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

甲状腺激素（TH）及其衍生物通过其他TH跨膜转运蛋白（THTT）和l型氨基酸转运蛋白（LAT）在细胞膜上进行转运。对Lat2如TH特异性和体内作用的了解最少。这促使我们在第一个资助期深入了解Lat2在TH摄取中的结构-功能关系。我们不仅证实了先前的发现，即Lat2普遍参与TH的摄取，而且我们在非洲爪蟾卵母细胞中发现Lat2主要运输3,3 ' -T2和少量T3，但既不运输rT3也不运输T4。Lat2的外向开放分子模型结合模型引导的Lat2变体和TH衍生物的摄取数据，成功地阐明了Lat2中TH摄取的决定因素，如i)细胞外3,3 ' -T2识别位点，ii) Lat2中心的底物穿越的不对称形状通道和iii)潜在的开关位点。根据这些数据，我们假设在Lat2的细胞外和细胞内有不同的识别机制。在初步的尝试中，我们实际上观察到亮氨酸流出，但没有由卵母细胞中的Lat2输出T2。最近有来自COS-1细胞的新的初步提示，T2， T1由LAT3/4输出。然而，出口的分子细节尚不清楚。为了探究LAT家族中TH的不同分子外排机制，我们将调查和比较LAT2、LAT3或LAT4介导的TH输出的结构-功能关系。具体而言，我们将i)生成向外和向内开放的模型，推导出细胞内合适的底物识别模式；ii)野生型和模型引导的LAT突变体在非洲爪蟾卵母细胞中的底物外排测量；iii)鉴定可能触发LAT2和LAT3或LAT4细胞内开关或转换功能的决定因素；iv)阐明TH或相反侧氨基酸的运输依赖性。为了了解LAT1/2和LAT3/4在TH输出和底物选择性方面的功能差异。关注T2作为非经典TH有助于阐明其作为替代TR配体的作用或可能介导TH对能量代谢的调节。我们的项目通过结合生物信息学、分子生物学和体外研究，跨越了结构、功能和生理方法，并有可能为未来新的治疗策略的发展做出贡献。合作伙伴的其他应用项目对甲状腺细胞、星形胶质细胞等多种细胞中LAT2、LAT3、LAT4的TH生理外排研究是对我们研究的补充。此外，关于LAT中T2摄取和外排的决定因素和分子机制的详细知识为未来对功能障碍进行药物干预以预防和靶向TH依赖性疾病提供了基础。

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.