Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

Abstract

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.

Structure of the human heterodimeric transporter 4F2hc-LAT2 in complex with Anticalin, an alternative binding protein for applications in single-particle cryo-EM

“…To heterologously overexpress human 4F2hc-LAT2, a previously characterized and reported P. pastoris clone was used 12 , 14 , 37 . The recombinant protein was produced in P. pastoris as described 23 , solubilized with GDN and purified according to published protocols 37 , 38 .…”

“…The HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is a Na + -independent obligatory exchanger that preferably transports neutral as well as small L-amino acids 9 – 12 and the thyroid hormones T3 and T4 13 , 14 . LAT2 is ubiquitously expressed in tissues 2 and has implications in human health.…”

Loss of LAT1 sex-dependently delays recovery after caerulein-induced acute pancreatitis

“…The knockout of LAT1 was efficient until the end of the experiment and did not cause compensatory upregulation of other AAT. LAT2 ( slc7a8 ), especially, which has a similar substrate specificity as LAT1[ 52 ] and the capacity to activate mammalian target of rapamycin (mTOR) signaling[ 53 ], was not upregulated in acinar cells. This stands in contrast to constitutive muscle-specific ablation of LAT1 ( MCK-cre slc7a5 ), where LAT2 mRNA was upregulated in muscle fibers[ 29 ].…”

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

“…To obtain purified proteins, the choice of the affinity tag added during the cloning procedure, according to the downstream applications, could be crucial. One of the most commonly used affinity tags used for the purification of human SLC transporters expressed in yeast is the polyhistidine tag (His-tag), consisting of 6His [ 30 , 58 ], 8His [ 103 , 180 , 189 ] or 10His residues [ 93 , 190 ]. During purification, there is the risk that the tag is inaccessible for binding to the affinity resin; thus, a linker between the tag and the target protein or a longer version of the His-tag may solve this problem.…”