Molecular biology entered the field of mammalian amino acid transporters in 1990-1991 with the cloning of the first GABA and cationic amino acid transporters. Since then, cDNA have been isolated for more than 20 mammalian amino acid transporters. All of them belong to four protein families. Here we describe the tissue expression, transport characteristics, structure-function relationship, and the putative physiological roles of these transporters. Wherever possible, the ascription of these transporters to known amino acid transport systems is suggested. Significant contributions have been made to the molecular biology of amino acid transport in mammals in the last 3 years, such as the construction of knockouts for the CAT-1 cationic amino acid transporter and the EAAT2 and EAAT3 glutamate transporters, as well as a growing number of studies aimed to elucidate the structure-function relationship of the amino acid transporter. In addition, the first gene (rBAT) responsible for an inherited disease of amino acid transport (cystinuria) has been identified. Identifying the molecular structure of amino acid transport systems of high physiological relevance (e.g., system A, L, N, and x(c)- and of the genes responsible for other aminoacidurias as well as revealing the key molecular mechanisms of the amino acid transporters are the main challenges of the future in this field.

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chlorideand pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodiumindependent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0'+ and carries cystine and dibasic amino acids with high affmity. The rBAT (bO'+ amino acid transporterrelated) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b°'+.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases l-arginine transport only through the y+ system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in l-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0°'-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y+-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (Mi) The stimulation of sodium-independent uptake of L-arge and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.Amino acids cross the plasma membrane of cells via sodiumindependent and sodium-dependent carriers. In eukaryotic cells, several carriers have been defined based on ion dependency, substrate specificity, and kinetic properties (1). Our knowledge of the structural identity of these carriers is limited. Recently, two cDNAs related to amino acid transport activity in mammalian cells have been isolated: (i) The murine receptor for the ecotropic murine leukemia virus has been identified as being responsible for system y+, a sodiumindependent cation-preferring amino acid carrier (2, 3). (ii) As described in the two preceding papers, kidney cortex cDNAs from rabbit and rat [named rBAT (b°+ amino acid transporter-related protein) (4) and D2 (5), respectively] have been cloned, which upon injection into oocytes induce system b°+ (6), a sodium-independent carrier for cationic and neutral amino acids (including L-cystine). The predicted protein for the ecotropic murine leukemia vir...

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-␤ 1 , 177-244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKB␣, PKB␥, and PKC activities. TPCK, an inhibitor of PDK1, abolished both HRG-and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKB␣. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKC abolished both HRG-and insulinstimulated glucose uptake. In soleus muscle, HRG induced PKC, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/ p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPK␣ 1 or AMPK␣ 2 activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKC, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKC pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b o,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and b o,+ AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. IntroductionThe heteromeric amino acid transporters (HATs) are the only known eukaryotic transporters of amino acids composed of two different subunits bound by a disul®de bridge: (i) a type II glycoprotein or heavy subunit (rBAT and 4F2hc), and (ii) a non-glycosylated light subunit with 12 putative transmembrane domains (LAT-1, LAT-2, asc1, y + LAT-1, y + LAT-2, xCT and b o,+ AT) (for reviews see Verrey et al., 1999Verrey et al., , 2000Deve Âs and Boyd, 2000;Chillaro Ân et al., 2001;Wagner et al., 2001). The heavy subunit has an ectodomain that is homologous to bacterial glucosidases (reviewed in Chillaro Ân et al., 2001). The light subunit is suspected to be the catalytic part of the holotransporter because it is considered to be a polytopic membrane protein and heterodimers of 4F2hc with different light subunits yield different amino acid transport systems: (i) with LAT-1 or LAT-2, variants of system L ( Kanai et al., 1998;Mastroberardino et al., 1998;Pineda et al., 1999;Rossier et al., 1999;Segawa et al., 1999) The light subunit of system b o,+ is fully functional in the absence of the heavy subunit