The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to as-The LIM domain is a Cys-rich domain composed of 50 -60 amino acid residues with the consensus sequence (Cys-X 2 -Cys-X 17-19 -His-X 2 -Cys) -X 2 -(Cys-X 2 -Cys-X 16 -20 -Cys-X 2 -His/Asp /Cys) (where X represents any amino acid) and is found in various proteins (1, 2): homeodomain-containing transcription factors, cytoskeletal proteins, LIM domain only proteins, protein kinases, and proteins of undefined function. Physicochemical and structural analyses have revealed that the LIM domain is composed of two independent zinc-coordinated fingers (3, 4). Although many zinc finger motifs bind to specific DNA or RNA sequences (5), the LIM domain has been proposed to participate in protein-protein interactions (1, 2). In fact, five proteins have been reported recently as a highly specific target of each LIM domain: the LIM1 domain of zyxin binds to the LIM-only protein CRP by LIM-LIM interaction (6); the CRP forms homodimer by LIM-LIM interaction (7); the LIM domain of RBTN2 binds to the bHLH (basichelix-loop-helix) domain of TAL1 protein (8); the LIM2 and LIM3 domains of Enigma interact with the Tyr-containing tight-turn motifs of the GDNF 1 receptor (GDNFR, known as a Ret Tyr kinase) and the insulin receptor (InsR), respectively (9, 10). Although most LIM domains adopt a similar zinc-coordinated finger consisting of well conserved amino acid sequences, no protein has been identified yet as a common target of LIM domains.The PKC family consists of at least 11 isoforms, which play distinct roles for many cellular functions but show subtle difference of substrate specificities by in vitro phosphorylation studies (11,12). Therefore, it is reasonable to assume that there are some mechanisms by which each PKC isoform recognizes its specific substrate proteins in vivo. Recently, several proteins associating with PKC have been emerged to govern the subcellular localization of the enzyme family (13-16).We report here a novel PKC-binding protein containing three LIM domains, designated ENH, and show the association of PKC with LIM domains of different proteins including this novel PKC-binding protein, suggesting that protein-protein interaction with PKC is a general property of LIM domains. EXPERIMENTAL PROCEDURESYeast Two-Hybrid Assay-The yeast two-hybrid assay (17) was conducted in the yeast strain CG-1945 , a derivative of HF7c (18), by using a fusion between GAL4 DNA binding domain and the regulatory domain of rat PKC I (residues 1-340) (19) as a bait. -Galactosidase activity in yeast cells was measured by plate assay methods. All measurements were repeated at least four times.Expression of Epitope-tagged ENH in COS-7 Cells-We constructed two parental vectors, pTB701-FLAG and pTB701-HA, for expression o...
Infusion of prostaglandin (PG) D2 into the lateral ventricle of the brain induced an increase in the amount of non-rapid eye movement sleep in wild-type (WT) mice but not in mice deficient in the PGD receptor (DP). Immunofluorescence staining of WT mouse brain revealed that DP immunoreactivity was dominantly localized in the leptomeninges (LM) of the basal forebrain but that PGD synthase immunoreactivity was widely distributed in the LM of the entire brain. Electron microscopic observation indicated that DPimmunoreactive particles were predominantly located on the plasma membranes of arachnoid trabecular cells of the LM. The region with the highest DP immunoreactivity was clearly defined as bilateral wings in the LM of the basal forebrain located lateral to the optic chiasm in the proximity of the ventrolateral preoptic area, one of the putative sleep centers, and the tuberomammillary nucleus, one of the putative wake centers. The LM of this region contained DP mRNA 70-fold higher than that in the cortex as judged from the results of quantitative reverse transcription-PCR. PGD2 infusion into the subarachnoid space of this region increased the extracellular adenosine level more than 2-fold in WT mice but not in the DP-deficient mice. These results indicate that DPs in the arachnoid trabecular cells of the basal forebrain mediate an increase in the extracellular adenosine level and sleep induction by PGD2. P rostaglandin (PG) D 2 is a potent endogenous sleeppromoting substance in monkeys and rats, and its somnogenic mechanism is the best characterized among those of various sleep-inducing substances (reviewed in ref. 1). In brief, PGD 2 infused into the subarachnoid space underlying the rostral basal forebrain was effective in inducing sleep but not when infused into most parts of the brain parenchyma of rats (2). The amount of PGD 2 -induced sleep was reduced by pretreatment with KF17837, the specific adenosine A 2A receptor antagonist, in a dose-dependent manner (3). Administration of CGS21680, a selective A 2A receptor agonist, into the subarachnoid space induced sleep (4), suggesting that PGD 2 -induced sleep is mediated by adenosine through the adenosine A 2A receptor system. Furthermore, PGD 2 infusion significantly increased Fos expression in the leptomeninges (LM) and neurons within the ventrolateral preoptic area, one of the putative sleep centers, and simultaneously decreased Fos expression in the tuberomammillary nucleus, one of the putative wake centers (5).PGD synthase (PGDS) is mainly produced in the LM and choroid plexus of the brain (6) and secreted into the cerebrospinal fluid (CSF) to become -trace, a major CSF protein (reviewed in ref. 7). We recently reported that human PGDSoverexpressing transgenic mice exhibited excessive amounts of non-rapid eye movement (NREM) sleep, but not rapid eye movement (REM) sleep, in response to the noxious stimulus by tail clipping, coupled with a significant increase in the PGD 2 content in the brain (8). Therefore, PGD 2 , PGDS, and the PGDS gene are considered...
The nectin–afadin system is a novel cell–cell adhesion system that organizes adherens junctions cooperatively with the cadherin–catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament–binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin–afadin system colocalizes with the cadherin–catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin–afadin system in the formation of synapses.
Dendrite arborization patterns are critical determinants of neuronal connectivity and integration. Planar and highly branched dendrites of the cerebellar Purkinje cell receive specific topographical projections from two major afferent pathways; a single climbing fiber axon from the inferior olive that extend along Purkinje dendrites, and parallel fiber axons of granule cells that contact vertically to the plane of dendrites. It has been believed that murine Purkinje cell dendrites extend in a single parasagittal plane in the molecular layer after the cell polarity is determined during the early postnatal development. By three-dimensional confocal analysis of growing Purkinje cells, we observed that mouse Purkinje cells underwent dynamic dendritic remodeling during circuit maturation in the third postnatal week. After dendrites were polarized and flattened in the early second postnatal week, dendritic arbors gradually expanded in multiple sagittal planes in the molecular layer by intensive growth and branching by the third postnatal week. Dendrites then became confined to a single plane in the fourth postnatal week. Multiplanar Purkinje cells in the third week were often associated by ectopic climbing fibers innervating nearby Purkinje cells in distinct sagittal planes. The mature monoplanar arborization was disrupted in mutant mice with abnormal Purkinje cell connectivity and motor discoordination. The dendrite remodeling was also impaired by pharmacological disruption of normal afferent activity during the second or third postnatal week. Our results suggest that the monoplanar arborization of Purkinje cells is coupled with functional development of the cerebellar circuitry.
Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) Phosphatidylethanol (PtdEtOH) and dioleoyl-PtdEtn were purchased from Avanti Polar Lipids. Dipalmitoyl-PtdCho, dipalmitoyl-PtdEtn, and L-a-palmitoyl-(3-linoleoyl-PtdEtn were obtained from Sigma. Phosphatidylserine (PtdSer), PtdEtn, phosphatidylinositol (Ptdlns), phosphatidic acid, PtdInsP2, sphingosine, ceramide, sphingomyelin, cholesterol, and plasmalogen-rich PtdEtn (60% plasmalogen) were purchased from
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