The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-␦1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1␣ (PP1c␣) as a p130-binding protein. The association between p130 and PP1c␣ was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1c␣ resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1c␣ also detected the presence of a complex of p130 and PP1c␣. The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1c␣, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca 2؉ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1c␣.D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ), 1 a product of receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) by phospholipase C (PLC), plays an important role as an intracellular second messenger by mobilizing Ca 2ϩ from nonmitochondrial stores (1). We previously isolated two Ins(1,4,5)P 3 -binding proteins with molecular masses of 130 and 85 kDa from rat brain (2, 3) with the use of an Ins(1,4,5)P 3 affinity column (4, 5). Partial amino acid sequencing revealed that the 85-kDa molecule was PLC-␦1 (2). Identification of the pleckstrin homology (PH) domain of PLC-␦1 as the site of Ins(1,4,5)P 3 binding helped to define the PH domain as an inositol compound binding module (6, 7). The Ins(1,4,5)P 3 -binding protein with a molecular mass of 130 kDa, termed p130, was a previously unidentified molecule (2, 3). The predicted amino acid sequence of rat p130 shares 38.2% identity with that of rat PLC-␦1; the five identified domains of PLC-␦1 (PH, EF-hand, putative catalytic (X and Y), and C2 domains) are all present in p130. The domain organization of p130 suggests that the protein is likely to possess a fold similar to that of PLC-␦1, a notion that is supported by the results of limited proteolysis with trypsin (8). However, p130 exhibits some distinct characteristics. It is larger than the PLC-␦ isozymes, and it possesses unique regions both at the NH 2 terminus, preceding the PH domain, and at the COOH terminus. Moreover, the residues within the catalytic domain of PLC-␦ that are critical for enzyme activity (His 356 and Glu 390 ) are not conserved in p130 (9). The PH domain of p130, like that of PLC-␦1, is important for the binding of Ins(1,4,5)P 3 (10). Other mol...

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3␣ and 3␤, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the ␣ form is replaced by Arg-89 in the ␤ form, making the latter ϳ2-fold more active in vitro. ). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40 -50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3␣; NUDT11 encodes hDIPP3␤) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.Hydrolases containing the GX 5 EX 7 REUXEEXGU 1 "Nudixtype" motif comprise a protein superfamily whose members have been proposed to act as "surveillance enzymes" (2) that function both to eliminate potentially toxic metabolites from the cell as well as to regulate concentrations and availability of substrates, cofactors, and signaling molecules (3). Almost all of the substrates for these hydrolases are nucleotide diphosphates. The unique exception is provided by a subgroup of phosphohydrolases (DIPPs) 2 that preferentially attack diphosphoinositol polyphosphates (4, 5).The diphosphoinositol polyphosphates provide the most extreme example of the degree to which phosphate groups can be clustered in high density around the inositol ring. These compounds are formed when specific monoester phosphate groups on Ins(1,3,4,5,6)P 5 and InsP 6 are converted to diphosphates, by enzymes known as diphosphoinositol polyphosphate synthases (DINS). For example, InsP 6 is phosphorylated to PP-InsP 5 and [PP] 2 -InsP 4 , which contain either one or two diphosphate groups, respectively (6, 7). The DIPPs rapidly degrade the diphosphates back to their monoester precursors. This phosphate release relieves some of the severity of the electrostatic and steric constraints imposed upon these polyphosphorylated molecules, and so a substantial and physiologically purposeful free-energy change ensues. Indeed, evidence has accumulated that this DINS-and DIPP-catalyzed substrate cycling contributes to the control of vesicle trafficking. Initially, this hypothesis arose from observations we made several years ago (8 -10) showing that the diphosphoinositol p...

We have derived the full-length sequences of the human and rat forms of the multiple inositol polyphosphate phosphatase (MIPP); their structural and functional comparison with a chick histidine acid phosphatase (HiPER1) has revealed new information: (1) MIPP is approximately 50% identical to HiPER1, but the ER-targeting domains are divergent; (2) MIPP appears to share the catalytic requirement of histidine acid phosphatases, namely, a C-terminal His residue remote from the RHGxRxP catalytic motif ; (3) rat MIPP mRNA is upregulated during chondrocyte hypertrophy. The latter observation provides a context for proposing that MIPP may aid bone mineralization and salvage the inositol moiety prior to apoptosis.z 1999 Federation of European Biochemical Societies.

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We previously described paralogous human genes [NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety 'X')-type motif, also known as the 'nudix'-type motif]] encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem. 277, 32730-32738]. Normally, gene duplication is redundant, and lacks biological significance. Is this true for the DIPP3 genes? We address this question by characterizing highly-conserved murine Nudt10 and Nudt11 homologues of the human genes. Thus these genes must have been duplicated prior to the divergence of primates and sciurognath rodents, approx. 115 million years ago, greatly exceeding the 4 million year half-life for inactivation of redundant paralogues; our data therefore indicate that the DIPP3 duplication is unusual in being physiologically significant. One possible functional consequence is gene neofunctionalization, but we exclude that, since Nudt10 and Nudt11 encode identical proteins. Another possibility is gene subfunctionalization, which we studied by conducting the first quantitative expression analysis of these genes. We demonstrated high Nudt10 expression in liver, kidney and testis; Nudt11 expression is primarily restricted to the brain. This differential, but complementary, expression pattern indicates that subfunctionalization is the evolutionary consequence of DIPP3 gene duplication. Our kinetic data argue that diphosphoinositol polyphosphates are more physiologically relevant substrates for DIPP3 than are either diadenosine hexaphosphate or 5-phosphoribosyl 1-pyrophosphate. Thus the significance of the Nudt10/Nudt11 duplication is specific hydrolysis of diphosphoinositol polyphosphates in a tissue-dependent manner.

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In protected strawberry (Fragaria × ananassa Duch.) cultivation, environmental control based on the process of photosynthate translocation is essential for optimizing fruit quality and yield, because the process of photosynthate translocation directly affects dry matter partitioning. We visualized photosynthate translocation to strawberry fruits non-invasively with 11CO2 and a positron-emitting tracer imaging system (PETIS). We used PETIS to evaluate real-time dynamics of 11C-labeled photosynthate translocation from a 11CO2-fed leaf, which was immediately below the inflorescence, to individual fruits on an inflorescence in intact plant. Serial photosynthate translocation images and animations obtained by PETIS verified that the 11C-photosynthates from the source leaf reached the sink fruit within 1 h but did not accumulate homogeneously within a fruit. The quantity of photosynthate translocation as represented by 11C radioactivity varied among individual fruits and their positions on the inflorescence. Photosynthate translocation rates to secondary fruit were faster than those to primary or tertiary fruits, even though the translocation pathway from leaf to fruit was the longest for the secondary fruit. Moreover, the secondary fruit was 25% smaller than the primary fruit. Sink activity (11C radioactivity/dry weight [DW]) of the secondary fruit was higher than those of the primary and tertiary fruits. These relative differences in sink activity levels among the three fruit positions were also confirmed by 13C tracer measurement. Photosynthate translocation rates in the pedicels might be dependent on the sink strength of the adjoining fruits. The present study established 11C-photosynthate arrival times to the sink fruits and demonstrated that the translocated material does not uniformly accumulate within a fruit. The actual quantities of translocated photosynthates from a specific leaf differed among individual fruits on the same inflorescence. To the best of our knowledge, this is the first reported observation of real-time translocation to individual fruits in an intact strawberry plant using 11C-radioactive- and 13C-stable-isotope analyses.