A cDNA encoding the new member of the multispecific organic anion transporter family, OAT3, was isolated by the reverse transcription-polymerase chain reaction cloning method. Degenerate primers were designed based on the sequences conserved among OAT1, OAT2, and organic cation transporter 1 (OCT1), and reverse transcription-polymerase chain reaction was performed using rat brain poly(A)؉ RNA. The 536-amino acid protein sequence encoded by OAT3 showed 49, 39, and 36% identity to those of OAT1, OAT2, and OCT1, respectively. Northern blot analysis revealed that rat OAT3 mRNA is expressed in the liver, brain, kidney, and eye. When

A cDNA was isolated from mouse testis which encodes a Na ؉ -dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40 -44% identity), Na ؉ -dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (K m ‫؍‬ 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na ؉ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na ؉ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na ؉ . Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na ؉ -dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.

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Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.

Reelin is a key mediator of ordered neuronal alignment in the brain. Here, we demonstrate that Reelin molecules assemble with each other to form a huge protein complex both in vitro and in vivo. The Reelin-Reelin interaction clearly is inhibited by the functionblocking anti-Reelin antibody, CR-50, at a concentration known to inhibit Reelin function. This assembly is mediated by electrostatic interaction of the CR-50 epitope region. Recombinant CR-50 epitope fragments spontaneously constitute a soluble, string-like homopolymer with a regularly repeated structure composed of more than 40 monomers. Mutated Reelin, which lacks the CR-50 epitope region, cannot form a homopolymer and fails to induce efficient tyrosine phosphorylation of Disabled 1 (Dab1), which should occur to transduce the Reelin signal. These data suggest that Reelin exerts its biological function by composing a large protein assembly driven by the CR-50 epitope region, proposing a novel model of the Reelin signaling in neurodevelopment. In the mammalian central nervous system (CNS), various classes of neurons migrate from their site of origin to the final positions, where they are arranged in elaborate laminar structures (1-3). Neocortical development starts from the preplate formation. The preplate resides near the surface of the cortex and is composed of a superficial plexus of corticopetal nerve fibers and earliest-generated neurons, including the CajalRetzius and prospective subplate neurons. Consecutively, the preplate is split by the cortical plate neurons into a superficial marginal zone, where the Cajal-Retzius neurons differentiate, and a deep subplate, in which the subplate neurons differentiate (4). The cortical plate neurons are born in the ventricular zone and migrate past the intermediate zone and subplate along radial glial fibers before reaching the cortical plate. The systematic migration of the later-generated neurons past those generated earlier results in an ''inside-out'' progression in the mammalian cortical plate (5, 6).The reeler is an autosomal recessive mouse mutant, in which neurons are generated normally but are abnormally placed, resulting in disorganization of cortical laminar layers in the CNS (7-12). In the reeler neocortex, for example, cortical plate neurons are aligned in a practically inverted fashion (''outsidein''). Therefore, the reeler mouse presents a good model in which to investigate the mechanisms of establishment of the precise neuronal network during development. We previously obtained a monoclonal alloantibody, CR-50, by immunizing reeler mice with homogenates of normal embryonic brains (13). This antibody was shown to react specifically with Cajal-Retzius neurons in the marginal zone of normal mice. It then was shown to recognize the Reelin protein itself (14), which is encoded by the reeler gene (reelin, Reln) (15, 16). Reelin is a secreted extracellular matrix protein composed of 3,461 aa with a relative molecular mass of 388 kDa (14, 15) (Fig. 1 A). The N terminus of Reelin has 25% identity with th...

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