Modulation of monocarboxylate transporter 8 oligomerization by specific pathogenic mutations

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   Figure 1

   Structural MCT8 model in an inward-facing conformation occupied by substrate. This MCT8 homology model (backbone ribbon) represents a potential 3D-structure of MCT8 that is subdivided into two halves: domain A (beige) and domain B (white). The N- and C-termini are localized intracellularly and each domain is constituted by six helices that are in a mirror-like arrangement (A) and with center-axis symmetry (dashed line). In addition, we propose a specific binding mode of T3 inside the substrate-translocation channel (B), with particular amino acids participating in the interaction with T3 (cyan sticks). This putative docking position is based on a combination of structural/functional data and results from bioinformatic procedures (see Materials and methods section in the Supplementary Material). T3 is most probably bound between the two domains (B, inner surface representation), exactly at the symmetry axis between the domains and helices (C). Details of putative MCT8/T3 interactions are represented in D. Hydrophilic and charged amino acids interact with the carboxy group of T3, whereby the positively charged side-chain of Arg445 might be a key player. The negatively charged side-chain of Glu422 participates in binding of T3 close to the bottom of the substrate translocation channel in this conformation, and also presumptively interacts with Arg445 (H-bonds shown as dotted yellow lines). Both residues are conserved within the MCT group. Our newly generated model indicates that Asp498 and Lys418 should function as counteracting charged residues, whereby interactions with the substrate (intermediates) are assumed. Moreover, in this arrangement, the two histidines His192 and His415 participate in substrate transport. On the basis of the occurrence of pathogenic mutations and results from bioinformatic studies (Friesema et al. 2010, Kinne et al. 2010, Kleinau et al. 2011), histidine 192 has previously been suggested to be of great importance for substrate translocation, as has been recently confirmed by the results of experimental in vitro studies (Braun et al. 2013, Groeneweg et al. 2013). In addition, His415 has also been predicted (Kleinau et al. 2011) and confirmed to be a determinant of substrate translocation in MCT8 (Braun et al. 2013). Further residues that may participate in this proposed T3-binding pocket are amino acids with hydrophobic and aromatic side-chain properties, such as Ile534, Phe189, Phe316, or Tyr419. They cover and adjust the substrate, as is also observed for those residues in crystal structures of T3-binding proteins (see also Supplementary Material). In addition, Ser317, Asn193, and Glu426 should make significant contributions to parameters of binding, translocation, and substrate selectivity.
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   Figure 2

   MCT8 mutants with a capacity for homodimerization similar to that of the WT. The capacity for dimerization was tested by sandwich ELISA. COS-7 cells were transiently co-transfected with N-HA and C-FLAG WT MCT8 (MCT8 WT) or mutant MCT8. Single transfection of N-HA-tagged WT served as a negative control (CTRL, white column). No significant differences compared with the MCT8 WT could be shown for the mutations ins189I, A224V, R271H, L471P, L512P, G558D, and L568P (grey columns). The mean absorption (492 nm/620 nm) was normalized to mg/ml protein concentration and given as percentage of WT/WT homodimer formation (black column, absorption (492/620)/mg/ml of protein: 0.27±0.6). Data are depicted as means+s.e.m. from three independent experiments carried out in triplicate.
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   Figure 3

   Modulation of MCT8 homodimerization by pathogenic mutations. The capacity of mutated MCT8 transporters to form dimers compared with MCT8 WT was measured using a sandwich-ELISA approach. N-HA-tagged MCT8 WT is defined as the negative control (CTRL, white column). The co-transfection of N-HA-tagged MCT8 WT and C-FLAG-tagged MCT8 WT serves as a positive control (black column). The mutations del230F, V235M, and ins236V increased dimerization of MCT8 (grey columns with black lines), whereas mutations S194F, A224T, L434W, and R445C (white columns with black lines) diminished the capacity for dimerization. The mean absorption (492 nm/620 nm) was calculated per mg/ml protein of transiently transfected COS-7 cells and shown as a percentage of the value for the WT/WT homodimer (black column, absorption (492/620)/ mg/ml of protein: 0.27±0.6). Data were assessed from three independent experiments, each performed in triplicate and represent mean+s.e.m. Statistical significance, as assessed by unpaired t-test with Welch's correction, is denoted as **P<0.01 and ***P<0.001.
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   Figure 4

   Positions of investigated pathogenic mutations in a three-dimensional model of MCT8. The positions of the pathogenic substitutions investigated here (atom spheres) are mapped onto the structural MCT8 model (white backbone ribbon and inner surface of the transport channel). Substitutions inhibiting MCT8 oligomerization are indicated in red and are localized close to the middle section of the transport channel between domains A and B. It is assumed that during transport of substrate, this ‘bottle-neck’ of the closed channel is opened by helical re-arrangements. A few of these WT amino acids are known to be involved in both substrate transport and intramolecular interaction, such as Arg445 (Kinne et al. 2010, Capri et al. 2013). Therefore, the tri-functionality of particular WT amino acids is important for: i) binding of substrate, ii) participating in intramolecular interaction network(s), and iii) switching the global protomer conformation. Notably, while a few mutations do not modify oligomerization of MCT8 (white spheres), three MCT8 mutations lead to an increase in oligomerization, and are localized exclusively at a specific section of TMH2 (green spheres). Insertion, deletion, or residue substitution between positions 230 and 236 increased the capacity for dimerization. The increase in oligomeric MCT8 molecules as a result of modifications at TMH2 might be related to optimization of a shape-fit of two MCT8 protomers to one another, most probably in close vicinity to this specific MCT8 region.
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   Figure 5

   Distribution of MCT8 dimers in COS-7 cells. Confocal laser scanning micrographs of COS-7 cells expressing MCT8 WT (A, B and C), the del230F (D, E and F), and the A224T (G, H and I) dimers are shown as multiple- (A, D and G) or single-channel fluorescence micrographs (B, E and H), or depicted as overlays with the corresponding phase-contrast images (C, F and I). Fluorescence indicates bimolecular complementation through dimerization of MCT8–YFP1 and MCT8–YFP2 fragments (green signals in A, C, D, F, G, I and white signals in B, E, H). Nuclei were counter-stained with DAPI (cyan signals in A, D and G). Cell-surface and vesicular occurrence of oligomerized transporter molecules are indicated by arrows and arrowheads respectively (B and E). Note the well-spread phenotype of COS-7 cells expressing MCT8 WT and del230F oligomers in the rough endoplasmic reticulum, the peri-nuclear Golgi-apparatus, and in numerous vesicles in transit to and from the plasma membrane (A, B, C, D, E and F). In contrast, the A224T dimers revealed an altered morphology and featured large aggregates accumulating in the peri-nuclear region, resulting from the dense packing of the mis-folded transporter mutant forms. N denotes nuclei, scale bars represent 50 μm in A, D, G and 20 μm in B, C, E, F, H, I.

• © 2015 Society for Endocrinology