The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology

Abstract

SLC1A5, known as ASCT2, is a neutral amino acid transporter belonging to the SLC1 family and localized in the plasma membrane of several body districts. ASCT2 is an acronym standing for Alanine, Serine, Cysteine Transporter 2 even if the preferred substrate is the conditionally essential amino acid glutamine, with cysteine being a modulator and not a substrate. The studies around amino acid transport in cells and tissues began in the '60s by using radiolabeled compounds and competition assays. After identification of murine and human genes, the function of the coded protein has been studied in cell system and in proteoliposomes revealing that this transporter is a Na⁺ dependent antiporter of neutral amino acids, some of which are only inwardly transported and others are bi-directionally exchanged. The functional asymmetry merged with the kinetic asymmetry in line with the physiological role of amino acid pool harmonization. An intriguing function has been described for ASCT2 that is exploited as a receptor by a group of retroviruses to infect human cells. Interactions with scaffold proteins and post-translational modifications regulate ASCT2 stability, trafficking and transport activity. Two asparagine residues, namely N163 and N212, are the sites of glycosylation that is responsible for the definitive localization into the plasma membrane. ASCT2 expression increases in highly proliferative cells such as inflammatory and stem cells to fulfill the augmented glutamine demand. Interestingly, for the same reason, the expression of ASCT2 is greatly enhanced in many human cancers. This finding has generated interest in its candidacy as a pharmacological target for new anticancer drugs. The recently solved 3D structure of ASCT2 will aid in the rational design of such therapeutic compounds.

Keywords: ASCT2; SLC1 family; drug design; glutamine; molecular docking; proteoliposomes.

Figure 1

Schematic representation of Amino Acid (AA) roles in cells. The arrows indicate the main processes in which AA are involved. On the plasma membrane of a generic cell, the different transport mechanisms of amino acid transporters are depicted: Na⁺– cotransport (dark gray), amino acid antiport (light gray) and, in the middle, ASCT2 (blue) that is a Na⁺ dependent antiporter with features overlapping both the co-transporters and the antiporters.

Figure 2

Schematic representation of human SLC1A5 gene. Exons and introns are depicted with solid boxes and black straight lines, respectively. UTR sequence is highlighted in gray while the coding region in red. Genbank accession number is reported below each of the transcripts. The scheme is realized using SnapGene tool.

Figure 3

Sketch of the transport mechanism of human ASCT2 homotrimer. The model summarizes the main findings on human ASCT2 collected in both intact cell systems and in proteoliposomes. Each monomer is depicted as a two-domain assembly composed of a fixed part embedded in the plasma membrane (blue) and a mobile part (light blue) that constitutes the elevator domain. The proposed model is a trimer in which the three monomers work independently allowing translocation of glutamine and Na⁺ in an exchange with other neutral amino acids. The red and green circles depict amino acids and Na⁺ binding sites, respectively. The oligomeric structure explains the random simultaneous mechanism in which the transporter exposes outward or inward faces at the same time on each membrane side. In the transport cycle, Na⁺ and Glutamine (Q), bound on the external side (out), are released in the internal side (in) upon moving of the mobile domain; simultaneously, neutral amino acids (AA⁰), bound on internal side (in), are released to the external side (out). The sidedness of membrane potential is indicated. The solid and dotted arrows indicate the loading or the release of the substrate(s) to or from the trimer, respectively.

Figure 4

Sketch of the three-dimensional structure of human ASCT2. The above panel shows the overall structure of trimeric human ASCT2 parallel to membrane; each monomer is represented with the fixed domain (blue) and elevator domain (light blue) as in Figure 3. In red, the residue C467 of the substrate binding site with glutamine (orange). N212 residues are depicted in yellow indicating the glycosylation sites; the N163 residues are missing because are not solved in the structure. The membrane is indicated by orange lines. The below panel shows the top view of the same structure. The picture is obtained using Chimera 1.11.2 and the 3D structure coordinates of hASCT2 (PDB: 6GCT) (Garaeva et al., 2018).

Figure 5

ON-OFF Regulation of human ASCT2. A single monomer of ASCT2 is depicted with the same shapes and colors of Figure 3 (fix domain, blue; mobile domain, light blue; amino acid substrate binding site red). C467 is represented in yellow and C308 or C309 residues close to C467 are depicted in blue. In “ON” state, the Cys residues are in a reduced (SH) form and the mobile part can slide across the membrane for substrate translocation. In “OFF” state, the C467 is oxidized by forming an S-S bond with C308 or C309; the mobility of the sliding moiety is impaired by the disulfide with consequent inactivation of the ASCT2 transport cycle.

Figure 6

A brief Summary of metabolic changes occurring in highly proliferative cells. In the scheme, uptake of glutamine (Gln, red) and glucose (green) occur mainly via membrane ASCT2 in a sodium-dependent reaction and via GLUT, respectively. The pathways are indicated in blue arrows (when related to Glutamine) in green (when related to glucose). Solid arrows indicate a single reaction(s), while dotted a multistep chain of reactions. The uptake of Gln across the plasma membrane is guaranteed by exchange with smaller amino acids such as threonine (Thr), asparagine (Asn) or serine (Ser). Carbon skeleton of asparagine (black) and serine (green) may derive from malate (black) and glucose (green), respectively. In the cytosol, Gln is used to produce glutathione for ROS scavenging. The entry of Gln in mitochondria is still questionable (“?” In the transporter across the inner mitochondrial membrane). In mitochondria, Gln is converted to Glu that enters the truncated TCA (tTCA) as α-ketoglutarate (αKG) which is also exported to the cytosol for fatty acid synthesis (boxed in light blue). The substrates of tTCA are indicated in black, malate (Mal) reaches cytosol where undergoes a chain of reactions (boxed in light blue) to restore NADPH and NAD⁺ pools. OXPHOS is also depicted in the inner mitochondrial membrane. ATP and reducing equivalent molecules produced by Gln and glucose metabolism are indicated in red.