Analysis of cellular localization and function of carboxy-terminal mutants of pendrin

Abstract

Background: Iodide uptake at the basolateral membrane and iodide efflux at the apical membrane of thyrocytes, essential steps in the biosynthesis of thyroid hormone, are stimulated by thyroid stimulating hormone (TSH). Pendrin (SLC26A4) is inserted into the apical membrane of thyrocytes and thought to be involved in mediating iodide efflux.

Methods: We determined the effects of carboxy-terminal mutations of pendrin on the cellular localization and the ability to transport iodide.

Results: After exposure to forskolin, the membrane abundance of wild type pendrin and iodide efflux increase. Truncation mutants lead to complete intracellular retention. Elimination of the distal part of the sulfate transporter and antisigma factor antagonist (STAS) domain with retention of the putative protein kinase A (PKA) phosphorylation site (RKDT 714-717) results in residual membrane insertion and a partial loss of function. Deletion of the PKA site results in decreased basal function and membrane insertion and abolishes the response to forskolin.

Conclusion: Pendrin membrane abundance and its ability to mediate iodide efflux increase after activation of the PKA pathway. Elimination of the PKA site abolishes the response to forskolin but partial basal function and membrane insertion are maintained.

Fig. 1

Secondary structure of pendrin and schematic presentation of the carboxy-terminal truncation mutants. (A) Current model of the secondary structure of the pendrin protein. Y = putative N-glycosylation sites, STAS = sulfate transporter and antisigma factor antagonist domain. (B) Schematic presentation of the carboxy-terminal truncation mutants of pendrin. TM1, transmembrane helix 1; TM12, transmembrane helix 12; PKA, putative phosphokinase A phosphorylation site. TM numbering based on Uniprot (http://www.uniprot.org/uniprot/O43511), STAS domain based on Aravind et al. [16].

Fig. 2

Intracellular iodide content of TSA cells expressing wild type and mutant pendrin proteins. A) Function of wild type and mutant pendrin proteins was determined by assessing intracellular iodide content using radiolabeled iodide. Cells were analyzed without forskolin treatment (white bars) or after exposure to forskolin (black bars); the forskolin treatment was performed without blocking NI S with perchlorate. TSA cells transiently expressing NIS alone show a 82% and 90% increase (p <0.05) in intracellular iodide content in the absence and in the presence of forskolin respectively, compared with control cells transfected with an empty vector (-FSK vs. +FSK p <0.05). Cotransfection of NIS and PDS leads to a significant decrease in iodide content in the absence and in the presence of forskolin (47% and 71%, respectively, p <0.001). Cotransfection of the previously characterized loss-of-function mutant L676Q and NIS does not result in a decrease in intracellular iodide content under both conditions, and serves as a positive control. Cells coexpressing NIS and a pendrin mutant with deletion of the distal STAS domain and an intact PKA site (ASTAS_dist/PKA) show a 28% decrease (p <0.001) in intracellular iodide content compared to cells expressing NIS in the absence and in the presence of forskolin, indicating that the mutant retains a partial ability to mediate iodide efflux but that it does not respond to forskolin. Contransfection of NIS and the PDS T717A mutant results in a 43% and 38% decrease in intracellular iodide content in the absence and in the presence of forskolin, respectively (-FSK vs. +FSK p >0.05), compared to cells expressing NIS only, suggesting that the mutant is partially functional but that it has a mitigated response to forskolin; the slight increase in intracellular iodide may be mediated by stimulation of NIS, which was not blocked by perchlorate. B) For comparison to cells transfected with NIS, the bars indicating the iodide accumulation in cells transfected with NIS are reproduced from panel 2A. Cells coexpressing the three truncation mutants (PDS 1-531+NIS; PDS 1-673+NIS; PDS 1-712+NIS) do not show a decrease (p >0.05) in intracellular iodide amount compared to cells expressing NIS only, indicating that these mutants lose their ability to mediate iodide transport. Cotransfection of NIS and the shortest truncation mutant 1-729 containing the putative PKA phosphorylation site, results in a 30% and 25% decrease in intracellular iodide accumulation in the absence and in the presence of forskolin, respectively (-FSK vs. +FSK p >0.05), but the reduction is significantly lower compared to the wild type (1-729 versus NIS/PDS: p<0.001; 1-729+FSK versus NIS/PDS+FSK: p<0.001). This suggests that the mutant retains a partial ability to mediate iodide efflux, but that it is unable to respond to stimulation by the PKA pathway. Data shown here are representative of at least three independent experiments. Values are the means of triplicates ± SEM.

Fig. 3

Subcellular distribution of the EGFP-tagged wild type pendrin in living PtK2 cells. PtK2 cells transiently expressing EGFP-tagged wild type pendrin were imaged using time-lapse confocal microscopy. Cells transfected with pEGFP empty plasmid served as control. In the absence of forskolin, the majority of the wild type pendrin is retained in intracellular compartments. Within 15 minutes after adding forskolin, the membrane abundance increases with a pattern that is suggestive for the formation of protein clusters within the lamellipodial protrusions of the cell (indicated by arrows). This pattern is observed for one hour following treatment with forskolin. These results suggest that plasma membrane targeting of pendrin is mediated, in part, by the PKA pathway.

Fig. 4

Subcellular distribution of the EGFP-tagged pendrin truncation mutants 1-531 and 1-673 in living PtK2 cells. PtK2 cells transiently expressing EGFP-tagged pendrin truncation mutants were imaged using time-lapse confocal microscopy. In the absence of forskolin, the majority of the truncation mutants 1-531 and 1-673 remain in intracellular compartments. The subcellular distribution of the truncation mutants remains unchanged following treatment with forskolin. These results indicate that these mutants fail to reach the plasma membrane following activation of the PKA pathway.

Fig. 5

Subcellular distribution of the EGFP-tagged pendrin truncation mutants 1-712 and 1-729 in living PtK2 cells. PtK2 cells transiently expressing EGFP-tagged pendrin truncation mutants were imaged using time-lapse confocal microscopy. In the absence of forskolin, the majority of the truncation mutants 1-712 and 1-729 remain in intracellular compartments. In the presence of forskolin, there is no visible change in membrane insertion. In the functional assay (Fig. 2B), 1-729 retains a partial basal function but does not respond to forskolin.

Fig. 6

Subcellular distribution of the EGFP-tagged pendrin mutant containing a modified putative PKA phosphorylation site (PDS T717A) and the mutant lacking the distal STAS domain but containing an intact PKA site and carboxy-terminus (ASTAS_dist/PKA) in living PtK2 cells. PtK2 cells transiently expressing the EGFP-tagged pendrin mutants were imaged using time-lapse confocal microscopy. In the absence of forskolin, the majority of the mutants PDS T717A and the ASTAS_dist/PKA are located in intracellular compartments. In the functional assay both mutants have a partially retained function, although significantly reduced compared to the wild type (Fig. 2A). In the presence of forskolin, there is no visible change in membrane insertion, which

Fig. 7

Cellular localization of wild type pendrin and mutants 1-531, 1-673, 1-712, 1-729, T717A, and ASTAS_dist/PKA. Twenty-four hours after transfection with the EGFP-tagged wild type and mutant pendrin proteins, CPAE cells were stained with an anti-calreticulin antibody. A significant amount of wild type pendrin showed ER localization whereas a small portion of the protein was present at the plasma membrane (shown by arrowheads). All mutant proteins showed predominant ER localization, as demonstrated by colocalization of the proteins with calreticulin.

Fig. 8

Alignment of the carboxy-terminal regions including the STAS domains of human SLC26A transporters. The carboxy-terminal STAS domains of 10 human SLC26A transporters were aligned using T-coffee and then shaded using the Boxshade program. The predicted domain boundaries of the SLC26A4 STAS domain according to Aravind et al. (residues 535 to 573 and 654 to 729) [16] are boxed. The asterisk indicates the position of known mutations causing Pendred syndrome and EVA, found in the SLC26A4 STAS domain.