Structure/function relationships of the human heterodimeric amino acids transporter 4F2hc/LAT1

Abstract

Amino acids transport in mammalian cells is mediated by different amino acid transporters whose activity allow the flow of an important source for metabolic need of cells. Moreover, some amino acids such as Gln, Arg and Leu work as signalling molecules and their availability and concentration represent key factors in the regulation of intracellular signalling pathways responsible of cellular growth. Thus, amino acids flow, which is important under physiological condition, becomes particularly relevant under pathological conditions such as in tumours cells to satisfy their unique metabolic and proliferative needs. Therefore, since in tumours upregulation of amino acids transporters is an important step to satisfy the increased demand for these nutrients, the same transporters are potential drug targets for cancer therapy. However, the certainty that a specific transporter could be a target in human therapy requires its functional characterization and the knowledge of the enchanting structure/function relationships. In this context, an important transporter that became of particular interest for its overexpression in many tumours is LAT1, and the aim of this work has been that to shed light on still unclear aspects of its function hLAT1 belongs to SLC7 family and into the plasma membrane forms heterodimers with the glycoprotein 4F2hc (also known as CD98 in mice), member of SLC3 family. Studies conducted in intact cells showed that 4F2hc/LAT1 complex catalyses amino acids transport; however, in this experimental model it was not possible to clarify whether one or both subunits are competent for transport activity and substrate recognition. Thus, aimed to unravel the dark side of 4F2hc/LAT1 mediated transport, different experimental strategies were adopted allowing to demonstrate that LAT1 is the sole transport competent unit of the heterodimer. Indeed, using western blot analyses and transport assays in liposomes reconstituted with proteins extracted from SiHa cells and in liposomes reconstituted with recombinant LAT1, it has been demonstrated that neither the covalent interaction nor the association of 4F2hc with LAT1 influence transport and specificity of LAT1. Moreover, the suitability of proteoliposome model used for reconstitution of recombinant LAT1, allowed to identify a functional asymmetry of this transporter which, on a physiological point of view, exhaustively elucidates the reciprocal correlation between the transport activity of LAT1 and that of another important amino acids transporter overexpressed in tumours cells, ASCT2. To the same extent, proteoliposome tool together with bioinformatics and site-directed mutagenesis have been useful to probe critical residues of the substrate binding site of LAT1. These results laid the groundwork for deciphering molecular mechanism of LAT1 function and for setting up studies aimed to identify new potent and specific inhibitors great for human health.