Localization of type I iodothyronine 5\u27-deiodinase to the basolateral plasma membrane in renal cortical epithelial cells

Type I iodothyronine 5\u27-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5\u27-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5\u27-deiodinase in rat kidney (Kohrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5\u27-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3\u27,5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone\u27s influence on arborization, axonal transport, and cell-cell contact in the developing brain

Affinity labeling of rat liver and kidney type I 5\u27-deiodinase. Identification of the 27-kDa substrate binding subunit

Extrathyroidal production of 3,3\u27,5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5\u27-deiodinases. Type I 5\u27-deiodinase (5\u27D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3\u27,5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5\u27D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5\u27D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5\u27D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5\u27D-I substrates and inhibitors specifically blocked affinity labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3\u27-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5\u27-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5\u27D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled p27. BrAcT3 was a less effective affinity label for p27 and minor labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5\u27D-I showed coincident peaks of p27 and 5\u27-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5\u27D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5\u27-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3

Selenium deficiency and type II 5\u27-deiodinase regulation in the euthyroid and hypothyroid rat: evidence of a direct effect of thyroxine

Selenium deficiency in rats is characterized by elevated serum T4 and decreased serum T3 concentrations, and low liver type I (5\u27D-I) and brain type II (5\u27D-II) iodothyronine 5\u27-deiodinase activities. These findings are partially explained by the demonstration that type I 5\u27D is a selenoprotein; however, 5\u27D-II does not contain selenium. Since 5\u27D-II varies inversely with serum T4 concentrations, and serum T4 is elevated in selenium deficiency, the decreased cerebrocortical 5\u27D-II activity may be secondary to the increased serum T4 levels. To determine the mechanism(s) by which selenium influences 5\u27D-II activity, we examined the effects of altered selenium intake on brain 5\u27D-II levels and enzyme turnover in euthyroid and thyroidectomized rats. Rats were fed a selenium-supplemented or selenium-deficient diet for 5 weeks from weaning; half of the animals were also thyroidectomized 3 weeks before death. Selenium deficiency was confirmed by decreased liver and brain glutathione peroxidase activities. In euthyroid rats, selenium deficiency caused a 38% increase in serum T4, and 91% and 39% decreases in 5\u27D-I and 5\u27D-II, respectively, compared to those in selenium-supplemented rats. In the thyroidectomized hypothyroid rats, selenium deficiency caused a 60% decrease in 5\u27D-I, but had no effect on 5\u27D-II activity, fractional turnover of the enzyme, or the calculated enzyme synthesis rate. The lack of effect of selenium deficiency on 5\u27D-II levels in hypothyroid rats is consistent with the finding that 5\u27D-II is not a seleno-enzyme. Thus, the decrease in brain and pituitary 5\u27D-II activity in selenium-deficient euthyroid rats is due to the T4-dependent increase in the turnover of the enzyme polypeptide