Catalytic Activity of Type II Iodothyronine 5′-Deiodinase Polypeptide Is Dependent upon a Cyclic AMP Activation Factor

Marjorie Safran,Alan P Farwell,Jack L Leonard

doi:10.1074/jbc.271.27.16363

Marjorie Safran, Alan P Farwell + Show 1 more

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https://doi.org/10.1074/jbc.271.27.16363

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Abstract

Type II iodothyronine 5'-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3'-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5'-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5'-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5'-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.

Highlights

Type II iodothyronine 5Ј-deiodinase (5ЈD-II)1 catalyzes the metabolism of T4 to its active metabolite, T3, in the brain [1, 2]
The following criteria were used to establish the identity of the p29 protein. (i) An increase in affinity labeling paralleled the cAMP-stimulated increase in 5ЈD-II catalytic activity; (ii) the quantity of BrAcT4 incorporated was directly proportional to 5ЈD-II activity; (iii) affinity labeling was competitively inhibited by substrates T4 and rT3, but not the product T3; and (iv) the rate of inactivation of 5ЈD-II by the affinity label equaled the rate of BrAcT4 incorporation into p29 [8, 11, 12]
Characterization of BrAcT4-labeled 29-kDa Protein(s) in Astrocytes—One criterion that established p29 as the substratebinding subunit of 5ЈD-II was the observation that the quantity of affinity-labeled p29 was directly proportional to 5ЈD-II catalytic activity [8]

Summary

Introduction

Type II iodothyronine 5Ј-deiodinase (5ЈD-II) catalyzes the metabolism of T4 to its active metabolite, T3, in the brain [1, 2]. Removal of thyroid hormone from the culture medium results in a 10-fold increase in 5ЈD-II activity in cAMP-stimulated astrocytes [7]. In addition to the well characterized p29 protein of 5ЈD-II, an additional 29-kDa protein was weakly labeled by BrAcT4 in astrocytes lacking 5ЈD-II catalytic activity [8]. Whether this protein was related to p29 or was another T4-binding protein or an artifact of affinity labeling [16] was not known. We re-examined the conditions of p29 affinity labeling, compared p29 with the 29-kDa polypeptide in unstimulated astrocytes, and evaluated the mechanism of cAMP activation of 5ЈD-II

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