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J. Biol. Chem., Vol. 280, Issue 12, 11093-11100, March 25, 2005

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Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases^*

Jack L. Leonard, Gregory Simpson, and Deborah M. Leonard

From the Molecular Endocrinology Laboratory, Department of Cell and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Received for publication, January 1, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D¹⁴⁸FL-YI-EAH-DGW¹⁶³-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD^GFP) to examine holoenzyme assembly of other family members. Overexpression of either the DDD^GFP or an inert D1 subunit (M4) into SeD2 (accession number U53505 [GenBank] )-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD^GFP subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Thyroid hormone is essential for the normal growth and development of vertebrates. Thyroxine (3',5',3,5-tetraiodothyronine) is the major secretory product of the thyroid gland and must be deiodinated to generate the transcriptionally active metabolite, T₃.¹ Type I iodothyronine deiodinase (D1; EC 3.8.1.4 [EC] ) is the best characterized member of a small family of membrane-bound enzymes that catalyze the production and disposal of T₃ (1–4). All deiodinase family members are composed of 27–30-kDa polypeptides that contain the novel amino acid, selenocysteine (SeC) that is required for full catalytic activity (3, 5–8). They also share an N-terminal membrane-spanning region and a central core of 14–16 residues surrounding the SeC, but little else is known about the polypeptide. Gel filtration and density gradient centrifugation of detergent-soluble enzyme activity and/or affinity radiolabeled D1 showed that catalytic activity has an M_r of 50,000 and that the 27-kDa D1 polypeptide co-migrated with a complex, suggesting that D1 is a dimer of 27-kDa subunits (9–11). The physiochemical properties of the other selenodeiodinase family members remain to be determined, although a recent report using transiently expressed, epitope-tagged D2 and D3 suggested that these family members also formed dimers, albeit at modest levels (12).

Direct analysis of D1 biosynthesis confirmed that the active enzyme was a homodimer formed by the post-translational assembly of two p27 subunits (9). Whether each subunit was catalytically functional or required post-translational assembly to become catalytically active was resolved by the finding that D1 dimers with only one functional subunit had 50% of the activity of the wild-type enzyme (9, 12). Importantly, these hybrid holoenzymes had a molecular mass of 54 kDa and an S_20,w of 3.5 S, identical to that of the native D1 holoenzyme (9). The ability of each D1 subunit to function as an independent catalytic center was recently confirmed by Curcio-Morelli et al. (12). Interestingly, deletion of the membrane anchor located between residues 9 and 37 of p27 resulted in the formation of catalytically active D1 heterodimers composed of a full-length, membrane-bound subunit and the N-terminal truncated partner lacking a transmembrane domain (9), indicating that insertion of both D1 subunits into the membrane was not required.

Whether holoenzyme assembly required covalent or noncovalent protein-protein interactions was recently examined using transient co-expression of epitope-tagged p27 constructs (12). A minor role for intermolecular disulfide bridging between p27 partners (ranging from 1 to 5% of the total) was recently proposed based on banding pattern(s) of epitope-tagged p27 found after SDS-PAGE analysis. Presumably because of sequence diversity among deiodinase family members, dimer complexes formed between different family members were not observed after SDS-PAGE analysis, suggesting that only like family members could associate (12).

In this study, we mapped the dimerization domain(s) required for deiodinase assembly by exploiting the ability of the native p27 present in LLC-PK1 cells to form dimers with truncation fragments of the immunologically unique, inert rat p27 (M4). We also determined the role of dimerization in the assembly of a catalytically active SeD2 enzyme using C6 cells that constitutively express the 30-kDa SeD2 subunit. The results of our studies show that the highly conserved sequence present in all deiodinase family members from frogs to humans, -DFL-YI-EAH-DGW-, serves as the dimerization domain for holoenzyme assembly. For the D1 family member, catalytic activity is preserved when only one subunit is catalytically competent, whereas the D2 isoform required two fully functional subunits to be catalytically active.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—All reagents were of the highest purity commercially available. Restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs (Beverly, MA). L-3'-¹²⁵I- or 5'-¹²⁵I-labeled rT₃ was prepared by radioiodination of 3,3'-diodothyronine using methods described previously (13). Synthetic oligonucleotides were prepared in house or purchased from either Invitrogen or Midland Scientific, Inc. All iodothyronines were of the L-configuration and were purchased from Henning Berlin GmbH. Dulbecco's modified Eagle's medium, antibiotics, Hanks' buffered salt solution, glucose, trypsin, and G418 were obtained from Invitrogen; supplemented bovine calf serum was from HyClone Laboratories (Boulder, CO); acrylamide and N,N'-methylenebisacrylamide were from U.S. Biochemical Corp.; ammonium persulfate and TEMED were from Bio-Rad; and dithiothreitol was from Calbiochem. Mouse monoclonal anti-GFP primary antibody and rabbit anti-mouse horseradish peroxidase secondary antibody were obtained from Molecular Probes, Inc. (Eugene, OR). Protein A-agarose and Protein G-Sepharose were obtained from RepliGen (Cambridge, MA) and Amersham Biosciences, respectively.

Cell Culture—LLC-PK1 and COS7 cells were grown in 25-cm² flasks in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C as described previously (9, 14). Complete growth medium was composed of Dulbecco's modified Eagle's medium containing 15 mM sodium bicarbonate, 25 mM HEPES (pH 7.2), 25 mM glucose, 1 mM sodium pyruvate, 3–10% (v/v) bovine calf serum, 50 milliunits/ml penicillin, and 90 µg/ml streptomycin. Cells were subcultured every 3–5 days by seeding 2 x 10⁴ cells/cm² into 25-cm² flasks. Culture medium was changed three times weekly.

SeD2 cells (C6 astrocytoma cells constitutively expressing an exogenous SeD2) were grown in the presence of 200 µg/ml G418 in 25-cm² flasks in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C as described previously (15).

Site-directed Mutagenesis and Deletion Constructs—Site-directed mutagenesis was used to substitute Ser for the active center SeC to inactivate the catalytic activity of rat p27 as detailed previously (9). M4, the catalytically inert rat p27 subunit (9), was subcloned into the KpnI-XhoI site of the eukaryotic expression vector, pcDNA3, and used as the template for truncation and deletion construct synthesis.

Progressive deletion of 19–30-residue long segments from both the N and C terminus of p27 was done by oligonucleotide-primed PCR. All primers used to create deletion and truncation mutants of D1 are listed in Table I. All upstream oligonucleotide primers have a 5'-end KpnI site for subcloning and an in-frame Met in the Kozak consensus translation initiation site to ensure optimal expression in mammalian cells. A common downstream primer complementary to nucleotides 895–914 (G21 cDNA; GenBank^TM accession number X57999 [GenBank] ) was used for all N-terminal truncation mutants.

Complementary synthetic oligonucleotides corresponding to nucleotides 445–496 of G21 cDNA were used to synthesize the D1 dimerization domain-GFP fusion protein (DDD^GFP). The 72-mer sense oligonucleotide (5'-TAGACCACCATGGTCGCTGACTTCCTCATCATTTACATTGAAGAAGCTCACGCCACAGATGGATGGGCTCTG-3') and its 72-mer complementary oligonucleotide (5'-GATCCAGAGCCCATCCATCTGTGGCGTGAGCTTCTTCAATGTAAATGATGAGGAAGTCAGCGACCATGGTGGT-3') were annealed, yielding a double-stranded 72-bp fragment with an N-terminal Met in a Kozak consensus start site, an NheI-compatible 5' overhang, and a BglII-compatible 3'-overhang. The annealed DDD encoding sequence was appended to the N terminus of GFP by cloning into the NheI BglII sites of pEGFP-N1, generating pDDD^GFP. Correct insertion was confirmed by DNA cycle sequencing.

Generation of M4- and DDD^GFP-expressing LLC-PK1 and SeD2 Cells—LLC-PK1 cells were grown in growth medium to 60% confluence and transfected with 10 µg of either pcDNA3 (empty vector control), pcDNA3-M4 deletion, and truncation construct(s), as indicated in individual figure legends, or pDDD^GFP using cationic liposomes (DOTAP®; Roche Applied Science) according to the manufacturer's instructions. Stable transfectants were selected at 1 mg/ml G418, and antibiotic-resistant cells were expanded without clonal isolation. Cells constitutively expressing M4 (inactivated D1 subunit), M4 deletion and M4 truncation mutants, and DDD^GFP were grown in 800 µg/ml G418 to maintain expression of the exogenous gene product.

SeD2-expressing C6 cells were grown to 60% confluence in growth medium containing 200 µg/ml G418 and transfected with 10 µg of either pEGFP (GFP control) or pcDNA3-M4 (D1 subunit) or 10 µg of pDDD^GFP fusion construct using cationic liposomes (DOTAP®; Roche Applied Science) according to the manufacturer's instructions. Cells expressing the GFP fusion proteins were amplified by selection with 1.5 mg/ml G418. Greater than 95% of the hyperresistant G418-selected SeD2 cells expressed GFP as judged by fluorescence microscopy.

DNA Sequencing—Double-stranded DNA sequencing was done by the dideoxynucleotide method of Sanger et al. (16) using cycle sequencing and iterative primers in the Nucleic Acid Core Facility at the University of Massachusetts Medical School. All sequence information was confirmed by sequencing both strands.

Antibody Depletion of Deiodinase Dimers—Anti-epitope antibodies directed against the C terminus of rat p27 or rat SeD2 were used for pull-down assays. Incubations were done for 90 min at 4 °C in a total volume of 100 µl that contained 50–100 units of tDOC-solubilized enzyme (D1 or D2) prepared from purified membranes (9, 10), 100 mM HEPES buffer (pH 8.0), 100 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM tDOC, 10 µl of either immobilized rProtein A^TM-agarose beads (RepliGen, Cambridge, MA) or immobilized rProtein G-Sepharose^TM beads (Amersham Biosciences), and 1 µl of either anti-rat p27 IgG (antibody 3050) or anti-SeD2 IgG (antibody 763). Where indicated, excess C-terminal p27 peptide (10 µg/ml, final concentration) was added to block antibody-binding sites prior to the pull-down assay.

Immune complexes were removed by centrifugation, and the deiodinase activity remaining in the clarified supernatant was determined as described below. Expression levels of the exogenous M4 deletion and truncation constructs in LLC-PK1 cells were determined by a competitive binding assay as described previously (9). The minimum detection limit of M4 and its derivatives is 3 fmol/assay tube (81 pg of p27/tube), and the maximum binding capacity of the anti-rat p27 antiserum is 22 nmol of M4/ml of antiserum (600 ng of p27/µl) as determined by Scatchard analysis of the binding data.

SeD2-specific immune complexes were removed by centrifugation and released from the beads by heating at 70 °C for 5 min in Laemmli sample buffer. Proteins were separated on 12.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-GFP monoclonal antibodies. Specific immune complexes were identified with horseradish peroxidase-conjugated, anti-mouse IgG (rabbit; Molecular Probes) and visualized by chemiluminescence.

Photomicroscopy—Cells expressing the DDD^GFP fusion protein were seeded onto poly-D-lysine (10 µg/ml)-coated glass coverslips (22 x 22 mm) and grown to 80% confluence. Cells were then fixed with 4% paraformaldehyde (w/v) in phosphate-buffered saline, and the excess cross-linker was quenched with 100 mM glycine. The distribution of GFP fusion protein in LLC-PK1 and SeD2 cells was then determined by fluorescence microscopy. Digital images were captured using a Spot CCD camera (Meyer Instruments, Inc., Houston, TX) and processed using Adobe Photoshop software. Photomicrographs shown are representative of 20–30 independent fields.

Analytical Procedures—D1 activity was determined in cell lysates by measuring the release of radioiodide from 10 µM [¹²⁵I]rT₃ (100 cpm/pmol) at 20 mM dithiothreitol (unless otherwise indicated) in a total volume of 100 µl. D1 reactions were done, in triplicate, in universal deiodinase buffer (100 mM boric acid, 100 mM HEPES, 100 mM glycine) (pH 5.5), 1 mM EDTA, with 10–25 µg of cell protein and incubated at 37 °C for 10–30 min. Product formation was measured as described previously (13), and the data are expressed as units/mg protein where 1 unit equals 1 pmol of I^– released per min for D1 and 1 fmol of I^– released per min for D2.

D2 activity was determined at substrate concentrations of 2 nM [¹²⁵I]rT₃ (500 cpm/fmol), 20 mM dithiothreitol in the presence of 1 mM 6-n-propyl-2-thiouracil as described previously (17, 18). All experiments were performed at least three times. Statistical analysis was done by Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The ability of an overexpressed catalytically inert D1 subunit to partner with >95% of the native p27 subunit(s) in LLC-PK1 cells (9) allowed us to use changes in catalytic activity as a measure of protein-protein interaction(s). Preliminary attempts to recover deiodinating activity from immune precipitated p27^wt:M4 heterodimers were unsuccessful due, in large part, to the >80% reduction in enzyme activity due to the nonspecific binding of the iodothyronine substrate to the immunoaffinity beads (data not shown). Since these nonspecific interactions are partially reversed under acidic conditions, we re-evaluated the pH dependence of the D1 reaction in an attempt to find conditions that would allow detection of immune precipitated enzyme. Shown in Fig. 1 is the pH profile of D1 activity present in detergent-solubilized COS7 cell lysates expressing a catalytically active D1 (G21 (5)). Contrary to traditional D1 assay conditions, deiodination showed a maximum at pH 5.5, identical to that of the pK_a of the reactive SeC nucleophile and 2 orders of magnitude lower than that used to measure D1 activity (19–21). At optimal pH, the catalytic potential of D1 was 2–3-fold greater than that measured at pH 7.0. All subsequent D1 assays were done at pH 5.5.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Effects of pH on D1 activity of wild type p27 (G21) expressed in COS7 cells. COS7 cells were transfected with rat p27 (G21) and grown for 72 h. Cell lysates were prepared, and D1 activity was determined at the pH indicated using a universal buffer composed of 100 mM boric acid, 100 mM HEPES, and 100 mM glycine. One unit is equivalent to 1 pmol I– released/min. Data are reported as the means of closely agreeing (±10) triplicate determinations

Cell membranes were then prepared for each X and C series M4 mutant by Percoll gradient centrifugation, and the enzyme activity in detergent extracts of the membrane fractions was measured after immune depletion with anti-rat p27 antisera with or without excess blocking peptide as described under "Experimental Procedures." As illustrated in Fig. 2A, >95% of the D1 activity in cells expressing the X series of M4 deletion mutants (X19 to X133) was specifically immune-depleted from detergent extracts, just as found previously with the full-length M4 (9). The percentages of D1 heterodimer formation, calculated from the difference in the quantity of residual D1 activity in immune depleted extracts measured in the absence and presence of blocking peptide, are summarized in Fig. 2B. More than half of the p27 could be deleted from the N terminus (up to residue 133, including the transmembrane anchor, the substrate specificity site, and the catalytic center) without altering the binding of the truncated M4 to p27^wt. These data indicate that the dimerization domain resides in the C-terminal 120 residues of p27.

View larger version (44K): [in this window] [in a new window]   FIG. 2. Mapping of the D1 dimerization domain. Lysates of LLC-PK1 cells constitutively expressing N-terminal and C-terminal M4 deletion mutants were immune precipitated with anti-rat p27 IgG, and D1 activity was determined in the immune depleted supernatant with or without blocking peptide (A) and in the initial tDOC extracts (B). D1 activity is expressed as units/mg protein. Data are reported as the mean ± S.E. of three separate experiments.

Kinetic Analysis of Engineered D1 Heterodimers Lacking a Transmembrane Anchor (X42), a Substrate Specificity Domain (X72), or One Catalytic Center (X133)—Shown in Fig. 3 are representative initial velocity reaction kinetics for the wild-type D1^wt, the full-length M4:p27^wt, and the X42:p27^wt dimers. All hybrid D1 heterodimers showed ping-pong reaction kinetics. Secondary replots yielded the limiting K_m and V_max values (Table III) and revealed that the loss of the N-terminal membrane anchor (X42), the iodothyronine specificity site (X101), and even the catalytic center (X133) resulted in a 2.5-fold decrease in the limiting K_m for the iodothyronine and a 75% fall in the molar activity compared with the native enzyme. Unexpectedly, loss of the membrane anchor increased the reduced thiol cofactor requirement 4-fold (Table III). These data show that kinetic properties of the D1 reaction are intrinsic to each subunit.

View larger version (34K): [in this window] [in a new window]   FIG. 3. Initial velocity reaction kinetics for D1 activity in control LLC-PK1 cells, LLC-PK1 cells constitutively expressing M4 or X42-M4. Steady-state reaction kinetics were done as detailed in Ref. 1. The hybrid D1 holoenzyme was obtained from cells detailed in Fig. 2. The enzyme concentration of functional D1 equaled that of the endogenous p27 (0.2 pmol/mg of cell protein). Data are representative of three separate studies. All lines were drawn by least squares linear regression analysis.

View larger version (65K): [in this window] [in a new window]   FIG. 4. Representative photomicrographs of LLC-PK1 cells expressing either GFP or DDDGFP. Cells were transfected with either pEGFP-N1 vector or pEGFP-N1 containing cDNA encoding DDDGFP and fixed 24 h later. Images were taken as detailed under "Experimental Procedures."

View larger version (41K): [in this window] [in a new window]   FIG. 5. Subcellular distribution of DDDGFP or GFP in LLC-PK1 cells carrying pDDDGFP or pGFP. DDDGFP or GFP was separated on 16% Percoll gradients, and 500-µl fractions were collected beginning at the top. Relative fluorescence was determined at 488-nm excitation and 512-nm emission. Data are representative of three experiments.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Analysis of endogenous D1-DDD interactions by immune depletion. Membranes from LLC-PK1 cells constitutively expressing pEGFP or pDDDGFP were solubilized with 5 mM tDOC, and the clarified extracts were incubated with 2 µg of mouse anti-GFP monoclonal antibody. Immune complexes were removed from the extracts with Protein G-Sepharose beads, and the D1 activity remaining in the immune depleted extract was determined as detailed under "Experimental Procedures." Control D1 activities were as follows: GFP, 15.6 units/mg protein; DDDGFP, 11.2 units/mg protein. Data represent the means ± S.E. of three individual membrane preparations.

View larger version (27K): [in this window] [in a new window]   FIG. 7. Effects of overexpression of the DDD domain on SeD2 activity. C6 astrocytoma cells constitutively expressing SeD2 were transfected with pEGFP-N1, pM4GFP, or pDDDGFP, and cells expressing these fusion proteins were amplified by G418 selection. D2 activity in cell lysates was determined as described under "Experimental Procedures." Data represent the mean ± S.E. of three different preparations.

View larger version (88K): [in this window] [in a new window]   FIG. 8. Representative photomicrographs of C6 and C6 cells constitutively expressing SeD2 transfected with cells expressing either pEGFP-N1 vector or pDDDGFP. Cells were fixed 24 h after transfection, and images were taken as detailed under "Experimental Procedures."

View larger version (18K): [in this window] [in a new window]   FIG. 9. Immune precipitation of SeD2:DDD dimers. Eight 75-cm2 flasks of confluent SeD2 cells were transfected with 20 µg of either pEGFP or pDDDGFP and grown for 2 days at 37 °C. Cell membranes were isolated as detailed under "Experimental Procedures" and solubilized with 0.5% Triton (v/v). Clarified extracts were incubated with anti-SeD2 antiserum (1:100 dilution), immune complexes were isolated with Protein A-agarose beads, and the immune precipitated proteins were separated on a 12.5% SDS-polyacrylamide gel. Separated proteins were electrotransferred to nitrocellulose membranes, and specifically immune precipitated DDDGFP was identified with a monoclonal anti-GFP IgG and visualized using chemiluminscent reagent. Ig HC, rabbit immunoglobulin heavy chain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To eliminate the technical challenges encountered when multiple, catalytically disabled constructs are transiently co-expressed in host cells (12), we used LLC-PK1 cells that constitutively express a full-length D1 selenoprotein and an exogenous, unique inert rat p27 to examine enzyme assembly. Earlier work done with this approach provided direct evidence that the D1 holoenzyme was a homodimer formed from p27 subunits and that the membrane anchor of one the subunits could be removed without altering dimerization (9). Interestingly, at least half of the p27 polypeptide, up to 133 residues, could be removed from the N terminus without altering the binding of the truncated D1 subunit to the native p27. Progressive truncation from the C terminus of the rat p27 revealed that residues from position 170 to 254 could also be deleted without altering dimerization but that removal of residues between positions 150 and 170 completely eliminated the binding of the truncated subunit to the native p27, suggesting that the domain responsible for enzyme assembly is located between residues 150 and 170.

Comparison of the primary amino acid sequence of this region across family members revealed that a 16-amino acid-long segment located between residues 150 and 170 of p27 is one of the most highly conserved domains in the family. As shown in Table IV, 10 of the 16 residues found between positions 147 and 163 of the rat p27 are identical in all deiodinase family members from fish to humans, and most, if not all, of the variant residues are conservative substitutions. Importantly, the distance between this conserved segment and the active center SeC is fixed at 22 residues in all family members. Because of this high conservation, we generated a domain-specific probe to study enzyme assembly by appending a 17-amino acid-long peptide corresponding to residues 147–163 of p27 to the N terminus of GFP forming the DDD^GFP fusion protein.

As expected, overexpression of the soluble DDD^GFP fusion protein in LLC-PK1 cells resulted in a 50% reduction in enzyme activity coincident with specific binding of the fluorescent DDD reporter to the native p27. Immune depletion analysis, subcellular fractionation, and photomicroscopy confirmed that this fusion protein was responsible for the decrease in deiodinating activity and was specifically bound to the full-length p27 in LLC-PK1 cells. These data obtained with the soluble DDD^GFP fusion protein are identical to those obtained with the full-length, M4 mutant of rat p27 reported previously (9) and establish that this highly conserved region contains all of the necessary structural requirements for assembly of the D1 holoenzyme.

Based on the finding that DDD^GFP fusion protein was a faithful mimic of the protein-protein interaction(s) required for holoenzyme assembly, we turned our attention to the role of this domain in the biosynthesis of catalytically active SeD2. To ensure sufficient quantities of catalytically active SeD2 for study, SeD2 cells (15), C6 astrocytoma cells that constitutively overexpress a functional SeD2, were used as the source of the membrane-bound SeD2 subunits. Unlike our findings for DDD: p27 heterodimers of D1, transient expression of either the DDD^GFP fusion or the full-length, M4 mutant of D1 in SeD2 cells completely inactivated SeD2. Since the SeD2:DDD^GFP heterodimers were catalytically inactive, we could not evaluate protein-protein interactions by measuring the loss of deiodinating activity by immune depletion. Instead, direct analysis of the binding between the DDD^GFP reporter and the 30-kDa SeD2 subunit was done by pull-down assays with anti-SeD2 antibodies and revealed that the complete loss of SeD2 activity was accompanied by the formation of a DDD:SeD2 heterodimer. The role of the DDD domain in SeD2 assembly was confirmed by subcellular fractionation and by co-localization with the full-length SeD2 in the astrocytoma host cells. Control experiments done with the unaltered GFP fluorescent reporter revealed that the assembly of a DDD^GFP-SeD2 heterodimer required the N-terminal DDD peptide. These data show that the DDD domain is essential for the post-translational assembly of holoenzymes of at least two deiodinase family members.

Using the immune depletion strategy modeled after that developed to study D1 biosynthesis, Curcio-Morelli et al. (12) used transient co-transfection of FLAG-tagged deiodinase constructs to confirm that a D1 dimer containing only one functional subunit was catalytically active (9, 12). Although more than 95% of D1 was immune precipitated by antibodies directed against a single subunit, they estimated that only 1–5% of the holoenzyme was a homodimer formed by intermolecular disulfide bridges based on SDS-PAGE analysis of incompletely reduced samples. Interestingly, the 50-kDa D1 band was only found after SDS-PAGE of inactive Cys mutants of D1; immune precipitates of radiolabeled wild-type D1 failed to show this additional band (12). Since the SeC Cys mutants of the p27 generate a divicinal thiol (-CXC-motif) in the active center of the mutant(s), a protein motif shown to have a strong propensity to aggregate during SDS-PAGE (23, 24), it seems likely that the 50-kDa protein aggregates found after SDS-PAGE resulted from the choice of Cys mutants rather than a specific role for disulfide bridging in the assembly of homodimers.

Whereas the results of our experiments make it unlikely that disulfide bridging participates in the assembly of the D1 homodimer, a role for noncovalent interactions is well documented. First, early work showed that 95% of detergent-solubilized D1 catalytic activity and the BrAc[¹²⁵I]thyroxine-labeled p27 co-migrated with a M_r of 50 kDa on a molecular sieve chromatography (11, 25). Importantly, the choice of detergent has a profound effect on the physiochemical properties of this membrane-bound enzyme and molecular sieve chromatography of D1 activity solubilized with nonionic detergents yielded M_r values as high as 400,000 (26, 27), presumably due to the size of the enzyme-detergent micelles. This problem can be overcome by the use of ionic detergents such as taurodeoxycholate at or near their CMD, where they contribute negligibly to the molecular size of the D1 as judged by sucrose density gradient centrifugation (10, 29). Second, that ability of p27 mutants lacking the membrane anchor to bind to the full-length p27 indicates that integration into the bilipid layer is not required for deiodinase assembly (9). Finally, the ability of the DDD^GFP fusion protein to bind specifically to the full-length p27 confirms that enzyme assembly is a post-translational event that is independent of membrane association (9).

The participation of selected residues located in the DDD domain in the catalytic reaction was recently proposed by Larsen and co-workers (30) based on hydrophobic cluster analysis coupled with position-specific BLAST protocols. Mutation of any one of three residues, Glu¹⁵⁶, His¹⁵⁸, or Glu¹⁶³, all located in the DDD domain, impaired catalysis of both D1 and D2 family members in transient expression assays (30). Whether this loss of catalytic function was due to improper enzyme assembly, direct participation in the catalytic reaction, or enhanced enzyme instability could not be evaluated. In light of the role of this domain in enzyme assembly and the improved deiodinase reaction conditions, the role of individual residues in the D1 catalytic reaction, especially in this important functional domain, needs to be re-evaluated.

In summary, assembly of a functional deiodinase holoenzyme requires a small 16-residue-long region that is one of the most highly conserved domains among family members. Identification of the domain responsible for enzyme assembly revealed that deiodinase family members retain the ability to interact with each other to form heterodimers. Whereas the consequence of heterodimer formation between different family members is unknown, it is possible that cells modulate the cellular levels of functional selenodeiodinase enzyme(s) family members by sequestering subunits in a pool of poorly functional heterodimers and thereby regulate production and disposal of bioactive thyroid hormone.

	FOOTNOTES

 
^* 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: Molecular Endocrinology Laboratory, Dept. of Cell and Molecular Physiology, University of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA 01655. Tel.: 508-856-6687; Fax: 508-856-5997; E-mail: jack.leonard{at}umassmed.edu.

¹ The abbreviations used are: T₃, 3',3,5-triiodothyronine; rT₃, 3',5',3-triiodothyronine; D1, D2, and D3, type I, II, and III iodothyronine deiodinase, respectively; GFP, green fluorescent protein; SeC, selenocysteine; TEMED, N,N,N',N'-tetramethylethylenediamine; tDOC, taurodeoxycholate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION 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. Kohrle, J. (2004) Z. Arztl. Fortbild. Qualitatssich. 98, Suppl. 5, 17–24
3. Bianco, A. C., Salvatore, D., Gereben, B., Berry, M. J., and Larsen, P. R. (2002) Endocr. Rev. 23, 38–89[Abstract/Free Full Text]
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