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Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

ABSTRACT: BACKGROUND: Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. EXPERIMENTAL APPROACH: Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. RESULTS: In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. CONCLUSIONS: Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

SUBMITTER: Sasaki S

PROVIDER: S-EPMC3729688 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

Sasaki Shotaro S Kobayashi Masaki M Futagi Yuya Y Ogura Jiro J Yamaguchi Hiroaki H Takahashi Natsuko N Iseki Ken K

PloS one 20130731 7

<h4>Background</h4>Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport fun ...[more]

PMID: 23935841

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Project description:Hypoxic regions are frequent in glioblastoma (GBM), the most common type of malignant adult brain tumor, and increased levels of tumor hypoxia have been associated with worse clinical outcomes. To unmask genes important in hypoxia, we treated GBM neurospheres in hypoxia and identified monocarboxylate transporter-4 (MCT4) as one of the most upregulated genes. To investigate the clinical importance of MCT4 in GBM, we examined clinical outcomes and found that MCT4 overexpression is associated with shorter patient survival. Consistent with this, MCT4 upregulation correlated with the aggressive mesenchymal subset of GBM, and MCT4 downregulation correlated with the less aggressive G-CIMP (Glioma CpG Methylator Phenotype) subset of GBM. Immunohistochemical analysis of tissue microarrays confirmed that MCT4 protein levels were increased in high-grade as compared with lower-grade astrocytomas, further suggesting that MCT4 is a clinically relevant target. To test the requirement for MCT4 in vitro, we transduced neurospheres with lentiviruses encoding short-hairpin RNAs (shRNAs) against MCT4, resulting in growth inhibition of 50-80% under hypoxia in two lines. MCT4 knockdown was associated with a decreased percentage of cells expressing the stem-cell marker CD133 and increased apoptotic fraction. We also found that flow-sorted CD133-positive cells had almost sixfold higher MCT4 levels than CD133-negative cells, suggesting that the stem-like population might have a greater requirement for MCT4. Most importantly, MCT4 silencing also slowed GBM intracranial xenograft growth in vivo. Interestingly, whereas MCT4 is a well-characterized lactate exporter, we found that both intracellular and extracellular lactate levels did not change following MCT4 silencing, suggesting a novel lactate export-independent mechanism for growth inhibition in GBMs. To identify this potential mechanism, we performed microarray analysis on control and shMCT4-expressing neurospheres and found a dramatic reduction in the expression of multiple Hypoxia-Inducible Factor (HIF)-regulated genes following MCT4 knockdown. The overall reduction in HIF transcriptional response was further validated using a hypoxia response element (HRE)-dependent green-fluorescent protein (GFP) reporter line.

Dataset Information

Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

Publications

Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

Similar Datasets

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