Catalytic Activity of Type II Iodothyronine 5 -Deiodinase Polypeptide Is ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 27, Issue of July 5, pp. 16363–16368, 1996 Printed in U.S.A.

Catalytic Activity of Type II Iodothyronine 5*-Deiodinase Polypeptide Is Dependent upon a Cyclic AMP Activation Factor* (Received for publication, November 16, 1995, and in revised form, March 1, 1996)

Marjorie Safran‡, Alan P. Farwell, and Jack L. Leonard From the Molecular Endocrinology Laboratory, Departments of Medicine, Nuclear Medicine, and Molecular and Cellular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Type II iodothyronine 59-deiodinase (59D-II)1 catalyzes the metabolism of T4 to its active metabolite, T3, in the brain (1, 2). In astrocytes, 59D-II is a multimeric protein with marked molecular asymmetry and has an apparent molecular mass of ;200 kDa (3). 59D-II-like catalytic activity has also been reported in the pituitary and brown adipose tissue (1); however, the hydrodynamic properties of the responsible protein(s) are unknown. One unique feature of brain 59D-II is the rapid, * This work was supported by National Institutes of Health Grant DK-38772 (to J. L. L.) and Clinical Investigator Award DK 02005 (to A. P. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Division of Endocrinology and Metabolism, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655-0308. Tel.: 508-856-3609; Fax: 508-856-6950. 1 The abbreviations used are: 59D-II, type II iodothyronine 59-deiodinase; T4, thyroxine; T3, 3,5,39-triiodothyronine; rT3 (reverse T3), 3,39,59triiodothyronine; BrAcT4, N-bromoacetyl-L-thyroxine; PAGE, polyacrylamide gel electrophoresis.

thyroxine-dependent regulation of catalytic activity observed both in the intact animal and in cell culture (4 –9). Cyclic AMP-stimulated astrocytes express abundant 59D-II catalytic activity (10) and faithfully mimic the thyroid hormone-induced changes seen in vivo. For example, removal of thyroid hormone from the culture medium results in a 10-fold increase in 59D-II activity in cAMP-stimulated astrocytes (7). This increase in enzyme activity results from slowed enzyme inactivation and not from increased enzyme synthesis (7, 11). Acute T4 replacement leads to a rapid loss of 59D-II activity that is independent of gene transcription or protein synthesis (7), but can be blocked by cytochalasin-induced disruption of the actin cytoskeleton (7, 12). The alkylating thyroid hormone analog N-bromoacetyl-T4 (BrAcT4) has proven invaluable for identifying deiodinase polypeptides (8, 13, 14). Under specific conditions, three to five astrocyte proteins contain .95% of the incorporated BrAcT4 label, and two of these have been identified. A 55-kDa affinitylabeled protein (p55) is the subunit monomer of protein-disulfide isomerase (15). The 29-kDa affinity-labeled protein (p29) was identified as the substrate-binding protein of 59D-II. 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 59D-II catalytic activity; (ii) the quantity of BrAcT4 incorporated was directly proportional to 59D-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 59D-II by the affinity label equaled the rate of BrAcT4 incorporation into p29 (8, 11, 12). Subsequently, the T4-dependent increase in 59D-II inactivation was shown to coincide with the migration of p29 from the plasma membrane to the endosomal storage pool, and the rate of loss of enzyme activity equaled that of the internalization of p29 (11). Cytochalasin-mediated depolymerization of the actin cytoskeleton blocked both T4-dependent enzyme inactivation and p29 internalization (11, 12). In addition to the well characterized p29 protein of 59D-II, an additional 29-kDa protein was weakly labeled by BrAcT4 in astrocytes lacking 59D-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 therefore 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 59D-II. EXPERIMENTAL PROCEDURES

Materials All chemicals were of the highest grade available. Dulbecco’s modified Eagle’s medium was obtained from Life Technologies, Inc., and supplemented bovine calf serum was from Hyclone Laboratories (Logan, UT). N-Bromoacetyl-L-T3 and BrAcT4 were obtained courtesy of Dr. Hans Cahnmann (National Institutes of Health, Bethesda, MD).

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Type II iodothyronine 5*-deiodinase is an ;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-bromoacetylL-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-bromoacetylL-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 10fold 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 ;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.

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cAMP Induction of Type II Iodothyronine 59-Deiodinase Methods

FIG. 1. Effect of increasing concentrations of BrAcT4 on labeled p29 from stimulated and unstimulated astrocytes. Confluent astrocyte monomers were grown overnight in serum-free medium with or without 1 mM dibutyryl cAMP and 100 nM hydrocortisone. Cells were incubated with increasing concentrations of BrAc[125I]T4, and the affinity-labeled proteins were identified by autoradiography after separation by SDS-PAGE. BrAc[125I]T4 incorporation into p29 was determined by scanning densitometry. Radiolabeled p29 proteins are shown above the quantification of affinity label incorporation into p29 from stimulated (M) and unstimulated (●) cells. AU, absorbance units. incubations included magnetic beads coated with bovine serum albumin. The beads were then collected; washed three times with 175 mM sodium phosphate buffer, pH 7.4, containing 5 mg/ml bovine serum albumin; and resuspended in SDS-PAGE sample buffer. Proteins were eluted from the beads by boiling for 5 min and resolved by SDS-PAGE. Immunocytochemical Analysis of p29 in Astrocytes—Astrocytes were grown on poly-D-lysine-coated coverslips as described previously (12) and treated as indicated in the figure legends. Cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100 as detailed previously (12). BrAcT4-labeled proteins and p29 were visualized by indirect immunofluorescence using rabbit anti-T4 IgG and rabbit antip29 IgG, respectively; immune complexes were identified with Texas Red-conjugated anti-rabbit IgG. Cells were imaged by confocal microscopy as described previously (12). Micrographs shown are representative of 30 – 40 independent fields. RESULTS

Characterization of BrAcT4-labeled 29-kDa Protein(s) in Astrocytes—One criterion that established p29 as the substratebinding subunit of 59D-II was the observation that the quantity of affinity-labeled p29 was directly proportional to 59D-II catalytic activity (8). However, in unstimulated cells lacking 59D-II activity, another 29-kDa protein was also weakly labeled with BrAcT4. To characterize this latter protein, we established conditions that optimized the BrAcT4 labeling of this T4-binding protein. The effect of increasing concentrations of BrAc[125I]T4 on affinity labeling of astrocyte polypeptides is shown in Fig. 1. At low concentrations, BrAcT4 incorporation into the 29-kDa protein in unstimulated cells was only 15% of that observed for p29 in cAMP-stimulated astrocytes as reported previously (8). At increasing concentrations of affinity ligand, this differential labeling pattern was progressively overcome, and little or no difference in affinity labeling of the 29-kDa protein(s) was observed at concentrations greater than ;2 nM BrAc[125I]T4. These data identify an affinity-labeled 29-kDa protein in astrocytes lacking 59D-II activity and establish the labeling conditions necessary to allow comparisons between this protein and the p29 protein in cAMP-stimulated cells. To establish the relationship(s) between these two 29-kDa polypeptides, we used limited proteolysis and peptide fingerprinting. Cyclic AMP-stimulated and untreated astrocytes were affinity-labeled with 10 nM BrAcT4 for 20 min, conditions that effectively label both p29 and the 29-kDa protein in unstimulated astrocytes (see Fig. 1). The 29-kDa proteins were then isolated by SDS-PAGE, fragments were prepared by

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Cell Culture—Astrocytes were obtained from 1-day-old neonatal rat cerebral hemispheres as described previously (10). Astrocytes were grown in a humidified atmosphere of 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium containing 15 mM sodium bicarbonate, 33 mM glucose, 1 mM sodium pyruvate, 15 mM HEPES, pH 7.4, 10% (v/v) supplemented bovine calf serum, 50 units/ml penicillin, and 90 mg/ml streptomycin. Cells were passaged weekly and used between passages 1 and 3. For all experiments, unless otherwise noted, maximal 59D-II activity was induced in confluent monolayers by growth in serum-free medium for 24 h, followed by an additional 16 h with 1 mM dibutyryl cAMP and 100 nM hydrocortisone (10). Affinity Labeling of 59D-II—BrAc[125I]T4 was prepared by radioiodination of N-bromoacetyl-L-T3 as described previously (13). Cells were affinity-labeled at 37 °C with 1.3 nM BrAc[125I]T4 (specific activity of 2200 Ci/mmol) in buffered Hanks’ solution containing 50 mM HEPES, pH 7.4, unless otherwise indicated. After a 20-min incubation, cells were washed free of unincorporated affinity ligand, scraped from the dish, and collected by centrifugation. Cells were resuspended in 10 mM HEPES, pH 7.0, containing 10 mM dithiothreitol, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (lysis buffer) and sonicated. Either cell lysates were used directly for SDS-PAGE analysis, or crude microsomal preparations were obtained by centrifuging the lysate for 30 min at 250,000 3 g through a 0.8 M sucrose cushion. Microsomes were resuspended by trituration in lysis buffer and used as described below. Gel Filtration of BrAc[125I]T4-labeled p29 —Microsomal preparations from affinity-labeled astrocytes were solubilized in 5 mM taurodeoxycholate, and the detergent extracts were clarified by centrifugation for 30 min at 250,000 3 g. The resultant supernatant was separated on a 90 3 1.5-cm Sephacryl S-300 column equilibrated in 50 mM NH4Ac, pH 7.0, containing 1 mM dithiothreitol, 0.1 mM EDTA, and 5 mM taurodeoxycholate at a flow rate of 10 ml/h, and 1-ml fractions were collected. The detergent-soluble extracts from microsomes isolated from 3 3 108 cells were used for each separation, and 100-ml aliquots of selected fractions were analyzed directly by SDS-PAGE. The distribution of p29 was quantitated either by densitometry or by counting the electrophoretically resolved p29 bands in a g-counter. The column was standardized using thyroglobulin, b-amylase, rabbit IgG, b-galactosidase, ovalbumin, and cytochrome c with dextran blue and 3H2O used for the void volume and total volume, respectively. Peptide Digests—Affinity-labeled 29-kDa proteins from cAMP-stimulated and unstimulated astrocytes were isolated from microsomal preparations by SDS-PAGE on 8 –14% gradient gels as described previously (3). The 29-kDa proteins were cleaved directly in the gel slices with Staphylococcus aureus V8 protease or cyanogen bromide using a modification of the Cleveland method (see Refs. 3, 17, and 18). Digestion products were separated on a 15% SDS-polyacrylamide gels. Peptide fingerprints were compared by autoradiography. Generation of Anti-p29 Antibody—Cyclic AMP-stimulated astrocytes (;109 cells) were affinity-radiolabeled with BrAc[125I]T4 (1 3 106 cpm/ pmol) for 15 min, followed by a 15-min incubation with excess 1 mM BrAcT4 to quantitatively label the p29 polypeptide (19, 20). Microsomes were prepared by discontinuous sucrose gradient centrifugation, and the membrane preparation was solubilized with 5 mM taurodeoxycholate. BrAc[125I]T4-labeled proteins were isolated by immunoprecipitation with anti-T4 IgG (12), and the radiolabeled proteins in the immune pellet were separated by preparative SDS-PAGE. The gel fragment containing p29 was homogenized in 125 mM Tris-HCl buffer, pH 6.8, mixed with an equal volume of complete Freund’s adjuvant and used to immunize 2.2-kg female New Zealand White rabbits. Rabbits were boosted 3 weeks later using the same preparation of p29 homogenized in 125 mM Tris buffer, pH 6.8, alone. Sera containing anti-p29 antibody were identified by Western blotting against purified p29 and used for immunoprecipitation of solubilized affinity-labeled p29- and 59D-II-containing vesicles. Subcellular localization of 59D-II was determined by immunocytochemistry as detailed in the figure legends. Endocytotic Vesicle Isolation—Affinity-labeled astrocytes grown in serum-free medium were treated with 10 nM T4 for 30 min to initiate endocytosis of the 59D-II-containing vesicles (11, 12). Cells were scraped from the dish, collected by centrifugation, and then lysed by three freeze-thaw cycles. Cell lysates were centrifuged (805,000 3 gmin) through 16% Percoll gradients in 250 mM sucrose in 10 mM HEPES buffer, pH 7, containing 10 mM dithiothreitol and 1 mM EDTA, and 0.5-ml fractions were collected. Fractions containing the endosomes (.80% of internalized 59D-II (12)) were pooled and then incubated for 30 min at 4 °C with magnetic beads (Dynabeads, Dynal, Inc.) coated with purified IgG according to the manufacturer’s instructions. Control

cAMP Induction of Type II Iodothyronine 59-Deiodinase

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FIG. 2. Peptide fragmentation patterns obtained after V8 protease (A) or CNBr (B) digestion of p29 from stimulated and unstimulated astrocytes. Gel slices containing affinity-labeled p29 were digested as described under “Experimental Procedures” and separated by SDS-PAGE, and the affinity-labeled proteins were identified by autoradiography. The protein maps shown are representative of three separate experiments.

CNBr cleavage or V8 protease proteolysis, and the peptides were separated by SDS-PAGE. As shown in Fig. 2, the peptide fingerprints of the p29 protein in cAMP-stimulated astrocytes were identical to those obtained for the 29-kDa protein in unstimulated cells, indicating that these two proteins are closely related, if not identical. Hydrodynamic Properties of p29 in cAMP-stimulated and Unstimulated Astrocytes—One possible explanation for the inability of unstimulated astrocytes to catalyze 59-deiodination is that the substrate-binding subunit (p29) is dissociated from the holoenzyme. Since p29 is part of an ;200-kDa multimeric complex, cAMP-induced protein-protein interactions could result in the formation of a functional enzyme by causing p29 to associate with the other, yet to be identified subunits in the holoenzyme. Microsomal preparations of affinity-labeled proteins from cAMP-stimulated and unstimulated astrocytes were solubilized in taurodeoxycholate and separated by gel filtration on a Sephacryl S-300 column, and the distribution of BrAc[125I]T4-labeled p29 was determined as described under “Experimental Procedures.” Shown in Fig. 3 is a representative chromatogram of p29 in unstimulated and cAMP-stimulated glial cells. The p29 subunit in cAMP-stimulated cells was associated with a complex of ;200 kDa, consistent with previous estimates of holoenzyme size (3). In contrast, in unstimulated cells, p29 was associated with a complex that was ;60 kDa smaller than that in cAMP-stimulated cells. This difference in chromatographic behavior of the p29 protein from the two cell populations was consistently observed (Table I). Since cAMP-

induced 59D-II catalytic activity requires both transcription and translation (10), these data suggest that cAMP induces the synthesis of an essential activating factor that associates with the inactive 59D-II complex. There was little or no evidence of p29 monomers or dimers in the chromatograms from either the cAMP-stimulated or unstimulated astrocytes, indicating that most, if not all, of p29 is contained in the multimeric holoenzyme. Characterization of Anti-p29 Antisera—To develop a 59D-IIspecific immunological probe, anti-p29 antibodies were generated against partially purified p29 from cAMP-stimulated cells as described under “Experimental Procedures.” Control studies showed that the anti-p29 antibody was not directed against T4 per se since anti-p29 IgG failed to immunoprecipitate T4 (Table II). This eliminated any potential problems of the cross-reactivity of this antibody with other BrAcT4-labeled proteins. The specificity of the anti-p29 antibody is shown in Fig. 4A. As expected, in control immunoprecipitations, anti-T4 IgG recognized all of the BrAcT4-labeled proteins (second lane) (11, 12), while anti-p29 IgG preferentially immunoprecipitated the p29 polypeptide (third lane). We then determined if anti-p29 IgG recognized the holoenzyme in its native environment. 59DII-containing endosomes were prepared from cAMP-stimulated astrocytes by density gradient centrifugation as detailed previously (11, 12) and incubated with immobilized anti-p29 IgG or immobilized normal rabbit IgG as described under “Experimental Procedures.” As shown in Fig. 4B (first lane), the total endosomal pool from cAMP-stimulated astrocytes contains three prominent BrAcT4-labeled proteins (p55, p29, and p18). Previous work has shown that both p55 and p18 are nonspecifically labeled with BrAcT4, while affinity labeling of p29 is selectively blocked by 59D-II substrates, but not products (8). Immunopurification with anti-p29 IgG-coated beads specifically isolated vesicles containing p29 (third lane), while normal rabbit IgG controls failed to enrich vesicles containing any BrAcT4-labeled protein (second lane). Consistent with previous reports (11, 12), both the p55 and p18 affinity-labeled proteins were also associated with the immunopurified endosome since the anti-p29 antibody does not recognize either p55 or p18 from detergent-solubilized preparations (see Fig. 4A). These data

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FIG. 3. Molecular sieve chromatography of BrAc[125I]T4-labeled 5*D-II from stimulated (M) and unstimulated (●) astrocytes. Taurodeoxycholate-solubilized astrocyte proteins were separated on Sephacryl S-300, and the fractions were analyzed by SDSPAGE as described under “Experimental Procedures.” The migration of 59D-II was determined from that of affinity-labeled p29. The following protein standards were used: thyroglobulin (Tg; 670,000 kDa), b-amylase (b-am; 200,000 kDa), rabbit IgG (158,000 kDa), b-galactosidase (b-gal; 130,000 kDa), ovalbumin (oval; 44,000 kDa), and cytochrome c (cyto-C; 12500 kDa). AU, absorbance units.

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cAMP Induction of Type II Iodothyronine 59-Deiodinase TABLE I Gel filtration analysis of BrAc[125I]T4-labeled p29 from unstimulated and cAMP-stimulated glial cells

Run No.

Column

Vt-V0

p29 (Kav) Unstimulated

cAMP-stimulated

0.25 0.23 0.29

0.20 0.19 0.11 0.22

(ml)

1 2 3 4

I I II II

51.5 68

TABLE II Immunoprecipitation of [125I]T4 IgG

Immunoprecipitated

Anti-p29 Preimmune rabbit Anti-T4

% 0.9 3.1 48.7

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FIG. 4. Autoradiograms of immunoprecipitated BrAc[125I]T4labeled astrocyte proteins. A, taurodeoxycholate-solubilized affinitylabeled astrocyte proteins (L) were immunoprecipitated using anti-T4 IgG (T4) and anti-p29 IgG (p29), and the purified proteins were separated by SDS-PAGE. B, Percoll fractions containing the endosomal pool (E) were obtained as described under “Experimental Procedures” and then incubated with either normal rabbit IgG-linked (IgG) or anti-p29linked (p29) magnetic beads. The isolated vesicle proteins were resolved by SDS-PAGE.

indicate that the anti-p29 antibody recognizes both detergentsoluble and membrane-bound forms of the p29 subunit of 59D-II. Immunocytochemical Localization of the p29 Subunit of 59D-II in cAMP-stimulated and Unstimulated Astrocytes—In cAMP-stimulated astrocytes, catalytically active 59D-II is a plasma membrane enzyme, and T4 regulates 59D-II activity by initiating the translocation of the enzyme from the plasma membrane to the perinuclear space, where it is catalytically inactive (11, 12). Since the p29 subunit is constitutively expressed and part of a multimeric complex in both cAMP-stimulated and unstimulated astrocytes, we determined whether subcellular location played a role in the cAMP induction of catalytically active 59D-II. A panel of representative photomicrographs of p29 immunoreactivity is shown in Fig. 5. Preimmune serum controls showed no specific immunostaining in cAMP-stimulated, BrAcT4-labeled astrocytes. Anti-p29 IgG yielded intense staining in cAMP-stimulated, BrAcT4-labeled astrocytes, presumably over the cell membranes since p29 is an integral membrane protein (8, 12). Interestingly, in the nonaffinity-labeled, cAMP-stimulated astrocytes, the immunoreactivity of p29 was much less than that in BrAcT4-labeled cells, suggesting that the epitope recognized by anti-p29 IgG requires affinity labeling for maximal exposure.

FIG. 5. Identification of p29 in control and BrAcT4-labeled cAMP-stimulated astrocytes. Dibutyryl cAMP-stimulated astrocytes were grown in the absence of thyroid hormone and affinity-labeled as indicated. Immunocytochemistry was done as described under “Experimental Procedures” using preimmune rabbit serum (NRS) and antip29 antisera in nonaffinity-labeled cells (2BrAcT4) and affinity-labeled cells (1BrAcT4).

Since p29 constitutes ;50% of the affinity-labeled protein in astrocytes (8) and the BrAcT4-labeled proteins are readily recognized by anti-T4 antibodies, we compared the distributions of anti-T4 and anti-p29 immunoreactivity in astrocytes. Shown in Fig. 6 are representative confocal micrographs of the effects of T4 on the subcellular distribution of immunoreactive p29 in BrAcT4-labeled, cAMP-stimulated astrocytes. As previously reported (11, 12), anti-T4 IgG shows punctate staining along the cell periphery. Since the nucleus of an astrocyte is elliptical with a long axis of ;8 –10 mm and the astrocyte, while polygonal in shape, is ;30 mm in diameter, the nucleus occupies only 25– 40% of the cross-sectional area of any given cell, and the remaining intracellular space is filled with other organelles and cell sap. Thus, anti-T4 IgG immunoreactivity is concentrated over the plasma membrane in the hypothyroid cAMPstimulated astrocyte, and little, if any, staining is present in

cAMP Induction of Type II Iodothyronine 59-Deiodinase

the cell interior (Fig. 6A). After 20 min of T4 treatment (Fig. 6B), the BrAcT4-labeled protein(s) were lost from the cell membrane and had migrated to the perinuclear space, with occasional punctates observed over the cell nucleus. Since p29 is a membrane-associated protein (8, 11, 12) and, when internalized, is a component of the endosomal vesicle, this pattern of immunoreactivity is consistent with the translocation of affinity-labeled 59D-II from the plasma membrane to the endosomal pool. Parallel astrocyte cultures stained with anti-p29 IgG exhibited identical patterns of rim immunostaining in hypothyroid cells (Fig. 6C) and showed the relocation of the immunoreactive protein to the perinuclear space in the T4-treated astrocytes (Fig. 6D). These data confirm that anti-p29 IgG recognizes the BrAcT4-labeled p29 subunit of the 59D-II holoenzyme in intact cells. Anti-p29 antiserum was then used to determine the subcellular localization of this 59D-II subunit in unstimulated astrocytes grown in thyroid hormone-free medium (Fig. 7). In unstimulated astrocytes, p29 is found in the perinuclear space (P), and little, if any, specific immunoreactivity is associated with the plasma membrane (see arrows). In contrast, in cAMPstimulated cells, the majority of immunoreactive p29 is found associated with the cell periphery, presumably the plasma

FIG. 7. Confocal micrographs of 5*D-II in stimulated and unstimulated astrocytes. Astrocytes were grown in the absence (2DBC) and presence (1DBC) of dibutyryl cAMP, affinity-labeled with BrAcT4, and stained with anti-p29 antisera as described for Fig. 5. Arrows point to the plasma membrane; the nucleus (N) and perinuclear space (P) are indicated. The bar equals 10 mm.

membrane (see arrows), and little remains in the perinuclear space. These data suggest that cAMP stimulation results in the translocation of p29 from the perinuclear space to the plasma membrane, coincident with the appearance of catalytically active 59D-II. DISCUSSION

In this study, we show that p29, the substrate-binding subunit of 59D-II, is constitutively expressed and resides, along with other 59D-II components, in membrane vesicles located in the perinuclear space of unstimulated astrocytes. Cyclic nucleotides induce the appearance of catalytically active 59D-II coincident with translocation of p29 to the plasma membrane. In addition, a cAMP-activating factor(s) is synthesized and becomes associated with the other enzyme components that are stored in vesicles located in the perinuclear space. The identification of p29 as the substrate-binding subunit of 59D-II was based upon multiple criteria, including a direct proportionality between the quantity of BrAcT4-labeled p29 and 59D-II activity and the reciprocal relationship between inactivation and p29 labeling. In fact, the rate of enzyme inactivation is identical to the rate of affinity labeling of the p29 subunit (8). At the concentrations of BrAcT4 used in the early studies (8), minimal background labeling of 29-kDa protein(s) was observed in catalytically inactive cells. In this study, we show that cAMP increases the avidity of p29 for BrAcT4 by .10-fold. This increase in apparent binding affinity may be

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FIG. 6. Effects of acute T4 treatment on distribution of immunoreactive 5*D-II. Dibutyryl cAMP-stimulated astrocytes were grown in the absence of thyroid hormone and affinity-labeled for 20 min. Cells in A and C were fixed immediately, while cells in B and D were incubated with 10 nM T4 at 37 °C for 20 min before fixation. All cells were fixed with 4% paraformaldehyde, permeabilized, and rehydrated, and immunocytochemistry was done with anti-T4 IgG (A and B) or anti-p29 IgG (C and D). The immune complexes were visualized using Texas Red-conjugated goat anti-rabbit IgG (12). The nucleus is indicated (N).

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cAMP Induction of Type II Iodothyronine 59-Deiodinase membrane identifies one of the other 59D-II subunit polypeptides required for functional 59D-II. In vivo, both the pineal gland (22–24) and the limbic system (25) in the brain show catecholamine-induced increases in 59D-II activity, suggesting that cAMP is likely to play an important role in regulating the expression of a polypeptide that is essential for 59D-II catalysis, both in vivo and in cell culture. Brain 59D-II is the largest of the deiodinases, with a calculated molecular mass of 200 kDa (3). Despite the recent cloning of an ;30-kDa frog selenoenzyme with kinetics similar to 59D-II (26), previous biological and biochemical evidence demonstrates that in the mammalian brain, 59D-II is not a selenoenzyme (27–30). Two of the required subunits of 59D-II are now known, the p29 substrate-binding subunit and an ;60kDa cyclic AMP-inducible factor. Whether the essential cyclic AMP-inducible factor is an integral part of the enzyme or is merely required to target the enzyme to the plasma membrane remains to be established. REFERENCES 1. Leonard, J. L., and Visser, T. J. (1986) in Thyroid Hormone Metabolism (Hennemann, G., ed) pp. 189 –229, Marcel Dekker, Inc., New York 2. Kaplan, M. M. (1986) in Thyroid Hormone Metabolism (Hennemann, G., ed) pp. 231–253, Marcel Dekker, Inc., New York 3. Safran, M., and Leonard, J. L. (1991) J. Biol. Chem. 266, 3233–3238 4. Silva, J. E., and Leonard, J. L. (1985) Endocrinology 116, 1627–1635 5. Leonard, J. L., Kaplan, M. M., Visser, T. J., Silva, J. E., and Larsen, P. R. (1981) Science 214, 571–573 6. Kaiser, C. A., Goumaz, M. O., and Burger, A. G. (1986) Endocrinology 119, 762–770 7. Leonard, J. L., Siegrist-Kaiser, C. A., and Zuckerman, C. J. (1990) J. Biol. Chem. 265, 940 –946 8. Farwell, A. P., and Leonard, J. L. (1989) J. Biol. Chem. 264, 20561–20567 9. Halperin, Y., Shapiro, L. E., and Surks, M. I. (1994) Endocrinology 135, 1464 –1469 10. Leonard, J. L. (1988) Biochem. Biophys. Res. Commun. 151, 1164 –1172 11. Farwell, A. P., DiBenedetto, D. J., and Leonard, J. L. (1993) J. Biol. Chem. 268, 5055–5062 12. Farwell, A. P., Lynch, R. M., Okulicz, W. C., Comi, A. M., and Leonard, J. L. (1990) J. Biol. Chem. 265, 18546 –18553 13. Ko¨hrle, 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 14. Schoenmakers, C. H. H., Pigmans, I. G. A. J., and Visser, T. J. (1995) Mol. Cell. Endocrinol. 70, 173–180 15. Safran, M., and Leonard, J. L. (1991) Endocrinology 129, 2011–2016 16. Wyss, M., Wallimann, T., and Ko¨hrle, J. (1993) Biochem. J. 291, 463– 472 17. Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102–1106 18. Nikodem, V., and Fresco, J. R. (1979) Anal. Biochem. 97, 382–386 19. de Duve, C. (1975) Science 189, 186 –193 20. Aronson, N. N., and Touster, O. (1974) Methods Enzymol. 31, 90 –102 21. Visser, T. J., Leonard, J. L., Kaplan, M. M., and Larsen, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5080 –5084 22. Guerrero, J. M., Puig-Domingo, M., and Reiter, R. (1988) Endocrinology 122, 236 –241 23. Osuna, C., Rubio, A., and Guerrero, J. M. (1993) Experientia (Basel) 49, 329 –331 24. Rubio, A., Menendez Pelaez, A., and Reiter, R. J. (1993) J. Pineal Res. 14, 53–59 25. Riskind, P. N., Kolodny, J. M., and Larsen, P. R. (1987) Brain Res. 420, 194 –198 26. Davey, J. C., Becker, K. B., Schneider, M. J., St. Germain, D. L., and Galton, V. A. (1995) J. Biol. Chem. 270, 26786 –26792 27. Behne, D., Hilmert, H., Scheid, S., Gessner, H., and Elger, W. (1988) Biochim. Biophys. Acta 966, 12–21 28. Safran, M., Farwell, A. P., and Leonard, J. L. (1991) J. Biol. Chem. 266, 13477–13480 29. Berry, M. J., Kieffer, J. D., and Larsen, P. R. (1991) Endocrinology 129, 550 –552 30. Chanoine, J. P., Safran, M., Farwell, A. P., Tranter, P., Ekenbarger, D. M., Dubord, S., Alex, S., Arthur, J. R., Beckett, G. J., Braverman, L. E., and Leonard, J. L. (1992) Endocrinology 131, 479 – 484 31. Farwell, A. P., Safran, M., Dubord, S., and Leonard, J. L. (1996) J. Biol. Chem. 271, 16369 –16374

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due, in part, to the translocation of the enzyme to the cell surface in close proximity to the sites of entry of the affinity ligand and/or to the low Km for T4 of the catalytically active enzyme (21). The presence of the p29 subunit of 59D-II in astrocytes lacking the catalytically active enzyme was confirmed by peptide mapping, gel filtration, and immunocytochemistry. Peptide fingerprints of the p29 proteins in unstimulated and cAMP-stimulated astrocytes were identical. Gel filtration revealed that p29 was a component of a larger membrane-associated complex and was not present as a monomer or dimer in either the unstimulated or cAMP-stimulated astrocyte. These data show that the substrate-binding subunit of 59D-II is constitutively synthesized and integrated into vesicular storage membranes of the cell. Interestingly, cAMP induction of catalytically active 59D-II was accompanied by an increase in the molecular mass of this p29-containing complex, suggesting that an additional cAMP-activating factor(s) is responsible for the generation of active 59D-II. The molecular events responsible for the cAMP-dependent generation of 59D-II activity in astrocytes are slowly emerging. Cyclic AMP induces expression of an activating factor that associates with the inactive 59D-II complex in storage vesicles and results in an increase in holoenzyme size. This is also accompanied by a marked difference in the subcellular location of the enzyme. Whether the cAMP-induced activation factor is integrated into the 59D-II complex prior to translocation to the plasma membrane or becomes associated with a 59D-II complex that is continually recycled to the plasma membrane is unknown. However, little, if any, immunoreactive p29 is located in the plasma membrane of unstimulated astrocytes, suggesting that recycling of the “inactive” 59D-II complex is minor. Thus, the cAMP-activating factor is required for the enzyme to relocate to its site of action at the plasma membrane. The regulation of 59D-II activity in cultured astrocytes is a complex process that requires cAMP-induced protein synthesis, cAMP-stimulated translocation of an inactive 59D-II complex from intracellular storage pools to the plasma membrane, and microfilament-based endocytosis. In T4-deficient astrocytes, the biological half-life of the catalytically active enzyme is identical to that of the p29 polypeptide (see accompanying paper (31)). However, in T4-replete cells, p29 is rapidly internalized by endocytosis, and the biological half-life of catalytically active 59D-II is equal to the rate of p29 endocytosis, while the degradation of the p29 polypeptide remains unchanged under hormone-free or hormone-containing conditions (11). Since the cellular levels of p29 are not regulated by thyroid hormone and p29 is present in astrocytes that do not catalyze 59-deiodination, then regulated expression of this substratebinding subunit is not the mechanism by which the levels of catalytically active 59D-II are controlled. Moreover, constitutive expression of p29 indicates that the p29 subunit is not the essential cAMP-induced polypeptide. From previous studies, we know that the cAMP-stimulated appearance of 59D-II activity requires the synthesis of an essential protein(s) (10), and it appears that this cAMP-activating factor may be the shortlived protein essential for 59D-II catalysis. The demonstration that an essential cAMP-dependent factor(s) is required to activate the enzyme and target it to its site of action on the plasma

Cell Biology and Metabolism: Catalytic Activity of Type II Iodothyronine 5 ′-Deiodinase Polypeptide Is Dependent upon a Cyclic AMP Activation Factor Marjorie Safran, Alan P. Farwell and Jack L. Leonard J. Biol. Chem. 1996, 271:16363-16368. doi: 10.1074/jbc.271.27.16363

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