Understanding the Regulation of the Human Amino Acid Transporter ASCT2

Glutamine is a very versatile source of energy and a precursor for many molecules which are essential to the growth and survival of actively dividing cells. Numerous amino acid transporters are involved in the transport and homeostasis of glutamine and ASCT2 is one of the most important transporters expressed in mammals, due to its moderate affinity for glutamine (Km = 70 - 90 M), its ubiquitous organ expression, and its correlation with aggressive forms of cancer. Yet, the regulation of ASCT2 in a non-pathological setting is poorly understood. In this study, I developed a new cell model for the study of ASCT2 by examining the molecular and functional characteristics of glutamine uptake in RWPE-1, an immortalized prostate epithelial human cell line. The expression of the canonical isoform of ASCT2 (SLC1A5) mRNA in RWPE-1 cells was confirmed by quantitative PCR and ASCT2 protein by western blotting. Immunofluorescence microscopy confirmed ASCT2 to be abundant and primarily localized to the plasma membrane. Radio-labelled glutamine uptake in RWPE-1 cells determined that ASCT2 is the major contributor (45 %) of glutamine transport in this cell line. Additionally, I have confirmed that EGF regulates the glutamine uptake via ASCT2, by regulating, through an unknown mechanism, the trafficking of the transporter to the plasma membrane. Thus, I conclude that the RWPE-1 cell-line is the first model to date used to study the detailed assessment of amino acid transporters role in the physiology of the prostate; moreover, RWPE-1 is a physiologically relevant model for the study of ASCT2 regulation. Moreover, I performed a largescale MYTH screening to target ASCT2 and discover potential interactors of the transporter which may help my future studies to further investigate and understand the regulation of the transporter.