Cloning and functional expression of a cDNA from rat jejunal epithelium encoding a protein (4F2hc) with system y+L amino acid transport activity.

Two different protein families, designated CAT (cationic amino acid transporter) and BAT (broad-specificity amino acid transporter) mediate the plasma membrane transport of cationic amino acids in animal cells. CAT transporters have 12-14 transmembrane domains and are selective for cationic amino acids. BAT proteins, in contrast, have one to four transmembrane domains and induce the transport of both cationic and zwitterionic amino acids when expressed in Xenopus oocytes. Mutations in the human BAT gene cause type I cystinuria, a disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. We have used functional expression cloning in oocytes to isolate a BAT-related cDNA from rat jejunal epithelium. The cDNA encodes the rat 4F2 heavy chain (4F2hc) cell-surface antigen, a 527-residue (60 kDa) protein that is 26% identical in amino acid sequence with rat renal BAT (also known as NBAT/D2). Expression of rat jejunal 4F2hc in oocytes induced the lysine-inhibitable Na+-dependent influx of leucine and the leucine-inhibitable Na+-independent influx of lysine. Lysine efflux was stimulated by extracellular (Na+ plus leucine). These characteristics identify the expressed amino acid transport activity as system y+L, a transporter that has been implicated in basal membrane transport of cationic amino acids in intestine, kidney and placenta.

Evidence that the transport-related proteins BAT and 4F2hc are not specific for amino acids: induction of Na+-dependent uridine and pyruvate transport activity by recombinant BAT and 4F2hc expressed in Xenopus oocytes.

Members of the BAT and 4F2hc gene family have one or, in the case of BAT, up to four transmembane domains and induce amino acid transport systems b(o,+) (BAT) and y+L (4F2hc) when expressed in Xenopus oocytes. System b(o,+) is a Na+-independent process with a broad tolerance for cationic and zwitterionic amino acids, whereas y+L exhibits Na+-independent transport of cationic amino acids (e.g., lysine) and Na+-dependent transport of zwitterionic amino acids (e.g., leucine). Mutations in the human BAT gene are associated with type I cystinuria, a genetic disease affecting the ability of intestinal and renal brush border membranes to transport cationic amino acids and cystine. An unresolved question is whether BAT and 4F2hc themselves have catalytic (i.e., transporting) activity or whether they operate as activators of other, as yet unidentified, transporter proteins. In this report, we have investigated the transport of representatives of four different classes of organic substrates in Xenopus oocytes following injection with rat BAT or 4F2hc RNA transcripts: leucine (a control amino acid substrate), uridine (a nucleoside), pyruvate (a monocarboxylate), and choline (an amine). Both recombinant proteins induced small, statistically significant Na+-dependent fluxes of uridine and pyruvate but had no effect on choline uptake. In contrast, control oocytes injected with transcripts for conventional nucleoside and cationic amino acid transporters (rat CNT1 and murine CAT1, respectively) showed no induction of transport of either leucine or pyruvate (CNT1) or uridine or pyruvate (CAT1). These findings support the idea that BAT and 4F2hc are transport activators and minimize the possibility that they have intrinsic transport capability. The transport-regulating functions of these proteins may extend to permeants other than amino acids.

Molecular cloning and functional characterization of nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins (rENT1 and rENT2) from rat tissues.

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

Poly(A)+ RNA from the mucosa of rat jejunum induces novel Na(+)-dependent and Na(+)-independent leucine transport activities in in oocytes of Xenopus laevis.

Complementary DNA clones have been isolated recently from rat (D2) and rabbit kidney (rBAT) which induce increased Na(+)-independent Leu and Lys transport activities (System b0, +) when expressed in oocytes of Xenopus laevis. These cDNAs encode type II membrane glycoproteins which show significant homology to the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Injection of human 4F2hc cRNA into oocytes also results in induction of Leu/Lys transport activity, but with differing cation requirements for the two amino acids (Na(+)-dependent for Leu, Na(+)-independent for Lys: system y+L). System y+L is a newly discovered zwitterionic/cationic amino acid transporter first described in human erythrocytes. Here we have examined the characteristics of Leu transport in Xenopus oocytes microinjected with mRNA from the mucosa of rat jejunum. L-Leu uptake during 10 min (0.2 mM, 20 degrees C) reached 20 pmol/oocyte compared with endogenous uptake by water-injected oocytes of typically 3-4 pmol/oocyte. The expressed transport activity was 80% Na(+)-dependent. The Na(+)-dependent component of the expressed flux was saturable (Km app 0.20 mM) and inhibited by Lys, but not by Ala or Phe. The minor Na(+)-independent component of expressed Leu transport activity was also saturable (Km app 0.10 mM). Amino acid inhibition studies resolved this flux into two main components, one of which was inhibited by Lys, Ala and Phe and another which was only inhibited by Lys. There was a small residual component of Na(+)-independent Leu transport which was insensitive to inhibition by Lys. Experiments utilizing polymerase chain reaction (PCR) demonstrated the presence of both D2 and 4F2hc message in rat jejunum. Hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to D2 had no effect on the expression of Na(+)-linked Leu transport activity, but reduced the smaller Na(+)-independent component of Leu transport by 40%, suggesting only a minor role of D2 in the expression of rat intestinal Leu transport activity. Although the properties of Na(+)-dependent Leu transport were, with the exception of a lack of inhibition by Ala and Phe, consistent with erythrocyte y+L, hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to 4F2hc had no detectable effect on the expressed transport activity. We conclude, therefore, that mRNA from rat jejunum encodes novel Na(+)-dependent and Na(+)-independent transport activities unrelated to the D2/4F2hc glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of rat intestinal L-lysine transport systems in isolated oocytes of Xenopus laevis.

The mechanisms by which cationic amino acids are transported across the intestinal epithelium are poorly understood. We show that isolated stage VI oocytes of Xenopus laevis can express lysine transport activity, which is due to the microinjection of mRNA from rat small intestine. L-Lysine transport activity (0.2 mM, 20 degrees C) reaches 400 pmol.oocyte-1.h-1, compared with a typical endogenous rate of 85 pmol.oocyte-1.h-1. Na(+)-dependence and amino acid inhibition studies resolved the expressed transport activity into three components: 1) a Na(+)-dependent transport system that can be inhibited by leucine with high affinity and also by alanine; 2) a Na(+)-independent system that can be inhibited by leucine with high affinity when Na+ is present, but this affinity is reduced in its absence; 3) a Na(+)-independent system that is inhibited by leucine with high affinity only when Na+ is present. Peak arginine-inhibitable lysine influx was found in a mRNA size fraction of 1.5-2.25 (median 2.0) kb.