Myosin V Plays an Essential Role in the Thyroid Hormone-dependent Endocytosis of Type II Iodothyronine 5′-Deiodinase

Abstract

In astrocytes, thyroxine modulates type II iodothyronine 5-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormonedependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC 50 levels ϳ1 nM) were >100-fold more potent than 3,5,3-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH 2 -terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxinedependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormonedependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.Type II iodothyronine deiodinase (D2) 1 is a membrane-bound enzyme that catalyzes the generation of T 3 from T 4 , and is the major source of T 3 in brain, pituitary, and brown adipose tissue (1, 2). In the brain, D2 levels are dynamically regulated by thyroid hormone (3-6). The thyroid hormone-dependent regulation of D2 observed in vivo is mimicked in cAMP-stimulated astrocytes in culture (4). In the absence of thyroid hormone, D2 levels are elevated, enzyme turnover is slow, and the microfilaments are disrupted (4, 7). Addition of T 4 or rT 3 , but not the transcriptionally active T 3 , causes the rapid restoration of the microfilaments and activates actin-based endocytosis of D2-containing vesicles, leading to a rapid fall in D2 levels in the cell activity (5,8,9). Importantly, repolymerization of the microfilaments in the absence of thyroid hormone does not alter D2 turnover or activate the actin-based endocytosis of D2-containing vesicles (5, 8, 9), suggesting that thyroid hormone independently regulates actin-based endocytosis. Inhibitor studies showed that disruption of the actin cytoskeleton by dihydrocytochalasin or depletion of cellular ATP stores block the T 4 -induced loss of D2 (4, 5) indicating that intact microfilaments and an energy source are required for the dynamic regulation of the turnover of this membrane-bound enzyme.One of the first steps in the T 4 -dependent regulation of astrocyte D2 activity is the hormone-induced binding of D2-containing vesicles to filamentous actin (F-actin). This is rapidly followed by the translocation of the actin-bound v...

Vesicle trafficking plays a novel role in erythroblast enucleation

“…A role for vesicle trafficking in enucleation is further supported by our observation that BDM, a nonspecific inhibitor of myosin ATPase activity, 26 but not BTS, a specific inhibitor of muscle myosin II subfragment 1 actin-stimulated ATPase activity, 27 blocked enucleation (supplemental Figure 4). These data suggest that myosins V and VI, which participate in vesicle trafficking [28][29][30] and are blocked by BDM but not by BTS, may be required for enucleation.…”

Myosin Va Controls Oligodendrocyte Morphogenesis and Myelination

“…We then determined that myosin Va is expressed by cortical neurons, astrocytes, microglia, and oligodendrocytes in culture ( Fig. 1) (data not shown), consistent with the known localization patterns of myosin Va in brain and CNS cell types (Espreafico et al, 1992;Bridgman, 1999;Stachelek et al, 2000;Rodriguez and Cheney, 2002;Tilelli et al, 2003).…”

The Hypothyroid Brain

“…In a hypothyroid state, DIO2 activity is increased to keep the supply of T3 at normal levels [77] . On the other hand, excess T4 inhibits the activity of DIO2 to posttranscriptional levels [78] and also acts on the mRNA [79] ; in this case the excess hormone also activates DIO3.…”