Benjamin Spindler

Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family

Amino-acid transport across cellular plasma membranes depends on several parallel-functioning (co-)transporters and exchangers. The widespread transport system L accounts for a sodium-independent exchange of large, neutral amino acids, whereas the system y+L exchanges positively charged amino acids and/or neutral amino acids together with sodium,. The molecular nature of these transporters remains unknown, although expression of the human cell-surface glycoprotein 4F2 heavy chain (h4F2hc; CD98 in the mouse), is known to induce low levels of L- and/or y+L-type transport. This glycoprotein is found in activated lymphocytes, together with an uncharacterized, disulphide-linked lipophilic light chain with an apparent relative molecular mass of 40,000 (Mr 40K),. Here we identify the permease-related protein E16 (ref. 12) as the first light chain of h4F2hc and show that the resulting heterodimeric complex mediates L-type amino-acid transport. The homologous protein from Schistosoma mansoni, SPRM1, also associates covalently with coexpressed h4F2hc glycoprotein, although it induces amino-acid transport of different substrate specificity. The coexpression of h4F2hc is required for surface expression of these permease-related light chains, which belong to a new family of amino-acid transporters that form heterodimers with cell-surface glycoproteins.

Amino acid transport of y+L‐type by heterodimers of 4F2hc/CD98 and members of the glycoprotein‐associated amino acid transporter family

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+‐independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease‐related protein (LAT1 or AmAT‐L‐lc) which for surface expression and function requires formation of disulfide‐linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are ∼48% identical with LAT1 and thus belong to the same family of glycoprotein‐associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+‐dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L‐type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1–4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.

Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia.

Abstract The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase). In order to contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved. Epithelia were treated for 1 h with 10–6 M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied. Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase α1 and β1 subunit mRNAs were slightly increased (1.2- to 1.4-fold). In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR). After 1 h of aldosterone treatment, ≈0.5% of these appeared to be regulated. Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned and for two of them cDNAs containing entire coding sequences were isolated by library screening. ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D. The K1/2 values were similar or slightly below those reported for stimulation of Na transport. These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.

Functional heterodimeric amino acid transporters lacking cysteine residues involved in disulfide bond

The protein mediating system L amino acid transport, AmAT‐L, is a disulfide‐linked heterodimer of a permease‐related light chain (AmAT‐L‐lc) and the type II glycoprotein 4F2hc/CD98. The Schistosoma mansoni protein SPRM1 also heterodimerizes with h4F2hc, inducing amino acid transport with different specificity. In this study, we show that the disulfide bond is formed by heavy chain C109 with a Cys residue located in the second putative extracellular loop of the multi‐transmembrane domain light chain (C164 and C137 for XAmAT‐L‐lc and SPRM1, respectively). The non‐covalent interaction of Cys‐mutant subunits is not sufficient to allow coimmunoprecipitation, but cell surface expression of the light chains is maintained to a large extent. The non‐covalently linked transporters display the same transport characteristics as disulfide bound heterodimers, but the maximal transport rates are reduced by 30–80%.

Identification of a new gene product (diphor-1) regulated by dietary phosphate

Chronic restriction of dietary Pi elicits an increased reabsorption of Pi in the kidney proximal tubules, which involves a stimulation of apical Na-Pi cotansport. This adaptation is in part a direct cellular response of which the mechanism(s) are poorly understood. In this study, the impact of dietary Pi restriction on the differential expression of rat kidney cortex mRNAs was visualized to identify gene products regulated by the Pistatus. When kidney cortex mRNAs of rats fed a low- or a high-Pi diet were compared by differential display-polymerase chain reaction (DD-PCR), thirty modulated cDNA bands were observed, of which four were confirmed as being regulated. We focused on one of the upregulated bands, dietary Pi-regulated RNA-1 (diphor-1). A cDNA containing an open reading frame encoding a 52-kDa protein was cloned by library screening. Diphor-1 exhibits a high degree of identity to the Na/H exchanger regulatory factor and to a tyrosine kinase activating protein. Highest expression of diphor-1 mRNA was detected in the kidney (proximal tubules) and in small intestine. Expression experiments showed that diphor-1 specifically increases Na-Picotransport in oocytes of Xenopus laevis coinjected with renal type II Na-Pi cotransporter cRNA. Further characterizations of diphor-1 will show whether diphor-1 is primarily or secondarily involved in the response to dietary Pi.

Aldosterone action: induction of p21 ras and fra-2 and transcription-independent decrease in myc, jun, and fos

Adrenal steroids induce an increase in transcellular Na+ reabsorption across Xenopus laevis A6 cell epithelia that requires the action of transcriptionally regulated gene products. In a previous study we identified K- ras2 as an aldosterone-upregulated mRNA in A6 epithelia. Here, we show that in vivo injection of aldosterone in Xenopus (2.5 h) increases K- ras2 mRNA specifically in the kidney (2.5-fold) and that in A6 epithelia aldosterone (2.5 h) increases Ras protein synthesis (∼6-fold). Xl- ras, another ras mRNA expressed at a low level in A6 cells, was also induced (2-fold). Aldosterone was shown to regulate the mRNA levels of several transcription factors as well. After 2 h of aldosterone treatment, fra-2 mRNA was upregulated by 130%, whereas c- myc, c- jun, c- fos, and glucocorticoid receptor mRNAs were downregulated by 23-43%. After 16 h, c- fos and GR mRNAs were further decreased, whereas levels of fra-2, c- jun, and c- myc began to return to control levels. Interestingly, the downregulation of the protooncogene mRNAs was independent of transcription. These results support the view that aldosterone exerts complex pleiotropic transcriptional and nontranscriptional actions that involve the regulation of signaling cascade elements (i.e., K-Ras2) as well as that of transcription factors.

Renal type II Na/Pi-cotransporter is strongly impaired whereas the Na/sulphate-cotransporter and aquaporin 1 are unchanged in cadmium-treated rats

The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly understood. In this study we investigated the cellular causes of the Cd-induced phosphaturia in the rat. Compared to controls, Cd-treated rats (2 mg Cd/kg body weight, s.c. for 14 days) showed a marked polyuria, proteinuria and phosphaturia. As studied by the rapid filtration technique in isolated cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptake of phosphate ([32Pi]) and of [3H] glucose were markedly decreased in Cd-treated rats, whereas uptake of sulphate ([35S]) remained unchanged. By Western blotting of BBMV proteins and by indirect immunocytochemistry in 4-μm thick frozen fixed kidney sections, using an antibody against the type II Na/Pi-cotransporter (NaPi-2), we found a diminished expression of this protein in the brush-border membrane from Cd-treated rats. However, the expression of the water channel aquaporin 1, estimated from the specific antibody staining in brush-border membranes, remained unchanged by Cd. Northern blot analysis showed a strong reduction of 2.7 kb NaPi-2-related mRNA in Cd-affected kidneys. Our data indicate that: (1) Cd may reduce reabsorption of Pi in proximal tubules by affecting the expression of the functional Na/Pi-cotransporters in the luminal membrane, and (2) Cd effects on brush-border transporters are selective.