System L-type transport of large neutral amino acids is mediated by ubiquitous LAT1-4F2hc and epithelial LAT2-4F2hc. These heterodimers are thought to function as obligatory exchangers, but only in¯ux properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, af®nity and exchange stoichiometry using the Xenopus oocyte expression system. Quanti®cation of amino acid in¯ux and ef¯ux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans-stimulations of amino acid in¯ux by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the ef¯ux selectivity range resembles that of in¯ux. Compared with high extracellular apparent af®nities, LAT1-and LAT2-4F2hc displayed much lower intracellular apparent af®nities (apparent K m in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1-4F2hc) and transcellular transport (LAT2-4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate af®nities such that the intracellular amino acid concentration controls their activity. Keywords: epithelial cell polarity/glycoproteinassociated amino acid transporter/LAT1-4F2hc/ LAT2-4F2hc IntroductionTwo heterodimeric transporters for large neutral amino acids that correspond to the Na + -independent system L have recently been identi®ed (Kanai et al., 1998;Mastroberardino et al., 1998;Pineda et al., 1999;Prasad et al., 1999;Rossier et al., 1999;Segawa et al., 1999;Rajan et al., 2000). These heterodimers contain catalytic subunits named LAT1 and LAT2, which belong to the family of glycoprotein-associated amino acid transporters (gpaATs) and are also called light chains (Verrey et al., , 2000. A disul®de bond covalently links these gpaATs to their associated glycoprotein 4F2hc/CD98.Functional experiments performed in expression systems suggest that the two L-type transporters function as exchangers (Mastroberardino et al., 1998;Pineda et al., 1999;Rossier et al., 1999). In the case of LAT2-4F2hc, some contradictory data have been published. Our laboratory and that of Palacin have shown that the ef¯ux of L-Phe and of L-Ile depend on the presence of an extracellular uptake substrate, as expected for an obligatory exchange (Pineda et al., 1999;Rossier et al., 1999). In contrast, an ef¯ux of L-Leu observed by Kanai and co-workers was interpreted as a facilitated diffusion (Segawa et al., 1999). Some functional differences between the two L-type transporters were reported, in particular the fact that LAT2-4F2hc has a broader selectivity range than LAT1-4F2hc in that it also mediates the uptake of smaller neutral amino acids.The tissue distribution and subcellular localization of the two L-type transporters suggest that they must play different roles. LAT1-4F2hc is found quite ubiquitously and is highly expressed in proliferating tissues, in particular also in tumors, sugges...

Hartnup disorder, an autosomal recessive defect named after an English family described in 1956 (ref. 1), results from impaired transport of neutral amino acids across epithelial cells in renal proximal tubules and intestinal mucosa. Symptoms include transient manifestations of pellagra (rashes), cerebellar ataxia and psychosis 1,2 . Using homozygosity mapping in the original family in whom Hartnup disorder was discovered, we confirmed that the critical region for one causative gene was located on chromosome 5p15 (ref. 3). This region is homologous to the area of mouse chromosome 13 that encodes the sodium-dependent amino acid transporter B 0 AT1 (ref. 4). We isolated the human homolog of B 0 AT1, called SLC6A19, and determined its size and molecular organization. We then identified mutations in SLC6A19 in members of the original family in whom Hartnup disorder was discovered and of three Japanese families. The protein product of SLC6A19, the Hartnup transporter, is expressed primarily in intestine and renal proximal tubule and functions as a neutral amino acid transporter.Despite molecular characterization of other proximal tubule transporters, the neutral amino acid carrier defective in Hartnup disorder (OMIM 2345000) has resisted genetic identification 2 . We carried out homozygosity mapping and fine mapping in ten members of two consanguineous families (the siblings in whom Hartnup disorder was originally discovered 1 ; family A; Fig. 1a) and in siblings from the US 5 (family B; Fig. 1a). We found linkage of Hartnup disorder to 5p15 only in family A, with a maximum combined multipoint lod score of 2.31 at 11.24 cM (P ¼ 0.01). This confirmed our previous results showing linkage to chromosome 5p15 (ref.3). In family B, we obtained a maximum multipoint lod score of À2.40 at 15.81 cM.We simultaneously pursued two mouse monoamine transporterrelated orphan genes, Slc6a18 (also called Xtrp2; ref. 6) and Slc6a19 (encoding B 0 AT1; ref. 4). These members of the SLC6 family of transporters map to the mouse chromosomal region that is homologous to human chromosome 5p15. Both Slc6a18 and Slc6a19 showed abundant expression in mouse kidney, as assessed by real time RT-PCR (Fig. 2a). Immunohistochemistry confirmed expression of mouse B 0 AT1 at the brush border of small intestine (data not shown) and kidney proximal tubule cells (Fig. 2b).The human homolog, B 0 AT1, is encoded by the predicted locus SLC6A19, with a 2,022-bp open reading frame. PCR amplification using human kidney cDNA produced a 1,905-bp product with 100% identity to SLC6A19 sequence. We next determined the genomic organization of SLC6A19, which has a stop codon 28 bases before the ATG in the 5¢ untranslated region. SLC6A19 has 12 coding exons. The B 0 AT1 protein contains 634 amino acids and 12 predicted transmembrane regions (Fig. 1b). In a panel of human cDNAs, we detected robust expression of SLC6A19 in kidney and small intestine, with minimal expression in pancreas (Fig. 2c). SLC6A19 was also expressed in stomach, liver, duodenum and ileocecum (data n...

This study identifies a GPCR, S1PR2, as a receptor for the Nogo-A-Δ20 domain of the membrane protein Nogo-A, which inhibits neuronal growth and synaptic plasticity.

The B 0 transport system mediates the Na ϩ -driven uptake of a broad range of neutral amino acids into epithelial cells of small intestine and kidney proximal tubule. A corresponding transporter was identified in 2004 (A. Broer, K. Klingel, S. Kowalczuk, J. E. Rasko, J. Cavanaugh, and S. Broer. J Biol Chem 279: [24467][24468][24469][24470][24471][24472][24473][24474][24475][24476] 2004) within the SLC6 family and named B 0 AT1 (SLC6A19). A phylogenetically related transporter known as XT3 in human (SLC6A20) and XT3s1 in mouse was shown to function as an imino acid transporter, to localize also to kidney and small intestine and renamed SIT1 or Imino B . Besides these two transporters with known functions, there are two other gene products belonging to the same phylogenetic B 0 AT-cluster, XT2 (SLC6A18) and rodent XT3 that are still "orphans." Quantitative real-time RT-PCR showed that the mRNAs of the four B 0 AT-cluster members are abundant in kidney, whereas only those of B 0 AT1 and XT3s1/SIT1 are elevated in small intestine. In brain, the XT3s1/SIT1 mRNA is more abundant than the other B 0 AT-cluster mRNAs. We show here by immunofluorescence that all four mouse B 0 AT-cluster transporters localize, with differential axial gradients, to the brushborder membrane of proximal kidney tubule and, with the possible exception of XT3, also of intestine. Deglycosylation and Western blotting of brush-border proteins demonstrated the glycosylation and confirmed the luminal localization of B 0 AT1, XT2, and XT3. In summary, this study shows the luminal brush-border localization of the Na ϩ -dependent amino and imino acid transporters B 0 AT1 and XT3s1/SIT1 in kidney and intestine. It also shows that the structurally highly similar orphan transporters XT2 and XT3 have the same luminal but a slightly differing axial localization along the kidney proximal tubule. B 0 AT1; XT2; SIT1; epithelial transporters; small intestine INGESTED DIETARY PROTEINS are cleaved into small oligopeptides and single amino acids that are then absorbed across small intestine enterocytes. Similarly, small oligopeptides and single amino acids are reabsorbed across kidney proximal tubule epithelial cells to prevent their loss in the urine. The first step of this transcellular transport is the influx across the luminal brush-border membrane that is mediated by symporters (cotransporters) and antiporters (exchangers; see Refs. 36). The basolateral efflux of amino acids from the epithelial cells into the extracellular space is mediated by facilitated diffusion pathway(s) and exchangers (36).

The membrane protein Nogo-A is known as an inhibitor of axonal outgrowth and regeneration in the CNS. However, its physiological functions in the normal adult CNS remain incompletely understood. Here, we investigated the role of Nogo-A in cortical synaptic plasticity and motor learning in the uninjured adult rodent motor cortex. Nogo-A and its receptor NgR1 are present at cortical synapses. Acute treatment of slices with function-blocking antibodies (Abs) against Nogo-A or against NgR1 increased long-term potentiation (LTP) induced by stimulation of layer 2/3 horizontal fibers. Furthermore, anti-Nogo-A Ab treatment increased LTP saturation levels, whereas long-term depression remained unchanged, thus leading to an enlarged synaptic modification range. In vivo, intrathecal application of Nogo-A-blocking Abs resulted in a higher dendritic spine density at cortical pyramidal neurons due to an increase in spine formation as revealed by in vivo two-photon microscopy. To investigate whether these changes in synaptic plasticity correlate with motor learning, we trained rats to learn a skilled forelimb-reaching task while receiving anti-Nogo-A Abs. Learning of this cortically controlled precision movement was improved upon anti-Nogo-A Ab treatment. Our results identify Nogo-A as an influential molecular modulator of synaptic plasticity and as a regulator for learning of skilled movements in the motor cortex.

Reabsorption of amino acids, similar to that of glucose, is a major task of the proximal kidney tubule. Various amino acids are actively transported across the luminal brush border membrane into proximal tubule epithelial cells, most of which by cotransport. An important player is the newly identified cotransporter (symporter) B0AT1 (SLC6A19), which imports a broad range of neutral amino acids together with Na+ across the luminal membrane and which is defective in Hartnup disorder. In contrast, cationic amino acids and cystine are taken up in exchange for recycled neutral amino acids by the heterodimeric cystinuria transporter. The basolateral release of some neutral amino acids into the extracellular space is mediated by unidirectional efflux transporters, analogous to GLUT2, that have not yet been definitively identified. Additionally, cationic amino acids and some other neutral amino acids leave the cell basolaterally via heterodimeric obligatory exchangers.

Most neutral l-amino acid acids are transported actively across the luminal brush-border membrane of small intestine and kidney proximal tubule epithelial cells by a Na(+) cotransport system named B(0) that has been recently molecularly identified (B(0)AT1, SLC6A19). We show here that the opossum kidney-derived cell line OK also displays a Na(+)-dependent B(0)-type neutral l-amino acid transport, although with a slightly differing substrate selectivity. We tested the hypothesis that one of the two B(0)AT1-related transporters, SLC6A18 (ortholog of orphan transporter XT2) or SLC6A20 (ortholog of the recently identified mammalian imino acid transporter SIT1), mediates this transport. Anti-sense RNA to OK SIT1 (oSIT1) but not to OK XT2 (oXT2) inhibited Na(+)-dependent neutral amino acid transport induced by OK mRNA injected in Xenopus laevis oocytes. Furthermore, inhibition of oSIT1 gene expression in OK cells by transfection of siRNA and expression of shRNA selectively reduced the Na(+)-dependent uptake of neutral l-amino acids. Finally, expression of OK cell oSIT1 cRNA in X. laevis oocytes induced besides the transport of the l-imino acid l-Pro also that of neutral l-amino acids. Taken together, the data indicate that in OK cells SIT1 (SLC6A20) is not only an apical imino acid transporter but also plays a major role as Na(+)-dependent neutral l-amino acid transporter. A similar double role could be envisaged for SIT1 in mammalian kidney proximal tubule and small intestine.

Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.