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• W2090985775 abstract "Thyroxine dynamically regulates levels of type II iodothyronine 5′-deiodinase (5′D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5′D-II is an ∼200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5′D-II. We show that T4 accelerates the rate of 5′D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5′D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane ∼2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5′D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways. Thyroxine dynamically regulates levels of type II iodothyronine 5′-deiodinase (5′D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5′D-II is an ∼200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5′D-II. We show that T4 accelerates the rate of 5′D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5′D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane ∼2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5′D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways. Type II iodothyronine 5′-deiodinase (5′D-II) 1The abbreviations used are: 5′D-IItype II iodothyronine 5′-deiodinaseT4thyroxineT33,5,3′-triiodothyroninerT3 (reverse T3)3,3′,5′-triiodothyronineBrAcT4N-bromoacetyl-L-thyroxineBSAbovine serum albuminDMEMDulbecco's modified Eagle's mediumBFAbrefeldin ABt2cAMPdibutyryl cAMP. is a multimeric integral membrane protein (∼200 kDa) that catalyzes T4 to T3 conversion in the brain (1Leonard J.L. Visser T.J. Hennemann G. Thyroid Hormone Metabolism. Marcel Dekker Inc., New York1986: 189Google Scholar, 2Visser T.J. Leonard J.L. Kaplan M.M. Larsen P.R. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5080-5084Crossref PubMed Scopus (204) Google Scholar, 3Visser T.J. Kaplan M.M. Leonard J.L. Larsen P.R. J. Clin. Invest. 1983; 71: 992-1002Crossref PubMed Scopus (164) Google Scholar, 4Safran M. Leonard J.L. J. Biol. Chem. 1991; 266: 3233-3238Abstract Full Text PDF PubMed Google Scholar). Thyroid hormone, specifically T4, dynamically regulates 5′D-II levels by modulating the biological half-life of this short-lived protein without altering enzyme synthesis (5Leonard J.L. Silva J.E. Kaplan M.M. Mellen S.A. Visser T.J. Larsen P.R. Endocrinology. 1984; 114: 998-1004Crossref PubMed Scopus (100) Google Scholar). In the absence of T4, enzyme levels are high, and 5′D-II inactivation is slow. T4 promotes interactions between the enzyme and the F-actin microfilaments that lead to 5′D-II inactivation and enzyme internalization, resulting in a rapid fall in enzyme levels (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). This action of T4 is independent of transcription or translation, indicating an extranuclear mode of action, and has been extensively characterized in cultured astrocytes that lack functional thyroid hormone receptors (9Leonard J.L. Farwell A.P. Yen P.M. Chin W.W. Stula M. Endocrinology. 1994; 135: 548-555Crossref PubMed Scopus (62) Google Scholar). Indeed, T4 is >100-fold more potent than the transcriptionally active T3 in regulating 5′D-II inactivation (1Leonard J.L. Visser T.J. Hennemann G. Thyroid Hormone Metabolism. Marcel Dekker Inc., New York1986: 189Google Scholar, 5Leonard J.L. Silva J.E. Kaplan M.M. Mellen S.A. Visser T.J. Larsen P.R. Endocrinology. 1984; 114: 998-1004Crossref PubMed Scopus (100) Google Scholar, 6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). type II iodothyronine 5′-deiodinase thyroxine 3,5,3′-triiodothyronine 3,3′,5′-triiodothyronine N-bromoacetyl-L-thyroxine bovine serum albumin Dulbecco's modified Eagle's medium brefeldin A dibutyryl cAMP. The 29-kDa substrate-binding subunit (p29) of 5′D-II is covalently modified by the alkylating affinity label N-bromoacetyl-L-T4 (BrAcT4), allowing the enzyme to be identified without measuring catalytic activity (10Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar). Utilizing cultured astrocytes that retain all of the 5′D-II regulatory aspects seen in the brain in vivo and that express high levels of 5′D-II activity in the presence of cyclic AMP (11Leonard J.L. Biochem. Biophys. Res. Commun. 1988; 151: 1164-1172Crossref PubMed Scopus (83) Google Scholar), we have previously shown that T4 dynamically regulates 5′D-II levels by directing the enzyme to use different pathways of internalization (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). Under T4-deficient conditions, p29 is internalized via the traditional endocytotic pathway and is slowly transported through the endosomes to the lysosomes. In contrast, T4 treatment activates specific protein-F-actin interactions involved in actin-mediated endocytosis and targets the enzyme to the endosomes, where the internalized 5′D-II-containing vesicle remains without subsequent transit to the lysosomes. Recycling of the catalytically active enzyme back to the plasma membrane has not been observed in the short time frames examined previously; thus, the fate of the endosomal pool of internalized 5′D-II remains uncertain in the presence of T4. The endosomes are a collection of vesicles located in the perinuclear space (12Helenius A. Mellman I. Wall D. Hubbard A. Trends Biochem. Sci. 1983; 8: 245-249Abstract Full Text PDF Scopus (380) Google Scholar, 13Mueller S.C. Hubbard A.L. J. Cell Biol. 1986; 102: 932-942Crossref PubMed Scopus (62) Google Scholar). Vesicles internalized via endocytosis initially exist as early endosomes and contain a mixture of polypeptides with differing fates. Vesicles containing proteins to be recycled, such as the transferrin and insulin receptors, are directed back to the plasma membrane (14Ciechanover A. Schwartz A.L. Dautry-Varsat A. Lodish H. J. Biol. Chem. 1983; 258: 9681-9689Abstract Full Text PDF PubMed Google Scholar), while vesicles containing proteins destined for degradation evolve into late endosomes and are targeted to the lysosomes. In the secretory pathway, vesicles containing newly synthesized membrane proteins pass from the endoplasmic reticulum to the Golgi stack before exiting through the trans-Golgi network, where they are targeted to the plasma membrane directly or routed through the endosomes (15Rothman J.E. Orci L. Nature. 1992; 355: 409-415Crossref PubMed Scopus (739) Google Scholar, 16Rothman J. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1993) Google Scholar). From the endosomes, the secretory vesicles may be directed to the plasma membrane, to the lysosomes, or to vesicle storage pools. The sorting mechanism that determines the destination of the differing vesicles that compose the endosomes is still unclear (17Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1991; 61: 471-516Crossref Scopus (368) Google Scholar). The T4-dependent regulation of the pathways of 5′D-II inactivation/internalization suggests a role for hormonal regulation in vesicle-mediated protein transport (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). In this study, we show that 1) the rate of degradation of the 29-kDa substrate-binding subunit of 5′D-II (p29) is unaffected by T4; 2) in the presence of T4, p29 is recycled back to its site of action on the plasma membrane; and 3) despite recycling of p29, 5′D-II catalytic activity requires de novo synthesis of additional enzyme components. Since T4 determines whether p29 is recycled back to the plasma membrane or is routed to the lysosomes, these data suggest that, in addition to regulating the internalization pathway of 5′D-II, T4 participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways. Pregnant (16–17-day gestation) rats were obtained from Charles River Laboratories (Kingston, NY). T4 and BSA was purchased from Sigma. T3 was obtained from Henning GmbH, and dihydrocytochalasin B was obtained from Calbiochem. Dulbecco's modified Eagle's medium (DMEM), antibiotics, Hanks' solution, and 0.25% trypsin were obtained from Life Technologies, Inc., and defined bovine calf serum (heat-inactivated) was from Hyclone Laboratories. Culture flasks were obtained from Nunc, and 24-well tissue culture plates were obtained from Falcon. All other reagents used were of the highest purity commercially available. Rat type I astrocyte cultures were obtained by enzymatic dispersion of neonatal rat brains as described previously (18McCarthy K.D. de Vellis J. J. Cyclic Nucleotide Res. 1978; 4: 15-26PubMed Google Scholar). Cells were grown in a humidified atmosphere of 5% CO2 and 95% air at 37°C in DMEM supplemented with 15 mM sodium bicarbonate, 33 mM glucose, 1 mM sodium pyruvate, and 15 mM HEPES, pH 7.4, with 10% (v/v) calf serum, 50 units/ml penicillin, and 90 µg/ml streptomycin. The culture medium was changed three times weekly, and cells were subcultured (2–3 × 104 cells/cm2) when they reached confluence (7–10 days). Confluent cells from passages 2–4, containing >95% astrocytes as determined by staining for the astrocyte-specific protein, glial fibrillary acidic protein (19McCarthy K.D. de Vellis J. J. Cell Biol. 1980; 85: 890-902Crossref PubMed Scopus (3364) Google Scholar), were utilized for experiments. Steady-state levels of 5′D-II were induced by incubating the cells for 8 h in a defined medium consisting of DMEM and 0.1% BSA ± thyroid hormone, followed by a 16-h stimulation with 1 mM Bt2cAMP and 100 nM hydrocortisone. Cells were harvested by scraping in ice-cold 8 mM sodium phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl and collected by centrifugation. 5′D-II activity was determined in cell sonicates by the iodide release method at 2 nM rT3 and 20 mM dithiothreitol in the presence of 1 mM propylthiouracil. Units are expressed as femtomoles of I− released per hour. The turnover of 5′D-II was determined by assaying enzyme activity 5–60 min after inhibition of protein synthesis with 100 µM cycloheximide in 50 mM HEPES and 1 mg/ml BSA in buffered Hanks' solution, pH 7.0, in the presence or absence of 10 nM T4. The turnover of 5′D-II in the presence of BFA was determined by preincubating cells with 5 µg/ml BFA for 15 min before the addition of 100 µM cycloheximide. Cells were then collected as described above and assayed for 5′D-II activity. The p29 polypeptide was affinity-labeled in confluent astrocytes expressing steady-state levels of 5′D-II in the absence of thyroid hormone by incubation for 20 min with either 0.4 or 10 nM BrAc[125I]T4, 1 mM dithiothreitol, and 50 mM HEPES in buffered Hanks' solution, pH 7.0. The labeling medium was removed, and the cells were washed free of unbound affinity label and then chased for 30–120 min with either 10 nM T4 or T3 or with no hormone in 50 mM HEPES and 1 mg/ml BSA in buffered Hanks' solution, pH 7.0. Cells were collected and sonicated. Affinity-labeled proteins were identified after SDS-polyacrylamide gel electrophoresis and autoradiography. The quantity of p29 was determined by scanning densitometry. Cells were seeded onto glass coverslips (22 × 22 mm) coated with poly-D-lysine (10 µg/ml) and grown for 24 h. 5′D-II activity was induced as described above. Cells were affinity-labeled for 20 min with 10 nM BrAcT4 in buffered Hanks' solution containing 10 mM dithiothreitol and 50 mM HEPES, pH 7.0, and washed and chased for 0–120 min in DMEM with 1 mg/ml BSA, 1 mM Bt2cAMP, and 100 nM hydrocortisone in the presence and absence of 10 nM T4. All cells were treated with 10 µM colchicine to relax the cell borders for 60 min prior to fixation. Cells were fixed in iced 4% paraformaldehyde and permeabilized with iced methanol. 5′D-II was identified by incubation with an antiserum raised against p29 (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)) or with an anti-T4 IgG that recognizes the BrAcT4 moiety, and immune complexes were identified by incubation with Texas Red-conjugated anti-rabbit IgG. Coverslips were then mounted and examined by laser scanning confocal microscopy with a depth of resolution of 0.5 µm. Where indicated, results were analyzed by unpaired Student's t test. The 29-kDa substrate-binding subunit of 5′D-II (p29) is selectively affinity-labeled by BrAcT4 (4Safran M. Leonard J.L. J. Biol. Chem. 1991; 266: 3233-3238Abstract Full Text PDF PubMed Google Scholar, 10Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar). In the absence of T4, p29 is internalized and targeted to the lysosomes, whereas in the presence of T4, p29 remains indefinitely in the endosomal pool localized in the perinuclear space of the cell (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). In light of the different metabolic fates of proteins residing in these two intracellular compartments, we examined the effect of thyroid hormone on the degradation of p29. Cells were grown in thyroid hormone-free medium and stimulated with Bt2cAMP and hydrocortisone to express high levels of 5′D-II (11Leonard J.L. Biochem. Biophys. Res. Commun. 1988; 151: 1164-1172Crossref PubMed Scopus (83) Google Scholar). P29 was affinity-labeled, and cells were then chased with either 10 nM T4 or T3 or with no hormone for up to 90 min. The cellular content of p29 and the levels of catalytically active 5′D-II were analyzed at 0–90 min after the addition of hormone. As shown in Fig. 1A, the disappearance of p29 over time was unaffected by thyroid hormone, while >80% of the catalytic activity of 5′D-II was lost due to the presence of T4 (Fig. 1B). Little or no other smaller affinity-labeled degradation products from the affinity-labeled p29 protein were observed over this time frame (data not shown). Shown in Table I are the steady-state levels of p29 in cells grown in the presence and absence of T4. As reported previously, the quantity of BrAcT4-labeled p29 (labeled at a concentration of 0.4 nM BrAcT4) (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)) in T4-deficient cells was ∼2-fold greater than that in T4-treated astrocytes. In contrast, 5′D-II activity in T4-deficient cells was ∼10-fold higher than that in T4-treated astrocytes (Table I) (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 10Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar). This apparent discrepancy between the p29 content and 5′D-II activity levels is likely to be due to different quantification techniques for catalytic activity and p29 (10Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar) and the ability of BrAcT4 to label the p29 subunit of catalytically inactive 5′D-II (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)). Since T4 has no effect on the fractional disappearance rate of p29 (Fig. 1A), the calculated appearance rate of this polypeptide in the presence of T4 is approximately half that seen in the absence of hormone (Table I). As shown previously (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 10Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar), the production rate of catalytic activity is unaffected by thyroid hormone (Table I). Thus, the production and disposal of the p29 substrate-binding subunit of 5′D-II differ from the production and disposal of 5′D-II catalytic activity.Table IEffect of thyroid hormone on the half-life of p29 and 5′D-II in astrocytes10 nM T4Thyroid hormone-deficientp29 contentSteady-state levels (OD/mg protein)17.735.1t1/2 (h−1)1.71.5Appearance rate (h−1)7.316.25′D-II activitySteady-state levels (units/mg protein)2002177t1/2 (h−1)0.22.2Production rate (h−1)692718 Open table in a new tab The differences between the effects of T4 on the respective t1/2 values of p29 and 5′D-II catalytic activity, our observation that p29 accumulates in the endosomal pool in the presence of T4 (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar), and the observation that p29 is found in the endosomal pool in catalytically inactive astrocytes (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)) raise the possibility that this multimeric enzyme is recycled. To examine this possibility, recycling of 5′D-II activity was determined in protein synthesis-blocked cells kept for extended periods of time. Cells expressing 5′D-II activity in T4-replete medium were incubated in the presence and absence of 100 µM cycloheximide for up to 4 h, and 5′D-II activity was assayed. As shown in Fig. 2, 5′D-II activity fell to undetectable levels in cycloheximide-blocked cells, with little or no recovery of catalytic activity observed at any time point. Cell viability, as determined by trypan blue exclusion, was unaffected over the 4-h treatment period with cycloheximide (data not shown). Enzyme levels remained constant in the presence of continued protein synthesis. Thus, the maintenance of 5′D-II catalytic activity in astrocytes requires ongoing protein synthesis. BFA is a fungal toxin that dramatically alters vesicle trafficking within the cell, causing fusion of the Golgi/endoplasmic reticulum/endosomal organelles and blocking transport of vesicle proteins to the lysosomes (20Klausner R.D. Finaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1530) Google Scholar, 21Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (673) Google Scholar, 22Damke H. Klumperman J. von Figura K. Braulkes T. J. Biol. Chem. 1991; 266: 24829-24833Abstract Full Text PDF PubMed Google Scholar). This leads to a functional separation of the secretory, recycling, and degradative pathways and also promotes membrane protein recycling (20Klausner R.D. Finaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1530) Google Scholar, 21Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (673) Google Scholar). For example, BFA increases the accumulation of the receptors for insulin-like growth factor II and insulin on the plasma membrane, two receptors that are recycled through the endosomes in fibroblasts (22Damke H. Klumperman J. von Figura K. Braulkes T. J. Biol. Chem. 1991; 266: 24829-24833Abstract Full Text PDF PubMed Google Scholar). To determine if 5′D-II activity could be recycled from the endosomes in astrocytes, we used BFA to maximize transit through the membrane protein recycling pathways. As shown in Fig. 2, the addition of BFA to cycloheximide-blocked, T4-replete cells had no effect on the fall of 5′D-II activity. In the absence of BFA, there was little or no recovery of catalytic activity observed at any time point. Next, we examined the effects of BFA on steady-state levels of 5′D-II in the presence of continued protein synthesis (Fig. 3). BFA had no effect on steady-state levels in T4-replete cells during treatment periods up to 60 min. In contrast, BFA treatment led to a steady increase in 5′D-II activity in T4-deficient cells, resulting in a doubling of enzyme activity by 60 min. Analysis of the disappearance kinetics of 5′D-II revealed that BFA prolonged the biological half-life of the catalytically active enzyme in both T4-deficient (t1/2 = 2.1 versus 3.0 h−1) (Fig. 4A) and T4-replete (t1/2 = 0.2 versus 0.3 h−1) (Fig. 4B) astrocytes. Since the biological half-life of 5′D-II activity in T4-replete cells was prolonged in the presence of BFA and the levels of enzyme remained unchanged, BFA treatment must cause a proportional fall in enzyme production. 5′D-II generation in T4-deficient cells was minimally affected by BFA, as there was a concordant increase in both t1/2 and enzyme levels. These data imply that the sources of the catalytically active holoenzyme are different in cells grown in the presence and absence of T4.Fig. 4Effect of brefeldin A on the turnover of 5′D-II activity in astrocytes. Steady-state levels of 5′D-II activity were induced in confluent astrocytes incubated in either the presence or absence of 10 nM T4 as described under “Experimental Procedures.” Cells were incubated in the presence (○) or absence (•) of BFA (5 µg/ml) for 15 min, and then protein synthesis was blocked by the addition of 100 µM cycloheximide. 5′D-II activity was assayed at increasing times after the addition of cycloheximide. Results are expressed as a percent of the starting 5′D-II activity (femtomoles of I− released per milligram of protein/hour) at the time of addition of cycloheximide and represent the means of triplicate values in at least two experiment. A, cells incubated in the absence of thyroid hormone; B, cells incubated in the presence of 10 nM T4.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One explanation for these differential effects of BFA on 5′D-II activity is that one or more of the enzyme-associated polypeptides are recycled in cells grown in the presence of T4. Thus, in T4-treated cells where 5′D-II is rapidly internalized to the endosomes (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar), BFA treatment should prevent the recycling enzyme polypeptide(s) from joining the de novo enzyme production pathway, leading to an accumulation of inactive enzyme precursors in the fused vesicle pool and thereby decreasing the observed enzyme production rate. To test this hypothesis, we determined the effects of BFA on the accumulation of catalytically active 5′D-II after depolymerization of the F-actin cytoskeleton in T4-treated astrocytes. Depolymerization of F-actin by dihydrocytochalasin B blocks T4-mediated endocytosis and results in the accumulation of 5′D-II activity at a rate equal to the production rate of enzyme catalytic activity (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar). An accelerated rate of accumulation of catalytic activity is expected if a pool of enzyme precursors is formed in the presence of BFA. As shown in Fig. 5, the addition of dihydrocytochalasin B to T4-replete astrocytes caused the steady accumulation of 5′D-II activity as reported previously (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar). Pretreating the T4-replete cells with BFA increased the initial rate of enzyme accumulation by ∼2-fold in dihydrocytochalasin B-blocked cells. This effect was transient in cells pretreated for only 30 min with BFA, while a 60-min pretreatment period led to a sustained increase in the rate of 5′D-II activity accumulation. These data suggest that BFA treatment results in an increase in the storage of enzyme-related polypeptides in an intracellular pool. Direct examination of the intracellular transit of the enzyme polypeptide(s) was performed by using a specific IgG directed against the affinity-labeled 29-kDa substrate-binding subunit of 5′D-II (anti-p29 IgG) (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)). T4-deficient astrocytes were grown on glass coverslips, pulse affinity-labeled with BrAcT4, and then chased with either 10 nMT4 or no hormone. Cells were fixed after increasing periods of time, stained with anti-p29 IgG, and examined by laser scanning confocal microscopy. As shown in Fig. 6A (arrows), punctate staining is present in a “rim” pattern at the periphery of the cell in the thyroid hormone-deficient, affinity-labeled astrocyte, while the interior of the cell is largely devoid of immunoreactive staining. This rim pattern is consistent with a plasma membrane location for the staining. After a 20-min treatment with T4, the majority of the staining is located in the perinuclear space within the cell (Fig. 6B, P), with little immunoreactive staining remaining on the plasma membrane (arrows). These patterns are consistent with previous studies done with an anti-T4 IgG that show that affinity-labeled polypeptide(s), predominantly p29, are located on the cytoplasmic leaflet of the plasma membrane in the thyroid hormone-deficient cell and are translocated to the perinuclear space within 20 min of exposure to T4 (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar). Similarly, previous studies have shown that the majority of affinity-labeled p29 after short-term exposure (20 min) to T4 resides in the endosomal pool (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). After a 2-h exposure to T4, prominent punctate staining is again found at the periphery of the cell in a rim pattern (Fig. 6C, white arrows), along with clusters of staining present diffusely throughout the cell (black arrow). Since anti-p29 IgG selectively recognizes affinity-labeled p29 (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)), these data show that pulse-labeled p29 was internalized and recycled back to the plasma membrane in T4-treated astrocytes. In contrast to the T4-treated cells, immunoreactive staining in the T4-deficient cell is clustered into discrete regions within the cell (Fig. 6D, black arrow), with little staining remaining on the plasma membrane (white arrows). Previous studies have shown that affinity-labeled p29 is transported from the plasma membrane to discrete intracellular pools identified as lysosomes in T4-deficient cells (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). The effects of BFA on the recycling of pulse-labeled p29 are shown in Fig. 7. Pulse-labeled p29 was identified with an anti-T4 IgG that recognizes the BrAcT4-labeled enzyme as described previously (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar). Punctate staining in a rim pattern is present in the cells treated with T4 for 2 h, indicating a plasma membrane location (Fig. 7, upper left panel, arrows). This pattern is identical to the pattern observed in Fig. 6C using anti-p29 IgG and is again consistent with the recycling of p29 back to the plasma membrane. Treatment with BFA blocked the reappearance of p29 on the plasma membrane in the presence of T4 since the immunofluorescence remains in clusters within the cell and rim staining is not present (Fig. 7, upper right panel). In the absence of T4, the immunoreactive staining is predominantly clustered within the cell at 2 h, with little effect of BFA observed on the distribution of immunofluorescence (Fig. 7, lower panels). In this study, we have characterized the intracellular trafficking of 5′D-II in cAMP-stimulated astrocytes and have shown that T4 regulates fundamental processes involved in the turnover of integral membrane proteins. Specifically, T4 shuttles plasma membrane proteins to the endosomes by switching from an actin-independent to an actin-mediated internalization pathway. Once in the endosomes, T4 modulates the sorting mechanism to select recycling pathways over degradation pathways. Thus, T4 participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways. The evidence the T4 shifts 5′D-II to a recycling pathway is 3-fold. First, there is a dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of the 29-kDa substrate-binding subunit (p29) in euthyroid cells, consistent with the reuse of this protein in the production of catalytically active 5′D-II. Second, there is an increased rate of accumulation of 5′D-II activity in brefeldin A-treated euthyroid cells after depolymerization of the F-actin microfilaments, consistent with the storage of enzyme-related polypeptides in an intracellular pool that is available to the synthetic pathway. Finally, using anti-p29 antisera, pulse-labeled p29 reappears on the plasma membrane ∼2 h after internalization in the presence of T4. In contrast, the 5′D-II-containing vesicles in T4-deficient cells are sorted through the endosomes to the degradation pathway, ending up in the lysosomes (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). Enzyme inactivation parallels p29 degradation in the absence of T4. Interestingly, the degradation rate of p29 is similar whether it is sorted directly to the lysosomes in the absence of T4 or is routed through one or more recycling sequences in the presence of T4. Since 5′D-II is a multimeric enzyme, there are two possibilities for recycling of this subunit. First, the holoenzyme may remain intact waiting for the synthesis of an accessory protein that either targets the enzyme to the plasma membrane or activates catalytic activity once it arrives at the plasma membrane. Consistent with this hypothesis, we showed that p29 in catalytically inactive astrocytes is part of a 180–200-kDa complex found in the endosomes (see accompanying paper (31Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 261: 16363-16368Abstract Full Text Full Text PDF Scopus (26) Google Scholar)). Cyclic AMP induces the transcription of a 5′D-II “activating factor,” leading to the translocation of 5′D-II to the plasma membrane, activation of catalytic activity, and an increase in the apparent molecular mass of 5′D-II to the 200–220-kDa range. Alternatively, the holoenzyme may be disassembled, with one or more subunits shuttled to the lysosomes and p29 combining with other newly synthesized subunits. At least one component of the catalytically active enzyme requires de novo synthesis since ongoing protein synthesis is required to maintain catalytic activity. It is likely that this latter component is the cAMP-inducible activating factor. Previous work on the T4-dependent regulation of 5′D-II activity identified the inactivation/internalization pathway as the primary site of action of T4 (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar, 23Farwell A.P. Leonard J.L. Endocrinology. 1992; 131: 721-728PubMed Google Scholar, 24Safran M. Farwell A.P. Rokos H. Leonard J.L. J. Biol. Chem. 1993; 268: 14224-14229Abstract Full Text PDF PubMed Google Scholar). Indeed, when measuring catalytic activity, T4 markedly increases the rate of inactivation of enzyme activity without affecting the production rate of 5′D-II activity (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar). However, analysis of the effects of T4 on the turnover of the p29 subunit revealed a more complex production pathway for catalytically active 5′D-II. Brefeldin A, a fungal toxin that disconnects the secretory, recycling, and degradation pathways for membrane proteins, decreased both the production rate and inactivation rate of the catalytically active enzyme in euthyroid cells, but had little effect on the production rate in hypothyroid cells. These data indicate that the production of catalytically active 5′D-II differs in the presence and absence of T4. One possible explanation for this dichotomy is that the production of 5′D-II in the presence of T4 uses both de novo synthesis and preformed subunits recycled from the endosomes. In euthyroid astrocytes, we found that BFA prevented the return of 5′D-II to the plasma membrane and led to an accumulation of 5′D-II polypeptide(s) in intracellular membrane pools (Fig. 7). In contrast, in hypothyroid cells, all components of catalytically active 5′D-II are produced de novo, and BFA did not affect the production rate. Since at least one component of catalytically active 5′D-II needs to be newly synthesized in both cases, it appears that fusion of vesicles from the Golgi and endosomal pool as well as de novo synthesis of all 5′D-II subunits contribute to the production of active 5′D-II in euthyroid cells, while only the latter process contributes to production of active 5′D-II in hypothyroid cells. Thus, our initial finding that steady-state production rates of enzyme activity are unaffected by T4 (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar) suggests that the rate-limiting synthetic step is the same in the presence and absence of T4. Our current model of the regulatory events that modulate 5′D-II activity is shown in Fig. 8. The synthetic pathway includes the production of 5′D-II polypeptides and a putative activating factor that leads to translocation of the enzyme to the plasma membrane and activation of catalytic activity. This activating factor may be a component of 5′D-II itself or simply an accessory/chaperon protein. As shown in Fig. 8A, T4 initiates inactivation of 5′D-II, located on the cytoplasmic surface of the plasma membrane (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar), by promoting the binding of the enzyme to the F-actin cytoskeleton (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar). The enzyme is then transported to the endosomes by an actin-mediated mechanism (7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar, 8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar), presumably involving a molecular motor protein (25Wolenski J.S. Cheney R.E. Forscher P. Mooseker M.S. J. Exp. Zool. 1993; 267: 33-39Crossref PubMed Scopus (19) Google Scholar, 26Vallee R.B. Sheptner H.S. Annu. Rev. Biochem. 1990; 59: 909-932Crossref PubMed Scopus (151) Google Scholar). From the endosomes, 5′D-II is shuttled back into the synthetic pathway, where it is recycled back to its active state on the plasma membrane. In hypothyroid cells or in cells treated with transcriptionally active T3, the 5′D-II polypeptide(s) are internalized by an actin-independent pathway (Fig. 8B) (6Leonard J.L. Siegrist-Kaiser C.A. Zuckerman C.J. J. Biol. Chem. 1990; 265: 940-946Abstract Full Text PDF PubMed Google Scholar, 7Farwell A.P. Lynch R.M. Okulicz W.C. Comi A.M. Leonard J.L. J. Biol. Chem. 1990; 265: 18546-18553Abstract Full Text PDF PubMed Google Scholar). The 5′D-II-containing vesicles are then sorted through the endosomes to the degradation pathway, ending up in the lysosomes (8Farwell A.P. Dibenedetto D.J. Leonard J.L. J. Biol. Chem. 1993; 268: 5055-5062Abstract Full Text PDF PubMed Google Scholar). The routing of vesicles through the endosomes to degradative, recycling, and synthetic pathways is regulated by targeting signals provided by vesicle-associated proteins located on the vesicle membrane. These vesicle proteins include regulatory coat proteins (17Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1991; 61: 471-516Crossref Scopus (368) Google Scholar, 20Klausner R.D. Finaldson J.G. Lippincott-Schwartz J. J. Cell Biol. 1992; 116: 1071-1080Crossref PubMed Scopus (1530) Google Scholar), small Ras-like GTP-binding proteins (17Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1991; 61: 471-516Crossref Scopus (368) Google Scholar), and proteins isolated primarily from synaptic vesicles, including the synapsins and the calcium-binding synaptotagmin (27Sudhof T.C. De Camilli P. Niermann H. Jahn R. Cell. 1993; 75: 1-4Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 28Sudhof T.C. Jahn R. Neuron. 1991; 6: 665-677Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 29Kelly R.B. Nature. 1993; 364: 487-488Crossref PubMed Scopus (34) Google Scholar). The binding of these proteins to, or removal from, vesicles results in the targeting of the vesicles to their ultimate destination. One potential mechanism by which T4 may regulate vesicle sorting is by binding to one of these regulatory proteins and thus modulating the targeting signals for the p29-containing vesicle. A potential candidate protein to be regulated by T4 is the synaptic vesicle protein synaptotagmin, which participates in vesicle recycling in the nerve terminal (27Sudhof T.C. De Camilli P. Niermann H. Jahn R. Cell. 1993; 75: 1-4Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 28Sudhof T.C. Jahn R. Neuron. 1991; 6: 665-677Abstract Full Text PDF PubMed Scopus (430) Google Scholar). In addition to promoting vesicle recycling, synaptotagmin also influences the morphology of the actin cytoskeleton (29Kelly R.B. Nature. 1993; 364: 487-488Crossref PubMed Scopus (34) Google Scholar, 30Feany M.B. Buckley K.M. Nature. 1993; 364: 537-540Crossref PubMed Scopus (53) Google Scholar), two cellular events regulated by T4 in astrocytes. In summary, the regulation of 5′D-II activity in cultured astrocytes suggests that thyroid hormone regulates the basic mechanisms by which cells synthesize, degrade, and recycle proteins." @default.
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