Studies for thyroid hormone transport mediated by the membrane transporters of the MCT and LAT families

Das Allan-Herndon-Dudley Syndrom (AHDS) ist eine schwere X-chromosomale Erkrankung, die sich phänotypisch in einer mentalen Retardation mit Sprach- und Bewegungsstörungen äußert. Im Gen des Schilddrüsenhormontransporters Monocarboxylat-Transporter 8 (MCT8) wurden bei betroffenen Patienten Mutationen identifiziert, die für die Störung ursächlich sind. Die vorliegende Arbeit charakterisiert den Pathomechanismus von ausgewählten Mutationen in Zellkulturstudien genauer. Die Ergebnisse weisen darauf hin, dass die gestörte Funktionalität der MCT8 Mutanten nicht nur auf einen Transportdefekt, sondern auch auf eine verminderte Zelloberflächenexpression zurückzuführen ist. Anhand dieser Resultate und der zelltypspezifschen Effekte wird ein Erklärungsversuch vorgenommen, warum die Schwere der Erkrankung unter den Betroffenen variiert. Bei den Analysen zur Substratspezifität von MCT8 mithilfe von Jodthyronin- Derivaten wurden die L-Aminosäurekonformation und das Vorhandensein von mindestens einem Jodatom pro aromatischem Ring als wichtige Voraussetzungen für einen effektiven Transport identifiziert. Studien zum Transportmechanismus von MCT8 zeigten die Aktivitätsrelevanz der geladenen und konservierten Aminosäuren R445 und D498. Dieser Zusammenhang bestätigt sich in einem ersten MCT8 Strukturmodell: Beide Aminosäuren sind in unmittelbarer Nähe des potenziellen Substratkanals lokalisiert. Im Gegensatz zum AHDS-Patienten führt der Mct8 Verlust im Mausmodell zu einem milderen neurologischen Phänotyp. Diese Diskrepanz lässt sich möglicherweise durch speziesspezifische Expressionsmuster alternativer Schilddrüsenhormontransporter erklären, die in der murinen Gehirnentwicklung einen Mct8 Mangel kompensieren können. Antidepressiva führen im Rattenmodell zu einem Schilddrüsenhormonmangel im Zentralen Nervensystem. Um den Mechanismus zu erforschen, haben wir Transportstudien durchgeführt und eine spezifische Inhibition von MCT8 durch Desipramin beobachtet. Dieses überraschende Ergebnis liefert einen Ansatzpunkt, die zentralen Effekte von Desipramin besser einzuordnen und zu kontrollieren.The Allan-Herndon-Dudley syndrome (AHDS) is a severe X-linked disease characterized by movement disorders and mental retardation with speech problems. The disease is associated with mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8). The objective of this thesis was to characterize the pathomechanisms of selected pathogenic MCT8 mutations in cell culture studies. The findings indicate that the diminished functionality of MCT8 mutants depends not only on a defect in substrate transport but also on a decreased cell surface expression of the proteins. These results and the observed cell type specific effects could help to explain the phenotypic variations among AHDS patients. The analysis of the substrate specificity of MCT8 using iodothyronine derivates shows that the L-amino acid side chain conformation and at least one iodine atom per aromatic ring are important for efficient substrate transport. Studies for the transport mechanism on MCT8 showed that both charged and conserved amino acids (R445 and D498) are involved in substrate recognition. This relationship was supported by a structural model for the MCT8 transporter: Both amino acids are flanking the potential substrate transport channel within the protein. In contrast to AHDS patients the loss of Mct8 in mice causes a milder phenotype. This difference may rely on species-specific expression profiles of alternative thyroid hormone transporters which are able to compensate the loss of Mct8 during brain development. Antidepressant drugs lead to a thyroid hormone deficiency in the central nervous system in rats. To address the underlying mechanism, substrate transport studies were performed for MCT8 in the presence of desipramine and a specific inhibition of the transporter could be demonstrated. This surprising result may be helpful to explain some of the central side effects of tricyclic antidepressant drugs in the future

Primary and secondary thyroid hormone transporters

Thyroid hormones (TH) are essential for the development of the human brain, growth and cellular metabolism. Investigation of TH transporters became one of the emerging fields in thyroid research after the discovery of inactivating mutations in the Monocarboxylate transporter 8 (MCT8), which was found to be highly specific for TH transport. However, additional transmembrane transporters are also very important for TH uptake and efflux in different cell types. They transport TH as secondary substrates and include the aromatic amino acid transporting MCT10, the organic anion transporting polypeptides (e.g. OATP1C1, OATP1A2, OPTP1A4) and the large neutral amino acid transporters (LAT1 and LAT2). These TH transporters characteristically possess 12 transmembrane spanners but due to the strong differing sequences between the three transporter families we assume an identical conformation is not very likely. In contrast to the others, the LAT family members form a heterodimer with the escort protein 4F2hc/CD98. A comparison of sequence proportions, locations and types of functional sensitive features for TH transport discovered by mutations, revealed that transport sensitive charged residues occur as conserved amino acids only within each family of the transporter types but not in all putative TH transporters. Based on the lack of highly conserved sensitive charged residues throughout the three transporter families as a common counterpart for the amino acid moiety of the substrates, we conclude that the molecular transport mechanism is likely organized either a) by different molecular determinants in the divergent transporter types or b) the counterparts for the substrates` amino acid moiety at the transporter are not any charged side chains but other proton acceptors or donators. However, positions of transport sensitive residues coincide at transmembrane helix 8 in the TH transporter MCT8, OATP1C1 and another amino acid transporter, the L-cystine and L-glutamate exchanger xCT, which is highly homologous to LAT1 and LAT2. Here we review the data available and compare similarities and differences between these primary and secondary TH transporters regarding sequences, topology, potential structures, trafficking to the plasma membrane, molecular features and locations of transport sensitive functionalities. Thereby, we focus on TH transporters occurring in the blood-brain barrier

Structural Insights Into Thyroid Hormone Transport Mechanisms of the L-Type Amino Acid Transporter 2

Thyroid hormones (THs) are transported across cell membranes by different transmembrane transporter proteins. In previous studies, we showed marked 3,3′-diiodothyronine (3,3′-T2) but moderate T3 uptake by the L-type amino acid transporter 2 (Lat2). We have now studied the structure-function relationships of this transporter and TH-like molecules. Our Lat2 homology model is based on 2 crystal structures of the homologous 12-transmembrane helix transporters arginine/agmatine antiporter and amino acid/polyamine/organocation transporter. Model-driven mutagenesis of residues lining an extracellular recognition site and a TH-traversing channel identified 9 sensitive residues. Using Xenopus laevis oocytes as expression system, we found that side chain shortening (N51S, N133S, N248S, and Y130A) expanded the channel and increased 3,3′-T2 transport. Side chain enlargements (T140F, Y130R, and I137M) decreased 3,3′-T2 uptake, indicating channel obstructions. The opposite results with mutations maintaining (F242W) or impairing (F242V) uptake suggest that F242 may have a gating function. Competitive inhibition studies of 14 TH-like compounds revealed that recognition by Lat2 requires amino and carboxylic acid groups. The size of the adjacent hydrophobic group is restricted. Bulky substituents in positions 3 and 5 of the tyrosine ring are allowed. The phenolic ring may be enlarged, provided that the whole molecule is flexible enough to fit into the distinctly shaped TH-traversing channel of Lat2. Taken together, the next Lat2 features were identified 1) TH recognition site; 2) TH-traversing channel in the center of Lat2; and 3) switch site that potentially facilitates intracellular substrate release. Together with identified substrate features, these data help to elucidate the molecular mechanisms and role of Lat2 in T2 transport

Insights into molecular properties of the human monocarboxylate transporter 8 by combining functional with structural information

<p>Abstract</p> <p>Background</p> <p>The monocarboxylate transporter 8 (MCT8) is a member of the major facilitator superfamily (MFS) and transports specificly iodothyronines. MCT8 mutations are the underlying cause of a syndrome of severe X-linked psychomotor retardation known as the Allan-Herndon-Dudley syndrome. This syndrome is characterized by abnormally high T3, low/normal T4 serum levels and slightly elevated serum TSH. To date, more than 25 pathogenic mutations in <it>hMCT8</it> are known and they are valuable indicators of important regions for structural and functional MCT8 properties.</p> <p>Methods</p> <p>We designed a structural human MCT8 model and studied reported pathogenic missense mutations with focus on the estimation of those amino acid positions which are probably sensitive for substrate transport. Furthermore, assuming similarities between determinants of T3 binding observed in the published crystal structure of the thyroid hormone receptor beta occupied by its ligand T3 and the structural MCT8 model, we explore potential T3 binding sites in the MCT8 substrate channel cavity.</p> <p>Results</p> <p>We found that all known pathogenic missense mutations are located exclusively in the transmembrane helices and to a high degree at conserved residues among the MCT family. Furthermore, mutations either of or to prolines/glycines are located mainly at helices 9-12 and are expected to cause steric clashes or structural misfolding. In contrast, several other mutations are close to the potential substrate channel and affected amino acids are likely involved in the switching mechanism between different transporter conformations. Finally, three potential substrate binding sites are predicted for MCT8.</p> <p>Conclusions</p> <p>Naturally occurring mutations of MCT8 provide molecular insights into protein regions important for protein folding, substrate binding and the switching mechanism during substrate transport. Future studies guided by this information should help to clarify structure-function relationships at MCT8 which may bear broader relevance for other members of the MCT family. This includes decoding of the complete set of transport-sensitive residue positions and description of structural re-arrangements during transport.</p

Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane

Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation

Transport of iodothyronines by human l-type amino acid transporters

Thyroid hormone (TH) transporters facilitate cellular TH influx and efflux, which is paramount for normal physiology. The L-type amino acid transporters LAT1 and LAT2 are known to facilitate TH transport. However, the role of LAT3, LAT4, and LAT5 is still unclear. Therefore, the aim of this study was to further characterize TH transport by LAT1 and LAT2 and to explore possible TH transport by LAT3, LAT4, and LAT5. FLAG-LAT1-5 constructs were transiently expressed in COS1 cells. LAT1 and LAT2 were cotransfected with the CD98 heavy chain. Cellular transport was measured using 10 nM 125I-labeled T4, T3, rT3, 3,3'-T2, and 10 μM [125I]3μ-iodotyrosine (MIT) as substrates. Intracellular metabolism of these substrates was determined in cells cotransfected with either of the LATs with type 1 or type 3 deiodinase. LAT1 facilitated cellular uptake of all substrates and LAT2 showed a net uptake of T3, 3,3μ-T2, and MIT. Expression of LAT3 or LAT4 did not affect transport of T4 and T3 but resulted in the decreased cellular accumulation of 3,3μ-T2 and MIT. LAT5 did not facilitate the transport of any substrate. Cotransfection with LAT3 or LAT4 strongly diminished the cellular accumulation of 3,3μ-T2 and MIT by LAT1 and LAT2. These data were confirmed by metabolism studies. LAT1 and LAT2 show distinct preferences for the uptake of the different iodocompounds, whereas LAT3 and LAT4 specifically facilitate the 3,3μ-T2 and MIT efflux. Together our findings suggest that different sets of transporters with specific influx or efflux capacities may cooperate to regulate the cellular thyroid state