Eiji Hirose

Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p ofSaccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [3H]GTP and [3H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotide-bound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.

Grit, a GTPase-Activating Protein for the Rho Family, Regulates Neurite Extension through Association with the TrkA Receptor and N-Shc and CrkL/Crk Adapter Molecules

ABSTRACT Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.

A Novel Human Nucleolar Protein, Nop132, Binds to the G Proteins, RRAG A/C/D

RRAG A (Rag A)/Gtr1p is a member of the Ras-like small G protein family that genetically interacts with RCC1, a guanine nucleotide exchange factor for RanGTPase. RRAG A/Gtr1p forms a heterodimer with other G proteins, RRAG C and RRAG D/Gtr2p, in a nucleotide-independent manner. To further elucidate the function of RRAG A/Gtr1p, we isolated a protein that interacts with RRAG A. This protein is a novel nucleolar protein, Nop132. Nop132 is associated with the GTP form, but not the GDP form, of RRAG A, suggesting that RRAG A might regulate Nop132 function. Nop132 is also associated with RRAG C and RRAG D. The Nop132 amino acid sequence is similar to the Saccharomyces cerevisiae nucleolar Nop8p, which is associated with Gtr1p, Gtr2p, and Nip7p. Nop132 also interacts with human Nip7 and is colocalized with RRAG A, RRAG C, and Nip7. RNA interference knockdown of Nop132 inhibited cell growth of HeLa cells.

The protoplasmic or exoplasmic face association of tight junction particles cannot predict paracellular permeability or heterotypic claudin compatibility.

Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na(+) but it did not change that of Cl(-). The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.

Claudin-7 Expressed on Lateral Membrane of Rat Epididymal Epithelium does not Form Aberrant Tight Junction Strands

Claudins are integral membrane proteins at tight junctions (TJs) and form TJ strands. In the present study, we found that claudin‐7 was localized along the entire lateral membranes of epididymal epithelium, including the apical junctional region throughout the epididymis, but claudin‐8 was restricted to the apical junctional region. This finding raises the possibility that aberrant TJ strands may be formed on lateral membranes. Thus, we focused on examining whether TJ strands exist on lateral membranes of epididymal epithelium. Freeze‐fracture electron microscopy showed that aberrant TJ strands were observed in only a few principal cells in all segments of the epididymis except for the initial segment, indicating that the occurrence of aberrant strands is very rare. Aberrant TJ strands were smooth and not subdivided into individual particles in the protoplasmic face, and complementary grooves in the extracellular face were almost free of particles. Aberrant TJ strands in the distal caput and corpus epididymis were accompanied by many vesicle‐like structures but those in the proximal caput and cauda epididymis were not. These results suggest that most of claudin‐7 in lateral membranes may exist in a nonpolymerized form and may play some different roles other than the formation of TJ strands, for example, in the formation of a pool of claudin proteins or in the reinforcement of cell adhesion. Anat Rec, 1431‐1438, 2007.

Postnatal development of mRNA specific for a metabotropic glutamate receptor in the rat brain

We examined the ontogenesis of a subtype of metabotropic glutamate receptors, termed mGluR1, which is linked to phosphoinositide metabolism, in various regions of rat brain during neonatal development. Northern blot analyses of mGluR1 mRNA indicated that mRNA increased monotonously or remained at plateau levels during the first 5 weeks after birth. In situ hybridization analyses supported this conclusion. The result is in contrast with the reported development of the activity in excitatory amino acid-stimulated phosphoinositide turnover during the same period. The latter increases during the first few weeks and then decreases sharply.

Differential expression of the tight junction proteins, claudin-1, claudin-4, occludin, ZO-1, and PAR3, in the ameloblasts of rat upper incisors.

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel‐forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle‐ended and smooth‐ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well‐developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less‐developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO‐1, and PAR3, a cell polarity‐related protein, in ameloblasts of rat upper incisors. ZO‐1 and claudin‐1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin‐4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs. Anat Rec, 291:577–585, 2008.

Freeze-fracture electron microscopic study of tight junction strands in HEK293 cells and MDCK II cells expressing claudin-1 mutants in the second extracellular loop

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLΔFY and 1CLΔPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH2-terminus (EGFP1CLΔFY and EGFP1CLΔPL) or the myc-epitope at the COOH-terminus (1CLΔFYmyc and 1CLΔPLmyc). The expression of EGFP1CLΔFY and EGFP1CLΔPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLΔPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLΔFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLΔPLmyc was localized at heterotypic cell–cell junctions but 1CLΔFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLΔFYmyc or 1CLΔPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.

MINIMUM ESSENTIAL REGION OF CCG1/TAFII250 REQUIRED FOR COMPLEMENTING THE TEMPERATURE-SENSITIVE CELL CYCLE MUTANTS, TSBN462 AND TS13 CELLS, OF HAMSTER BHK21 CELLS

CCG1/TAFII250, the largest subunit of theTFIID complex, is mutated in ts cell cycle mutants of BHK21 cells, ts13 and tsBN462, which have a promoter-selective transcriptional defect. A series of deletion mutants ofCCG1 cDNA were prepared and transfected into these mutants, in order to identify functional domains of CCG1 required for the complementation of ts 13/BN462 mutation. We determined the minimum size of CCG1: CCG1ME, essential for complementing the ts mutation, which possessed one proline cluster, an HMG1-like domain, and a nuclear localization signal, but which lacked the bromo domains and the acidic phosphorylation sites for casein kinase II common to transcriptional activators. It encodes a protein of 140 kDa. These characteristics of CCG1ME correspond to yeast TAFII145, the yeast homolog of human TAFII250. CCG1ME bound to TBP, creating its own TFIID complex different from that of the endogenous mutated CCG1 in ts+ transformants of tsBN462 cells.

Loss of RanGEF/Pim1 activity abolishes the orchestration of Ran-mediated mitotic cellular events in S. pombe.

RCC1, a conserved chromosomal protein with a seven‐bladed propeller is a GDP/GTP nucleotide exchange factor for RanGTPase that mediates various cellular events. We isolated 16 temperature‐sensitive (ts) mutants of S. pombe RCC1‐homolog, pim1+, by error‐prone PCR. Five pim1ts mutants had a single mutation. The obtained pim1ts mutations and previously reported mutations were localized on similar sites in seven RCC1 repeats. Those mutations resulted in a reduced binding of Pim1 with Spi1. All pim1ts mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1ts examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, the septum was formed being uncoupled with nuclear division and abnormally widened, thus resulting in chromosomal DNA mis‐segregation and the accumulation of enucleated cells. As a result, a defect of RanGEF/Pim1 abolished the orchestration of sequential mitotic events, spindle formation, septation and cytokinesis that are essential to produce two identical daughter cells.