Identification of Monocarboxylate Transporter 8 as a Specific Thyroid Hormone Transporter

Edith C.H Friesema,Sumita Ganguly,Amal Abdalla,Jocelyn E.Manning Fox,Andrew P Halestrap,Theo J Visser

doi:10.1074/jbc.m300909200

Edith C.H Friesema, Sumita Ganguly + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m300909200

Copy DOI

Abstract

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.

Highlights

Transport of thyroid hormone across the cell membrane is required for its action and metabolism
MCT8 cRNA induced an ϳ10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3؅,5-triiodothyronine (T3), 3,3؅,5؅-triiodothyronine and 3,3؅-diiodothyronine
The MCT8 gene consists of 6 exons coding for a protein with 12 putative transmembrane domains

Summary

EXPERIMENTAL PROCEDURES

Materials—Nonradioactive L-iodothyronines, 3,3Ј,5-triiodothyroacetic acid (Triac) and N-bromoacetyl-3,3Ј,5-triiodothyronine (BrAcT3) were obtained from Henning, Berlin. Insertion of a FLAG-tagged MCT8 Construct into the Oocyte Expression Vector pGEM-HeJuel—For expression in oocytes it was decided to append an 8-amino acid FLAG epitope to the C terminus of MCT8 to allow detection of expression by immunofluorescence microscopy and Western blotting For this purpose the stop codon of MCT8 was removed by performing PCR using the same sense primer as above, but with the antisense primer, 5Ј-ACAGCGGCCGCAAATGGGCTCTTCAGGTGTTG-3Ј, which lacks the stop codon. Transport was tested by incubation of groups of 5–10 oocytes for 2– 60 min at 25 °C with 10 nM 125I-labeled iodothyronines or 10 ␮M 3H-labeled amino acids in 0.1 ml of standard uptake solution. Transport Kinetics—Saturation of iodothyronine uptake in MCT8 cRNA-injected oocytes was analyzed in incubations containing labeled and unlabeled T4, T3, or rT3 at final concentrations of 1 nM–30 ␮M. Statistical significance was determined using the Student’s t test for unpaired observations

RESULTS

TABLE I

DISCUSSION