DESCRIPTION (provided by applicant): Thyroid hormone (TH) is essential for the normal growth and development of the brain, but the molecular events that mediate these actions are poorly understood. While TH-regulated gene expression is widely assumed to play a key role in this process, the recent revelation that both TH receptor genes (TRa and TRb) can be deleted without affecting brain development raises the possibility that other nongenomic TH-dependent processes contribute to the actions of TH in brain. One of the best-characterized, nongenomic actions of TH is the dynamic regulation of brain type 2 deiodinase (D2). Both thyroxine (T4) and the metabolically inert TH, reverse T3 (rT3), dynamically regulate brain D2 levels in vivo by modulating the rate of D2 removal from the plasma membrane through actin-based endocytosis. A key component of this regulatory cascade is a high affinity TH Initiator Protein (TIP) that facilitates the docking of the D2 containing endosome to the actin based molecular motor, Myosin 5a (Myo5a). We hypothesize the high affinity T4-binding protein(s) (TIP) that tethers the endocytotic vesicle to Myo5a is derived from the C-terminus of the TRa gene, known as TRAa. We will establish the role of TRAa(s) in TH-dependent vesicle trafficking using TH-displacement analysis, in vitro actin-binding assays, real time analysis of TH-initiated vesicle trafficking and pull-down assays with the vesicle binding domain of Myo 5a (SA #1). We will also survey the cell lysate for additional TIP proteins using bait:prey with the C-terminus of Myo5a, expression cloning and TH-binding assays (SA#II). We will then develop targeted gene constructs designed to delete the TRAdelta-alphas using homologous recombination and the LoxP/Cre system. We will examine the consequences of this targeted TRa intron 7 deletion on TH-dependent :actin-based endocytosis in neurons and glial cells derived in vitro from TRAa-nul ES cells and create TRda-nul :mice (SA#III). The biology of T4-dependent actin-based endocytosis of p29 is a unique experimental model for understanding the nongenomic actions(s) of TH in brain and uses cellular machinery essential to normal synaptic function which may account for the neurological abnormalities of hypothyroidism.