C. A. Richard Boyd

PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids.

Growth in normal and tumour cells is regulated by evolutionarily conserved extracellular inputs from the endocrine insulin receptor (InR) signalling pathway and by local nutrients. Both signals modulate activity of the intracellular TOR kinase, with nutrients at least partly acting through changes in intracellular amino acid levels mediated by amino acid transporters. We show that in Drosophila, two molecules related to mammalian proton-assisted SLC36 amino acid transporters (PATs), CG3424 and CG1139, are potent mediators of growth. These transporters genetically interact with TOR and other InR signalling components, indicating that they control growth by directly or indirectly modulating the effects of TOR signalling. A mutation in the CG3424 gene, which we have named pathetic (path), reduces growth in the fly. In a heterologous Xenopus oocyte system, PATH also activates the TOR target S6 kinase in an amino acid-dependent way. However, functional analysis reveals that PATH has an extremely low capacity and an exceptionally high affinity compared with characterised human PATs and the CG1139 transporter. PATH and potentially other PAT-related transporters must therefore control growth via a mechanism that does not require bulk transport of amino acids into the cell. As PATH is likely to be saturated in vivo, we propose that one specialised function of high-affinity PAT-related molecules is to maintain growth as local nutrient levels fluctuate during development.

How to Make Drugs Orally Active: A Substrate Template for Peptide Transporter PepT1

By building key structural features into hydrophilic drugs, they can be recognized by the PepT1 transporter system of the small intestine and rendered orally active. The model shown provides, for the first time, a 3D template for all known substrates of PepT1.

IDO Induces Expression of a Novel Tryptophan Transporter in Mouse and Human Tumor Cells

IDO is the rate-limiting enzyme in the kynurenine pathway, catabolizing tryptophan to kynurenine. Tryptophan depletion by IDO-expressing tumors is a common mechanism of immune evasion inducing regulatory T cells and inhibiting effector T cells. Because mammalian cells cannot synthesize tryptophan, it remains unclear how IDO+ tumor cells overcome the detrimental effects of local tryptophan depletion. We demonstrate that IDO+ tumor cells express a novel amino acid transporter, which accounts for ∼50% of the tryptophan uptake. The induced transporter is biochemically distinguished from the constitutively expressed tryptophan transporter System L by increased resistance to inhibitors of System L, resistance to inhibition by high concentrations of most amino acids tested, and high substrate specificity for tryptophan. Under conditions of low extracellular tryptophan, expression of this novel transporter significantly increases tryptophan entry into IDO+ tumors relative to tryptophan uptake through the low-affinity System L alone, and further decreases tryptophan levels in the microenvironment. Targeting this additional tryptophan transporter could be a way of pharmacological inhibition of IDO-mediated tumor escape. These findings highlight the ability of IDO-expressing tumor cells to thrive in a tryptophan-depleted microenvironment by expressing a novel, highly tryptophan-specific transporter, which is resistant to inhibition by most other amino acids. The additional transporter allows tumor cells to strike the ideal balance between supply of tryptophan essential for their own proliferation and survival, and depleting the extracellular milieu of tryptophan to inhibit T cell proliferation.

The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro

Four hydrolysis-resistant dipeptides (D-phenylalanyl-L-alanine, D-phenylalanyl-L-glutamine, D-phenylalanyl-L-glutamate and D-phenylalanyl-L-lysine) were synthesized to investigate the effects of net charge on transmural dipeptide transport by isolated jejunal loops of rat small intestine. At a luminal pH of 7.4 and a concentration of 1 mM the two dipeptides with a net charge of -1 and +1 were transported at substantially slower rates (18 +/- 1.3 and 8.4 +/- 1.3 nmol min(-1)(g dry wt.)(-1), respectively) than neutral D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine (87 +/- 0.2 and 197 +/- 14 nmol min(-1)(g dry wt.)(-1), respectively). We investigated the effects of luminal pH on dipeptide transport by varying the NaHCO3 content of Krebs Ringer perfusate equilibrated with 95% 02/5% CO2. The pH changes did not affect water transport, but serosal glucose appearance increased significantly at pH 6.8. Transmural transport of D-phenylalanyl-L-alanine and D-phenylalanyl-L-glutamine at pH 6.8 was stimulated (P < 0.01) by 61% and 49%, respectively, whereas the lower pH increased the rate for negatively charged D-phenylalanyl-L-glutamate by 306% (P < 0.01) and decreased that for positively charged D-phenylalanyl-L-lysine by 46% (P < 0.05). Increasing luminal pH to 8.0 inhibited D-phenylalanyl-L-alanine transport by 60%, whereas D-phenylalanyl-L-lysine transport was 60% faster.

Tripeptide transport in rat lung

Transport of L-alanyl-D-phenylalanyl-L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 +/- 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 +/- 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could be detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl-D-phenylalanyl-L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl-L-phenylalanyl-D-alanine, was taken up significantly more slowly by the vesicles. D-phenylalanyl-L-alanine and D-phenylalanyl-D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl-D-phenylalanyl-L-alanine whereas D-phenylalanyl-D-alanine appeared more slowly and was not hydrolysed.

PREFERENTIAL RECOGNITION OF ZWITTERIONIC DIPEPTIDES AS TRANSPORTABLE SUBSTRATES BY THE HIGH-AFFINITY PEPTIDE TRANSPORTER PEPT2

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. D-Phe-L-Gln (neutral), D-Phe-L-Glu (anionic), and D-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H+-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H+:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.

Both the H13 gene product and 4F2 antigen are involved in the induction of system y+ cationic amino-acid transport following activation of human peripheral blood mononuclear cells (PBM).

Prior transfection with antisense oligonucleotides to the H13 and 4F2 hc genes, singly or in combination, was found to inhibit phytohaemagglutinin-induced activation of cationic amino-acid transport system y+ in human peripheral blood mononuclear cells (mostly circulating lymphocytes). These effects on system y+ function or expression mean that 4F2 hc cannot only be the molecular basis of system y+L (Fei, Y.-J., Prasad, P.D., Leibach, F.H. and Ganapathy, V. (1995) Biochemistry 34, 8744-8751).

Dipeptide transport and hydrolysis in rat small intestine, in vitro

A range of natural and mixed D-/L-stereoisomer phenylalanine dipeptides was used to investigate peptide uptake and hydrolysis by isolated rings of rat jejunum. Characterisation of dipeptide hydrolysis by the brush border fraction revealed apparent Km values in the 0.1-1.0 mM range which, except for the charged dipeptides, were significantly higher than those for hydrolysis by the cytosolic fraction. Uptake of L-/L-dipeptides into jejunal rings, which was followed by HPLC, was unaffected by the presence of peptidase inhibitors in the incubation medium although the absorbed peptides were completely hydrolysed in the cytosol; comparison of the effects of excess leucine on dipeptide uptake and on the uptake of the two constituent amino acids were also consistent with absorption of intact dipeptide followed by cytosolic hydrolysis. The uptake of hydrolysis-resistant mixed D-/L-dipeptides was also studied and confirmed that peptide uptake preceded hydrolysis; D-alanyl-L-phenylalanine accumulated within the rings to twice the medium concentration.