The rapid effects of thyroid hormone on the activity of Kv11.1 channels in rat pituitary cells were recently shown (Storey et al 2006) to be mediated by the classical nuclear receptor for thyroid hormone, TRbeta;, acting at the plasma membrane through the phosphoinositide 3 kinase (PI3K) and the Rac GTPase, which is a well-known effector of PIP3 dependent Rac exchange factors. Signaling was reconstituted in CHO cells by heterologous expression of human Kv11.1 channels and the human TRbeta, but not the TRalpha receptor. We have continued to investigate the mechanism of PI3K stimulation by TRbeta and the consequences of PI3K signaling for the physiological effects of thyroid hormone. We have used a fluorescent PIP3 binding domain from the Akt protein kinase coupled with cyan and yellow fluorecent proteins to detect PIP3 production by fluorescence resonance energy transfer (FRET) in live cells in real time. Fret signals are blocked by inhibition of TRbeta with the nuclear receptor antagonist,1-850, and by wortmannin, an active site inhibitor of PI3K. PIP3 production is also blocked completely by 10 min preexposure to 100 nM TCDD, an environmental toxicant. We have used immunoprecipitation to show that TRbeta associates with the regulatory p85 subunit of PI3K in the absence of ligand but dissociates in the presence of thyroid hormone. We have also observed rapid thyroid hormone-dependent phosphorylation of the Akt protein kinase and recruitment of Rac to the plasma membrane confirming that thyroid hormone stimulates PI3K-dependent effectors. Finally we have discovered that the Src family kinase, Lyn, is part of the signaling complex and identified its binding site with mass spectrometry. Other proteins interact with p85 through two Src homology (SH2) domains which recognize phosphotyrosine. TRbeta but not TRalpha contains a consensus SH2-binding domain with high affinity for p85. Mutating the tyrosine in that consensus site to phenylalanine prevents reconstitution of Kv11.1 regulation by thyroid hormone in CHO cells but not activation of a heterologous transcription of a luciferase gene driven by a thyroid hormone receptor response element (TRE). A second tyrosine that is responsible for Lyn binding has been identified and is also required for PI3K stimulation. Thus, this mechanism provides a direct link for integrating growth signals through thyroid hormone and receptor tyrosine kinases.
| Hönes, G Sebastian; Rakov, Helena; Logan, John et al. (2017) Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo. Proc Natl Acad Sci U S A 114:E11323-E11332 |
| Martin, Negin P; Marron Fernandez de Velasco, Ezequiel; Mizuno, Fengxia et al. (2014) A rapid cytoplasmic mechanism for PI3 kinase regulation by the nuclear thyroid hormone receptor, TR?, and genetic evidence for its role in the maturation of mouse hippocampal synapses in vivo. Endocrinology 155:3713-24 |
| Storey, Nina M; Gentile, Saverio; Ullah, Hemayet et al. (2006) Rapid signaling at the plasma membrane by a nuclear receptor for thyroid hormone. Proc Natl Acad Sci U S A 103:5197-201 |
