Critical role of glutamine 252 in the hormone-dependent transcriptional activity of the thyroid hormone beta1 nuclear receptor.

To understand the molecular basis of the ligand-dependent transcriptional activity of thyroid hormone nuclear receptors (TRs), we investigated the effect of mutation of glutamine 252 (Q252) on the function of human TR subtype beta1 (wTRbeta1). Q252 is conserved in TRs in all species and is located in a region of the hormone binding domain that has been shown to undergo 3,3',5-triiodo-L-thyronine (T3) induced conformational changes. Q252 was mutated to Gly (Q252G) or Asn (Q252N) and their immunoreactivity, hormone, and DNA binding activities were characterized. Mutants Q252G and Q252N bound to T3 with similar affinity as the wTRbeta1. However, they failed to interact with a monoclonal anti-wTRbeta1 antibody whose epitope is located in the region of amino acids 248-256, suggesting that mutation of Q252 to Gly or Asn resulted in local structural alteration in TRbeta1. In addition, mutation of Glu to Gly or Asn led to increases in their binding to the thyroid hormone response elements (TREs) as homodimers and as heterodimers with the retinoid X receptor. Mutants Q252G and Q252N were more effective as repressors in the absence of T3, while both had a 1.5-2-fold higher T3-dependent transcriptional activity mediated by three TREs than the wTRbeta1. The increases in the transcriptional activity were not due to an increase in the expression of the mutant receptor proteins because the in vivo expression level of the mutant receptor proteins was identical to that of the wTRbeta1. Our data indicate that the T3-dependent transcriptional activity is not entirely dependent on the T3 binding activity of the receptor. The interplay of ligand and DNA binding domains plays a pivotal role in the transcriptional activity of the TRs.