STRUCTURAL REQUIREMENTS OF IODOTHYRONINES FOR THE RAPID INACTIVATION AND INTERNALIZATION OF TYPE-II IODOTHYRONINE 5'-DEIODINASE IN GLIAL-CELLS

3,3'5,5'-Tetraiodothyronine (T4), but not 3,3'5-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, t ype II iodothyronine 5'-deiodinase (5'D-II), by inducing internalizati on of the enzyme through an extranuclear, energy-dependent mechanism t hat requires an intact actin cytoskeleton. The affinity label, N-bromo acetyl-L-T4, binds to 5'D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural element s of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5'D-I I. The rank order of potency for enzyme inactivation was T4 > COOH-blo cked analogs > NH3 and COOH blocked analogs >> NH3 blocked analogs (EC 50 values range from 1 to > 1000 nM). In contrast, all T4 analogs test ed and T4 were excellent competitive inhibitors of 5'D-II with respect to substrate (K(i) values ranged from 4 to 27 nM). The differential c apability of iodothyronines to inactivate the enzyme was not related t o their ability to enter the cell, since K(i) values measured in intac t glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5'D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5'D-II binding to F-actin. The data show that modification of the alanine sid e chain markedly alters the ability of T4 analogs to induce 5'D-II ina ctivation and actin polymerization. A net negative charge on the alani ne side chain of T4 is detrimental for the hormone-dependent inactivat ion of 5'D-II and polymerization of actin, whereas uncharged or positi vely charged molecules retain significant activity.