering with the normal peripheral melanosome distribution, whereas when myosin VIIa alone was expressed in melanocytes, it was not localized on the melanosomes. Moreover, Slac2-c ectopically expressed in melanocytes did not rescue the perinuclear aggregation phenotype induced by the knockdown of endogenous Slac2-a with a specific small interfering RNA, whereas the expression of the Slac2-c x myosin VIIa complex supported the normal melanosome distribution in Slac2-a-depleted melanocytes, indicating that Slac2-c functions as a myosin VIIa receptor rather than a myosin Va receptor in melanosome transport. Based on these findings, we propose that Slac2-c acts as a functional myosin VIIa receptor and that the Rab27A.Slac2-c x myosin VIIa tripartite protein complex regulates the transport of retinal melanosomes in pigment epithelium cells.

Animals↗
PubMed2005

Classical protein kinase C(s) regulates targeting of synaptotagmin IX to the endocytic recycling compartment.

Neuronal and non-neuronal tissues show distinctly different intracellular localization of synaptotagmin (Syt) homologues. Therefore, cell type-specific mechanisms are likely to direct Syt homologues to their final cellular destinations. Syt IX localizes to dense core vesicles in PC12 cells. However, in the rat basophilic leukemia (RBL-2H3) mast cell line, as well as in CHO cells, Syt IX is localized at the endocytic recycling compartment (ERC). We show that targeting of Syt IX to the ERC involves constitutive trafficking to the plasma membrane followed by internalization and transport to the ERC. We further show that internalization from the plasma membrane and delivery to the ERC are dependent on phosphorylation by Ca(2+)-dependent protein kinase Calpha or beta. As such, correct targeting of Syt IX is facilitated by the phorbol ester TPA but prevented by the cPKC inhibitor Go 6976.

Animals↗
PubMed2005

Functional analysis of slac2-a/melanophilin as a linker protein between Rab27A and myosin Va in melanosome transport.

Slac2-a/melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Analysis of the role of Rab27 effector Slp4-a/Granuphilin-a in dense-core vesicle exocytosis.

Slp4-a/granuphilin-a is a member of the synaptotagmin-like protein (Slp) family and consists of an N-terminal Slp homology domain (SHD) and C-terminal tandem C2 domains. Slp4-a is specifically localized on secretory granules in some endocrine and exocrine cells through its SHD, and it attenuates Ca(2+)-dependent dense-core vesicle (DCV) exocytosis when transiently expressed in endocrine cells. Although the SHD of Slp4-a interacts with three distinct Rab species (Rab3A, Rab8A, and Rab27A) in vitro, in contrast to other Slp members, which only recognize Rab27 isoforms, Slp4-a functions as a Rab27A effector during DCV exocytosis under physiological conditions. This chapter describes various approaches that have been used to characterize the function of Slp4-a as a Rab27A effector, rather than a Rab3A or Rab8A effector, both in in vitro and in neuroendocrine PC12 cells. Specifically, the methods that have been used to analyze (1) the physical interaction between Slp4-a and Rab27A, including pull-down assay and cotransfection assay in COS-7 cells; (2) the localization of Slp4-a-Rab27A complex on DCVs in PC12 cells; and (3) the involvement of Slp4-a and Rab27A in DCV exocytosis by neuropeptide Y (NPY) cotransfection assay combined with site-directed mutagenesis are described.

Amino Acid Sequence↗
PubMed2005

Assay and functional interactions of Rim2 with Rab3.

Rim was originally identified as a protein that contains a putative Rab3A-effector domain at the N-terminus, the same as rabphilin, and two forms of Rim, Rim1 and Rim2, have been reported in mammals. The putative Rab3A-binding domain (RBD) of Rim consists of two alpha-helical regions (named RBD1 and RBD2) separated by two zinc finger motifs, and several alternative splicing events occur in the RBD1 of both Rims that result in the production of long forms and short forms of RBD. The short forms of Rim2 RBD are capable of interacting with Rab3A with high affinity in vitro, and it is recruited to dense-core vesicles (DCVs) in neuroendocrine PC12 cells through interaction with endogenous Rab3A, whereas the long forms of Rim2 RBD show dramatically reduced Rab3A-binding activity in vitro (more than a 50-fold decrease in affinity compared with the short forms of Rim2 RBD), and it is mainly present in the cytoplasm and nucleus. Expression of the shortest form of Rim2 RBD, but not its Rab3A binding-defective mutant (E36A/R37S), promotes high-KCl-dependent neuropeptide Y secretion from PC12 cells, suggesting that the Rim2 containing the short forms of RBD functions as a Rab3A effector during DCV exocytosis. In this Chapter, I describe several assay methods that have been used to determine the physiological significance of the alternative splicing event in the RBD1 of Rim2, including assays for the in vitro interaction between Rim2 RBD and Rab3A and for the localization of Rim2-RBD on DCVs in PC12 cells.

Animals↗
PubMed2005

Assay of the Rab-binding specificity of rabphilin and Noc2: target molecules for Rab27.

Rabphilin and Noc2 were originally described as Rab3A effector proteins involved in the regulation of secretory vesicle exocytosis in neurons and certain endocrine cells. Both proteins share the conserved N-terminal Rab-binding domain (RBD) that consists of two alpha-helical regions separated by two zinc finger motifs. However, the RBD of rabphilin and Noc2 has been shown to bind Rab27A (the closest homologue of Rab3 isoforms) in preference to Rab3A, both in vitro and in vivo. Rabphilin and Noc2 are recruited to dense-core vesicles (DCVs) in neuroendocrine PC12 cells and regulate their exocytosis through interaction with Rab27A rather than with Rab3A. Rab3A-binding-defective mutants of rabphilin(E50A) and Noc2(E51A) retain the ability to target DCVs in PC12 cells, the same as the wild-type proteins, whereas Rab27A-binding-defective mutants of rabphilin(E50A/I54A) and Noc2(E51A/I55A) do not (i.e., they are present throughout the cytoplasm). Expression of the wild-type or the E50A mutant of rabphilin-RBD, but not the E50A/I54A mutant of rabphilin-RBD, in PC12 cells significantly attenuated DCV exocytosis monitored by high-KCl-stimulated neuropeptide Y secretion. In this chapter we describe various assay methods that have been used to characterize the RBD of rabphilin and Noc2 as "RBD27 (Rab-binding domain for Rab27)."

Adaptor Proteins, Signal Transducing↗
PubMed2005

Versatile role of Rab27 in membrane trafficking: focus on the Rab27 effector families.

Rab27A was the only Rab protein whose dysfunction was found to cause human immunodeficiency. Since Griscelli syndrome patients (i.e., Rab27A-deficient) exhibit silvery hair color (i.e., pigmentary dilution) in addition to loss of cytotoxic killing activity by cytotoxic T lymphocytes, and Rab27A protein is expressed in a wide variety of secretory cells, Rab27A (or its closely related isoform Rab27B) has been implicated in the regulation of different types of membrane trafficking, including melanosome transport and various regulated secretion events. How does Rab27 protein regulate these different types of membrane trafficking? Recent discoveries of three different families of Rab27-binding proteins (a total of eleven distinct proteins) have supplied an important clue to the answer of this question: different types of Rab27 effectors function in different cell types. In this review I describe the literature on the identification of Rab27-binding proteins (i.e., the synaptotagmin-like protein (Slp) family with tandem C2 Ca(2+)-binding motifs, the Slac2 family without any C2 motifs, and Munc13-4, a putative priming factor for exocytosis) and the current state of our understanding of the molecular mechanism of the Rab27-dependent membrane trafficking.

Animals↗
PubMed2004

Vesicular reuptake inhibition by a synaptotagmin I C2B domain antibody at the squid giant synapse.

Synaptotagmin (Syt) I, a ubiquitous synaptic vesicle protein, comprises a transmembrane region and two C2 domains. The C2 domains, which have been shown to be essential for both synaptic vesicle exocytosis and endocytosis, are also seen as the Ca(2+) sensors in synaptic vesicular release. In a previous study, we reported that a polyclonal antibody raised against the squid (Loligo pealei) Syt I C2B domain, while inhibiting vesicular endocytosis, was synaptic release neutral at the squid giant synapse. Recent reports concerning the C2B requirements for synaptic release prompted us to readdress the role of C2B in squid giant synapse function. Presynaptic injection of another anti-Syt I-C2B antibody (using recombinant whole C2B domain expressed in mammalian cell culture as an antigen) into the presynaptic terminal reproduced our previous results, i.e., reduction of vesicular endocytosis without affecting synaptic release. This set of results addresses the issue of the geometrical arrangement of the Ca(2+) sensor, allowing the C2B domain antibody to restrict Ca(2+)-dependent C2B self-oligomerization without modifying the Ca(2+)-dependent release process.

Animals↗
PubMed2004

Rab27A-binding protein Slp2-a is required for peripheral melanosome distribution and elongated cell shape in melanocytes.

The synaptotagmin-like protein (Slp) family is implicated in regulating Rab27A-mediated membrane transport, but how it might do this is unknown. Here we report that Slp2-a, a previously uncharacterized Rab27A-binding protein in melanocytes, controls melanosome distribution in the cell periphery and regulates the morphology of melanocytes. Slp2-a is the most abundantly expressed of the Slp- and Slac2-family proteins in melanocytes and colocalizes with Rab27A on melanosomes. Knockdown of endogenous Slp2-a protein by small-interfering RNAs (siRNAs) markedly reduced the number of melanosomes in the cell periphery of mouse melanocytes ('peripheral dilution'). Expression of siRNA-resistant Slp2-a (Slp2-a(SR)) rescued the peripheral dilution of melanosomes induced by Slp2-a siRNAs, but Slp2-a(SR) mutants, which failed to interact with either phospholipids or Rab27A, did not. Loss of Slp2-a protein also induced a change in melanocyte morphology, from their normal elongated shape to a more rounded shape, which depended on the phospholipid-binding activity of Slp2-a, but not on its Rab27A-binding activity. By contrast, knockdown of Slac2-a (also called melanophilin), another Rab27A-binding protein in melanocytes, caused perinuclear aggregation of melanosomes alone without altering cell shape. These results reveal the differential and sequential roles of Rab27A-binding proteins in melanosome transport in melanocytes.

Adaptor Proteins, Signal Transducing↗