Effective cellular uptake and efflux of thyroid hormone by human monocarboxylate transporter 10

Abstract

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[(125)I]T(3), 3) a marked stimulation of cellular T(4) and, particularly, T(3) uptake, 4) a significant inhibition of T(3) uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T(4) and T(3) by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein micro-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T(4) and T(3). In the absence of CRYM, hMCT10 and hMCT8 increased T(3) uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T(4) than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Fig. 1.

Alignment of the Amino Acid Sequences of hMCT8 and hMCT10 Identical amino acids occupying corresponding positions in these proteins are indicated in red. The putative 12 TMDs are indicated by blue shading.

Fig. 2.

Phylogenetic Tree of the MCT8/MCT10 Protein Family The construction of the tree is based on amino acid sequences from the following species: hs, Homo sapiens (human); rn, Rattus norvegicus (rat); md, Monodelphis domestica (opossum); gg, Gallus gallus (chicken); xt, Xenopus tropicalis (frog); dr, Danio rerio (zebrafish); tr, Takifugu rubripes (pufferfish); gmm, Glossina morsitans morsitans (tsetse fly); dm, Drosophila melanogaster (fruitfly); ag, Anopheles gambiae (mosquito); am, Apis mellifera (honey bee); tc, Tribolium castaneum (beetle); bm, Bombyx mori (silkworm)

Fig. 3.

Immunoblot of hMCT8 and hMCT10 Protein in Transfected COS1 Cells Left, Staining with polyclonal anti-hMCT10 antibody 1758. Specific bands of approximately 55 and 240 kDa are detected in hMCT10 but not in hMCT8 transfected cells, irrespective of cotransfection with hCRYM. Right, Staining with polyclonal anti-hMCT8 antibody 1306. Specific bands of approximately 60 and 240 kDa are detected in hMCT8 but not in hMCT10 transfected cells, irrespective of cotransfection with hCRYM. Ab, Antibody.

Fig. 4.

Immunocytochemistry of COS1 Cells Transfected with hMCT10 Left panel, hMCT10 protein detected with polyclonal antibody 1758 and stained with goat antirabbit Alexa Fluor 488. Middle panel, Plasma membrane staining using antibody against tight junction protein ZO-1 and goat antimouse Alexa Fluor 633. Right panel, Merged images of hMCT10, plasma membrane marker, and nuclear marker (DAPI staining).

Fig. 5.

Affinity-Labeling of hMCT8, hMCT10, hCRYM, and/or rD1 (Co)transfected COS1 Cells with BrAc[¹²⁵I]T₃ A band of approximately 60 kDa is present in control plasmid-transfected cells. This signal is greatly increased in hMCT8- but not in hMCT10-transfected cells, irrespective of cotransfection with hCRYM or rD1. rD1-transfected cells show affinity labeling of a protein of approximately 30 kDa, the appropriate size for rD1. Cotransfection of hMCT8 and hMCT10 increases labeling of rD1, indicating transport of BrAcT₃ by hMCT8 as well as hMCT10.

Fig. 6.

Uptake of T₃ and T₄ by COS1 Cells Transfected with hMCT8 or hMCT8 cDNA without or with hCRYM cDNA Cells were transfected with control cDNA (○), hMCT8 cDNA (•) or hMCT10 cDNA (▴) without (A and B) or with (C–F) hCRYM cDNA, and incubated for 5–30 min at 37 C with 1 nm [¹²⁵I]T₃ (A–D) or [¹²⁵I]T₄ (E and F) in DMEM/F12 (A, C, and E) or D-PBS medium (B, D, and F) containing 0.1% BSA. After incubation, cells were processed and cellular radioactivity was determined as described in Materials and Methods. Results are presented as means ± sd (n = 2–4).

Fig. 7.

Effects of hCRYM on Efflux of Cellular T₃ COS1 cells were transfected with control cDNA (○), hMCT8 cDNA (•), or hMCT10 cDNA (▴) without (A and C) or with (B and D) hCRYM cDNA, and incubated for 10 min at 37 C with 1 nm [¹²⁵I]T₃ in D-PBS medium containing 0.1% BSA. After brief washing, cells were incubated with fresh D-PBS medium plus 0.1% BSA without (A and B) or with (C and D) 10 μm unlabeled T₃. After incubation for 2–10 min at 37 C, cells were processed, and cellular radioactivity was determined as described in Materials and Methods. Results are presented as means ± sd (n = 2–4).

Fig. 8.

Effects of hCRYM on Efflux of Cellular T₄ COS1 cells were transfected with control cDNA (○), hMCT8 cDNA (•), or hMCT10 cDNA (▴) without (A and C) or with (B and D) hCRYM cDNA, and incubated for 10 min at 37 C with 1 nm [¹²⁵I]T₄ in D-PBS medium containing 0.1% BSA. After brief washing, cells were incubated with fresh D-PBS medium plus 0.1% BSA without (A and B) or with (C and D) 10 μm unlabeled T₄. After incubation for 2–10 min at 37 C, cells were processed and cellular radioactivity was determined as described in Materials and Methods. Results are presented as means ± sd (n = 2–4).

Fig. 9.

Uptake of Aromatic Amino Acids by COS1 Cells Transfected with Control cDNA (○), hMCT8 cDNA (•), or hMCT10 cDNA (▴) Cells were incubated for 0.5–30 min at 37 C with 10 μm [³H]Phe (A), [³H]Tyr (B), or [³H]Trp (C) in D-PBS medium without BSA. After incubation, cells were processed and cellular radioactivity was determined as described in Materials and Methods. Results are presented as means ± sd from a representative experiment.

Fig. 10.

Influence of Aromatic Amino Acids on the Uptake of T₃ COS1 cells were transfected with hMCT8 (A) or hMCT10 (B) cDNA and incubated for 10 min with 1 nm [¹²⁵I]T₃ in D-PBS medium containing 0.1% BSA and with or without 1 mm Phe, Tyr, or Trp. Results are presented as uptake minus uptake from control transfected COS1 cells. Results are presented as means ± sd from a representative experiment.

Fig. 11.

Influence of Trp on the Efflux of Cellular T₃ COS1 cells were transfected with hMCT8 cDNA (A) or hMCT10 (B) cDNA without or with hCRYM cDNA and incubated for 10 min at 37 C with 1 nm [¹²⁵I]T₃ in D-PBS medium containing 0.1% BSA. After brief washing, cells were incubated with fresh D-PBS medium plus 0.1% BSA without (open symbols) or with (solid symbols) 1 mm Trp. After incubation for 2–10 min at 37 C, cells were processed and cellular radioactivity was determined as described in Materials and Methods. Results are presented as means ± sd (n = 3).

Fig. 12.

Effects of hMCT8 and hMCT10 on Intracellular Metabolism of T₃ and T₄ by hD3 COS1 cells were cotransfected with hD3 cDNA and control, hMCT8, or hMCT10 cDNA and incubated for 2 h or 24 h at 37 C with 1 nm [¹²⁵I]T₃ (A) or [¹²⁵I]T₄ (B), respectively, in DMEM/F12 medium containing 0.1% BSA. Metabolism of T₃ and T₄ was analyzed by HPLC as described in Materials and Methods. Results are the means of duplicate determinations from a representative experiment.