scholarly journals Histidines in Potential Substrate Recognition Sites Affect Thyroid Hormone Transport by Monocarboxylate Transporter 8 (MCT8)

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2553-2561 ◽  
Author(s):  
Doreen Braun ◽  
Iva Lelios ◽  
Gerd Krause ◽  
Ulrich Schweizer
Keyword(s):  
Thyroid Hormone ◽  
Ring Structure ◽  
Psychomotor Retardation ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Monocarboxylate Transporter ◽  
Transport Channel ◽  
Hormone Transport ◽  
Bonding Properties ◽  
Major Facilitator

Abstract Mutations in monocarboxylate transporter 8 (MCT8; SLC16A2) cause the Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation syndrome. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and transports thyroid hormones across the blood-brain barrier and into neurons. How MCT8 distinguishes thyroid hormone substrates from structurally closely related compounds is not known. The goal of this study was to identify critical amino acids along the transport channel cavity, which participate in thyroid hormone recognition. The fact that T3 is bound between a His-Arg clamp in the crystal structure of the T3 receptor/T3 complex prompted us to investigate whether such a motif might potentially be relevant for T3 recognition in MCT8. We therefore replaced candidate histidines and arginines by site-directed mutagenesis and performed activity assays in MDCK-1 cells and Xenopus oocytes. Histidines were replaced by alanine, phenylalanine, and glutamine to probe for molecular properties like aromatic ring structure and H-bonding properties. It was found that some mutations in His192 and His415 significantly changed substrate transport kinetics. Arg301 at the intracellular end of the substrate channel is at an ideal distance to His415 to participate in a His-Arg clamp and mutation to alanine-abrogated hormone transport. Molecular modeling demonstrates a perfect fit of T3 poised into the substrate channel between His415 and Arg301 and observing the same geometry as in the T3 receptor.

scholarly journals Importance of His192 in the Human Thyroid Hormone Transporter MCT8 for Substrate Recognition

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2525-2532 ◽  
Author(s):  
Stefan Groeneweg ◽  
Elaine C. Lima de Souza ◽  
W. Edward Visser ◽  
Robin P. Peeters ◽  
Theo J. Visser
Keyword(s):  
Thyroid Hormone ◽  
Transmembrane Domain ◽  
Substrate Recognition ◽  
Inhibitory Effect ◽  
Psychomotor Retardation ◽  
Site Directed Mutagenesis ◽  
Monocarboxylate Transporter ◽  
Human Thyroid ◽  
Extracellular Loop

Abstract Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). So far, functional domains within MCT8 are not well defined. Mutations in MCT8 result in severe psychomotor retardation due to impaired neuronal differentiation. One such mutation concerns His192 (H192R), located at the border of transmembrane domain (TMD) 1 and extracellular loop (ECL) 1, suggesting that this His residue is important for efficient TH transport. Here, we studied the role of different His residues, predicted within TMDs or ECLs of MCT8, in substrate recognition and translocation. Therefore, we analyzed the effects of the His-modifying reagent diethylpyrocarbonate (DEPC) and of site-directed mutagenesis of several His residues on TH transport by MCT8. Reaction of MCT8 with DEPC inhibited subsequent uptake of T3 and T4, whereas T3 and T4 efflux were not inhibited. The inhibitory effect of DEPC on TH uptake was prevented in the presence of T3 or T4, suggesting that TH blocks access to DEPC-sensitive residues. Three putative DEPC target His residues were replaced by Ala: H192A, H260A, and H450A. The H260A and H450A mutants showed similar TH transport and DEPC sensitivity as wild-type MCT8. However, the H192A mutant showed a significant reduction in TH uptake and was insensitive to DEPC. Taken together, these results indicate that His192 is sensitive to modification by DEPC and may be located close to a putative substrate recognition site within the MCT8 protein, important for efficient TH uptake.

scholarly journals Importance of Cysteine Residues in the Thyroid Hormone Transporter MCT8

Endocrinology ◽  
2013 ◽  
Vol 154 (5) ◽  
pp. 1948-1955 ◽  
Author(s):  
Elaine C. Lima de Souza ◽  
Stefan Groeneweg ◽  
W. Edward Visser ◽  
Robin P. Peeters ◽  
Theo J. Visser
Keyword(s):  
Thyroid Hormone ◽  
Double Mutant ◽  
Substrate Recognition ◽  
Psychomotor Retardation ◽  
Site Directed Mutagenesis ◽  
Time Dependent ◽  
Monocarboxylate Transporter ◽  
Double Mutation ◽  
Functional Roles ◽  
Specific Reagent

Abstract The thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) is crucial for brain development as demonstrated by the severe psychomotor retardation in patients with MCT8 mutations. MCT8 contains 10 residues of the reactive amino acid cysteine (Cys) whose functional roles were studied using the Cys-specific reagent p-chloromercurybenzenesulfonate (pCMBS) and by site-directed mutagenesis. Pretreatment of JEG3 cells with pCMBS resulted in a dose- and time-dependent decrease of subsequent T3 uptake. Pretreatment with dithiothreitol did not affect TH transport or its inhibition by pCMBS. However, pCMBS inhibition of MCT8 was reversed by dithiothreitol. Inhibition of MCT8 by pCMBS was prevented in the presence of T3. The single and double mutation of C481A and C497A did not affect T3 transport, but the single mutants were less sensitive and the double mutant was completely insensitive to pCMBS. Similar effects on MCT8 were obtained using HgCl2 instead of pCMBS. In conclusion, we have identified Cys481 and Cys497 in MCT8 as the residues modified by pCMBS or HgCl2. These residues are probably located at or near the substrate-recognition site in MCT8. It remains to be investigated whether MCT8 function is regulated by modification of these Cys residues under pathophysiological conditions.

scholarly journals Few Amino Acid Exchanges Expand the Substrate Spectrum of Monocarboxylate Transporter 10*

2016 ◽  
Vol 30 (7) ◽  
pp. 796-808 ◽  
Author(s):  
Jörg Johannes ◽  
Doreen Braun ◽  
Anita Kinne ◽  
Daniel Rathmann ◽  
Josef Köhrle ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aromatic Amino Acids ◽  
Homology Model ◽  
Amino Acid Identity ◽  
Psychomotor Retardation ◽  
Major Facilitator Superfamily ◽  
Monocarboxylate Transporter ◽  
Amino Acid Substitutions ◽  
Major Facilitator

Monocarboxylate transporters (MCTs) belong to the SLC16 family within the major facilitator superfamily of transmembrane transporters. MCT8 is a thyroid hormone transporter mutated in the Allan-Herndon-Dudley syndrome, a severe psychomotor retardation syndrome. MCT10 is closely related to MCT8 and is known as T-type amino acid transporter. Both transporters mediate T3 transport, but although MCT8 also transports rT3 and T4, these compounds are not efficiently transported by MCT10, which, in contrast, transports aromatic amino acids. Based on the 58% amino acid identity within the transmembrane regions among MCT8 and MCT10, we reasoned that substrate specificity may be primarily determined by a small number of amino acid differences between MCT8 and MCT10 along the substrate translocation channel. Inspecting the homology model of MCT8 and a structure-guided alignment between both proteins, we selected 8 amino acid positions and prepared chimeric MCT10 proteins with selected amino acids changed to the corresponding amino acids in MCT8. The MCT10 mutant harboring 8 amino acid substitutions was stably expressed in Madin-Darby canine kidney 1 cells and found to exhibit T4 transport activity. We then successively reduced the number of amino acid substitutions and eventually identified a minimal set of 2–3 amino acid exchanges which were sufficient to allow T4 transport. The resulting MCT10 chimeras exhibited KM values for T4 similar to MCT8 but transported T4 at a slower rate. The acquisition of T4 transport by MCT10 was associated with complete loss of the capacity to transport Phe, when Tyr184 was mutated to Phe.

scholarly journals Minireview: Thyroid Hormone Transporters: The Knowns and the Unknowns

2011 ◽  
Vol 25 (1) ◽  
pp. 1-14 ◽  
Author(s):  
W. Edward Visser ◽  
Edith C. H. Friesema ◽  
Theo J. Visser
Keyword(s):  
Thyroid Hormone ◽  
Organic Anion ◽  
Psychomotor Retardation ◽  
Cellular Level ◽  
Monocarboxylate Transporter ◽  
Causative Role ◽  
Organic Anion Transporting Polypeptide ◽  
Neurological Abnormalities ◽  
Knockout Animals

The effects of thyroid hormone (TH) on development and metabolism are exerted at the cellular level. Metabolism and action of TH take place intracellularly, which require transport of the hormone across the plasma membrane. This process is mediated by TH transporter proteins. Many TH transporters have been identified at the molecular level, although a few are classified as specific TH transporters, including monocarboxylate transporter (MCT)8, MCT10, and organic anion-transporting polypeptide 1C1. The importance of TH transporters for physiology has been illustrated dramatically by the causative role of MCT8 mutations in males with psychomotor retardation and abnormal serum TH concentrations. Although Mct8 knockout animals have provided insight in the mechanisms underlying parts of the endocrine phenotype, they lack obvious neurological abnormalities. Thus, the pathogenesis of the neurological abnormalities in males with MCT8 mutations is not fully understood. The prospects of identifying other transporters and transporter-based syndromes promise an exciting future in the TH transporter field.

scholarly journals Structural and Functional Study of the Phenicol-Specific Efflux Pump FloR Belonging to the Major Facilitator Superfamily

2005 ◽  
Vol 49 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
Martine Braibant ◽  
Jacqueline Chevalier ◽  
Elisabeth Chaslus-Dancla ◽  
Jean-Marie Pagès ◽  
Axel Cloeckaert
Keyword(s):  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Efflux Transporters ◽  
Chloramphenicol Resistance ◽  
Active Efflux ◽  
Transmembrane Segments ◽  
Efflux Mechanism ◽  
Major Facilitator

ABSTRACT The florfenicol-chloramphenicol resistance gene floR from Salmonella enterica was previously identified and postulated to belong to the major facilitator (MF) superfamily of drug exporters. Here, we confirmed a computer-predicted transmembrane topological model of FloR, using the phoA gene fusion method, and classified this protein in the DHA12 family (containing 12 transmembrane domains) of MF efflux transporters. We also showed that FloR is a transporter specific for structurally associated phenicol drugs (chloramphenicol, florfenicol, thiamphenicol) which utilizes the proton motive force to energize an active efflux mechanism. By site-directed mutagenesis of specific charged residues belonging to putative transmembrane segments (TMS), two residues essential for active efflux function, D23 in TMS1 and R109 in TMS4, were identified. Of these, the acidic residue D23 seems to participate directly in the affinity pocket involved in phenicol derivative recognition. A third residue, E283 in TMS9, seems to be necessary for correct membrane folding of the transporter.

Intermittent Esotropia in 4 Patients With Allan-Herndon-Dudley Syndrome

2018 ◽  
Vol 33 (8) ◽  
pp. 525-527 ◽  
Author(s):  
Cole J. Swiston ◽  
David L. Nash
Keyword(s):  
Cognitive Impairment ◽  
Thyroid Hormone ◽  
Clinical Finding ◽  
Nonsense Mutation ◽  
Monocarboxylate Transporter ◽  
Spastic Paraplegia ◽  
Severe Cognitive Impairment ◽  
Hormone Transport ◽  
Ocular Findings

Allan-Herndon-Dudley syndrome is a rare X-linked neurologic condition caused by mutations in monocarboxylate transporter 8 ( MCT8), which leads to deficient thyroid hormone transport. Typical features include severe cognitive impairment, truncal hypotonia, spastic paraplegia, weakness, and speech difficulties. Minimal literature exists describing the ocular findings in patients with Allan-Herndon-Dudley syndrome. We describe 4 male siblings affected with Allan-Herndon-Dudley syndrome with a novel nonsense mutation (Q90X) in the MCT8 protein. All affected siblings presented with classic findings of Allan-Herndon-Dudley syndrome, and each of the siblings also developed intermittent esotropia. This group of affected siblings represents the first consistent documentation of strabismus in Allan-Herndon-Dudley syndrome, suggesting a possible association between this clinical finding and the neurologic syndrome.

scholarly journals Evidence for a Homodimeric Structure of Human Monocarboxylate Transporter 8

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 5163-5170 ◽  
Author(s):  
W. Edward Visser ◽  
Nancy J. Philp ◽  
Thamar B. van Dijk ◽  
Wim Klootwijk ◽  
Edith C. H. Friesema ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Psychomotor Retardation ◽  
Monocarboxylate Transporter ◽  
Membrane Association ◽  
Transport Function ◽  
Wild Type ◽  
Transfected Cells ◽  
Physiological Relevance ◽  
Dimerization Process

The human monocarboxylate transporter 8 (hMCT8) protein mediates transport of thyroid hormone across the plasma membrane. Association of hMCT8 mutations with severe psychomotor retardation and disturbed thyroid hormone levels has established its physiological relevance, but little is still known about the basic properties of hMCT8. In this study we present evidence that hMCT8 does not form heterodimers with the ancillary proteins basigin, embigin, or neuroplastin, unlike other MCTs. In contrast, it is suggested that MCT8 exists as monomer and homodimer in transiently and stably transfected cells. Apparently hMCT8 forms stable dimers because the complex is resistant to denaturing conditions and dithiothreitol. Cotransfection of wild-type hMCT8 with a mutant lacking amino acids 267–360 resulted in formation of homo-and heterodimers of the variants, indicating that transmembrane domains 4–6 are not involved in the dimerization process. Furthermore, we explored the structural and functional role of the 10 Cys residues in hMCT8. All possible Cys>Ala mutants did not behave differently from wild-type hMCT8 in protein expression, cross-linking experiments with HgCl2 and transport function. Our findings indicate that individual Cys residues are not important for the function of hMCT8 or suggest that hMCT8 has other yet-undiscovered functions in which cysteines play an essential role.

scholarly journals Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8

2010 ◽  
Vol 285 (36) ◽  
pp. 28054-28063 ◽  
Author(s):  
Anita Kinne ◽  
Gunnar Kleinau ◽  
Carolin S. Hoefig ◽  
Annette Grüters ◽  
Josef Köhrle ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Monocarboxylate Transporter ◽  
Hormone Transport ◽  
Molecular Determinants

scholarly journals Molecular aspects of thyroid hormone transporters, including MCT8, MCT10, and OATPs, and the effects of genetic variation in these transporters

2009 ◽  
Vol 44 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Wendy M van der Deure ◽  
Robin P Peeters ◽  
Theo J Visser
Keyword(s):  
Genetic Variation ◽  
Thyroid Hormone ◽  
Organic Anion ◽  
Elevated Serum ◽  
Tissue Level ◽  
Psychomotor Retardation ◽  
Monocarboxylate Transporter ◽  
Organic Anion Transporting Polypeptides ◽  
The Central Nervous System ◽  
Molecular Aspects

Thyroid hormone is a pleiotropic hormone with widespread biological actions. For instance, adequate levels of thyroid hormone are critical for the development of different tissues such as the central nervous system, but are also essential for the regulation of metabolic processes throughout life. The biological activity of thyroid hormone depends not only on serum thyroid hormone levels, but is also regulated at the tissue level by the expression and activity of deiodinases, which activate thyroid hormone or mediate its degradation. In addition, thyroid hormone transporters are necessary for the uptake of thyroid hormone into target tissues. With the discovery of monocarboxylate transporter 8 (MCT8) as a specific thyroid hormone transporter and the finding that mutations in this transporter lead to a syndrome of severe psychomotor retardation and elevated serum 3,3′,5-tri-iodothyronine levels known as the Allan–Herndon–Dudley syndrome, the interest in this area of research has greatly increased. In this review, we will focus on the molecular aspects of thyroid hormone transporters, including MCT8, MCT10, organic anion transporting polypeptides, and the effects of genetic variation in these transporters.

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