Endocrine Abstracts

Objectives: The canonical function of thyroid hormone receptors (TRs) as mediators of thyroid hormone action on target gene expression is well known. However, the physiological function of the thyroid hormone receptor α splice variant TRα2, that does not bind thyroid hormones, remains elusive. Initial studies addressing the function of TRα2 indicated that it might act as a TRα1 antagonist, but the mechanisms underlying the dominant-negative activity of TRα2 are poorly understood. Further functional studies to characterize TRα2 have been impaired by the lack of reliable isoform-specific antibodies.

Methods and Results: In order to allow specific detection of TRα2, we added different fusion tags to the C-termini of TRα1 and TRα2. To gain first insights into isoform-specific actions, we initially used ectopically expressed TRα isoforms in HEK293 cells for Co-immunoprecipitation (Co-IP) followed by mass spectrometry to obtain isoform-specific protein interactions. By this, we could identify several binding partners interacting with both isoforms. Moreover, in subsequent Co-IP experiments we gained insights into homo- and heterodimerization properties of TRα2 and identified the C-terminal part of TRα2 as the main protein-binding domain by using different TRα1 and 2 protein fragments. Currently, we are investigating whether the interaction of TRα2 with selected binding partners exert T3-independent transcriptional activities and if these functional protein complexes explain the dominant-negative activity described for TRα2.

Conclusions and Outlook: Our preliminary data suggest that TRα2 shares several binding partners with TRα1, and, in contrast to TRα1, thyroid hormones do not affect complex formation of TRα2 with other proteins. We will next further dissect the effect of TRα2 and its binding partners on TRα1 action and expand our analyses to other e.g. cardiac and neuronal cell types. Moreover, we are currently finalizing the generation of induced pluripotent stem cells expressing endogenously tagged TRα isoforms generated by a CRISPR/Cas9-based approach. IPSCs will be differentiated into selected cell types for further analyses to obtain isoform-specific protein interactions and genome-wide DNA binding profiles of the endogenous receptors. Combining these data sets will give us new insights into tissue-specific activities of TRα2 and its role in thyroid hormone action.