Molecular determinants of different thyroid hormone uptake/ efflux mechanisms for L-type amino acid transporter subtypes

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Thyroid hormones (TH) and their derivatives are transported across the cell membrane, amongst other TH transmembrane transporter proteins (THTT), also by the L-type amino acid transporters (LAT). Least was known about Lat2 such as TH specificity and the unclear role in vivo. This motivated us in the first funding period to gain insight into structure-function relationships of Lat2 in TH uptake. We not only affirmed previous findings of a general involvement of Lat2 in the uptake of TH, as important new finding we showed in Xenopus laevis oocytes that Lat2 is mainly transporting 3,3`-T2 and somewhat less T3 but neither rT3 nor T4. Outward-open molecular models of Lat2 combined with uptake data of model-guided Lat2-variants and TH-derivatives enabled a successful elucidation of determinants for TH uptake in Lat2 such as i) the extracellular 3,3`-T2 recognition site, ii) an asymmetrically shaped channel for substrate traversing in the center of Lat2 and iii) a potentially switch site. From these data we hypothesize different recognition mechanisms at the extra and intracellular side of Lat2. In preliminary attempts we in fact observed leucine efflux but no T2 export by Lat2 in oocytes. There are recent new initial hints from COS-1 cells that T2, T1 are exported by LAT3/4. However molecular details for export are unknown.Thus exploring the different molecular efflux mechanisms of TH in the LAT family, we will survey and compare the structure-function relationships of TH export mediated by LAT2, LAT3 or LAT4. In detail we will i) generate outward and inward open models deducing appropriate substrate recognition patterns at the intracellular sides, ii) substrate efflux measurements in Xenopus laevis oocytes, for wild type and model-guided LAT mutants, iii) identification of determinants potentially triggering a switch or changeover function at the intracellular side of LAT2 and LAT3 or LAT4 and iv) clarify transport dependence of TH or amino acids at the counter-side. In order to understand the functional differences between LAT1/2 and LAT3/4 in TH export and substrate selectivity. Focusing on T2 as nonclassical TH contributes to elucidate its role assumed to be an alternative TR ligand or might mediate TH regulation on the energy metabolism.Our project straddles structural, functional and physiological approaches by combining bioinformatics molecular biological and in vitro studies and has the potential to contribute to the future development of new therapeutic strategies. Physiological TH efflux studies for LAT2, LAT3 and LAT4 in diverse cells such as thyrocytes and astrocytes by other applying projects of cooperating partners are complementing our studies. Moreover, detailed knowledge about determinants and molecular mechanisms for both uptake and efflux of T2 in LAT provides the basis for future pharmacological interventions of malfunctions to prevent and target TH dependent diseases.