Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxylate transporter 8

Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe Xlinked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carriermediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. In order to gain insight into structure-function relations in TH transport, we chose human MCT8 as paradigm. We systematically performed conventional and liquid chromatographytandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for Liodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tertraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T3 transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate
interaction. Indeed, alanine mutation of either Arg445 (helix 8) or Asp498 (helix 10) abrogated T3 transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon- Dudley syndrome.