Thyroxine-Thyroid Hormone Receptor Interactions

Abstract

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs α and β) that bind triiodothyronine (T3, 3,5,3′-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T4, 3,5,3′,5′-tetraiodo-l-thyronine) with lower affinity. T4 contains a bulky 5′ iodine group absent from T3. Because T3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5′ substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T4 complexes adopt a conformation that differs from TR-T3 complexes in solution. Nonetheless, T4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T3 does not contribute to agonist activity. We determined x-ray crystal structures of the TRβ LBD in complex with T3 and T4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TRβ accommodates T4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5′ iodine and complete the coactivator binding surface. While T3 is the major active TH, our results suggest that T4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5′ extension should be considered in TR ligand design. Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs α and β) that bind triiodothyronine (T3, 3,5,3′-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T4, 3,5,3′,5′-tetraiodo-l-thyronine) with lower affinity. T4 contains a bulky 5′ iodine group absent from T3. Because T3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5′ substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T4 complexes adopt a conformation that differs from TR-T3 complexes in solution. Nonetheless, T4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T3 does not contribute to agonist activity. We determined x-ray crystal structures of the TRβ LBD in complex with T3 and T4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TRβ accommodates T4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5′ iodine and complete the coactivator binding surface. While T3 is the major active TH, our results suggest that T4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5′ extension should be considered in TR ligand design. Thyroid hormone (TH) 1The abbreviations used are: TH, thyroid hormone; LBD, ligand binding domain; TR, thyroid receptor; T3, triiodothyronine (3,5,3′-triiodo-l-thyronine); T4, thyroxine (3,5,3′,5′-tetraiodo-l-thyronine); R.m.s., root mean square; MIBRT, 3,5-dibromo-4-(3′isopropyl-4′-hydroxyphenoxy)benzoic acid); DIBRT, 3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)benzoic acid; HIC, hydrophobic interaction columns; Dimit, 3, 5-dimethyl-3′isopropyl-l-thyronine; NR, nuclear receptor.1The abbreviations used are: TH, thyroid hormone; LBD, ligand binding domain; TR, thyroid receptor; T3, triiodothyronine (3,5,3′-triiodo-l-thyronine); T4, thyroxine (3,5,3′,5′-tetraiodo-l-thyronine); R.m.s., root mean square; MIBRT, 3,5-dibromo-4-(3′isopropyl-4′-hydroxyphenoxy)benzoic acid); DIBRT, 3,5-dibromo-4-(3′,5′-diisopropyl-4′-hydroxyphenoxy)benzoic acid; HIC, hydrophobic interaction columns; Dimit, 3, 5-dimethyl-3′isopropyl-l-thyronine; NR, nuclear receptor. plays important regulatory roles in metabolism, homeostasis, and development by binding and altering the transcriptional regulatory properties of two related nuclear receptors (NRs), the thyroid hormone receptors (TRs) α and β (1Zhang J. Lazar M.A. Annu. Rev. Physiol. 2000; 62: 439-466Crossref PubMed Scopus (572) Google Scholar, 2Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1529) Google Scholar). Most TH produced in the thyroid gland is secreted in the form of thyroxine (T4; 3,5,3′,5′-tetraiodo-l-thyronine) (2Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1529) Google Scholar, 3Braverman L.E. Utiger R.D. 8th Ed. Werner's and Ingbar's The Thyroid: A Fundamental and Clinical Text. Lippincott Williams & Wilkins, Philadelphia, PA2000Google Scholar). The thyroid gland also produces smaller amounts of triiodothyronine (T3; 3,5,3′-triiodo-l-thyronine) and reverse T3 (rT3; 3,3′,5′-triiodo-l-thyronine), and 80% of T4 is converted to T3 and rT3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4Kohrle J. Mol. Cell. Endocrinol. 1999; 151: 103-119Crossref PubMed Scopus (284) Google Scholar). Current beliefs are that T3 is the dominant active form of TH; T3 binds the TRs with an affinity about 20-30 times higher than that of T4 (5Samuels H.H. Stanley F. Casanova J. J. Clin. Investig. 1979; 63: 1229-1240Crossref PubMed Scopus (97) Google Scholar, 6Apriletti J.W. Eberhardt N.L. Latham K.R. Baxter J.D. J. Biol. Chem. 1981; 256: 12094-12101Abstract Full Text PDF PubMed Google Scholar, 7Ichikawa K. DeGroot L.J. Refetoff S. Horwitz A.L. Pollak E.R. Metabolism. 1986; 35: 861-868Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 8Schueler P.A. Schwartz H.L. Strait K.A. Mariash C.N. Oppenheimer J.H. Mol. Endocrinol. 1990; 4: 227-234Crossref PubMed Scopus (123) Google Scholar, 9Apriletti J.W. Baxter J.D. Lau K.H. West B.L. Protein Expr. Purif. 1995; 6: 363-370Crossref PubMed Scopus (35) Google Scholar), and some studies suggest that T3 is present at higher concentrations in the nucleus than T4 (10Surks M.I. Oppenheimer J.H. J. Clin. Investig. 1977; 60: 555-562Crossref PubMed Scopus (92) Google Scholar, 11Silva J.E. Larsen P.R. J. Clin. Investig. 1978; 61: 1247-1259Crossref PubMed Google Scholar). Nonetheless, the question of whether T4 is simply a prohormone or an active TH species is not completely resolved. T4 exerts rapid nongenomic effects at several loci distinct from TRs (12Davis P.J. Tillmann H.C. Davis F.B. Wehling M. J. Endocrinol. Investig. 2002; 25: 377-388Crossref PubMed Scopus (82) Google Scholar). Moreover, saturating levels of T4 activate transcription of TH-responsive genes in cell culture (see for example Ref. 5Samuels H.H. Stanley F. Casanova J. J. Clin. Investig. 1979; 63: 1229-1240Crossref PubMed Scopus (97) Google Scholar). Whereas it is possible that at least some of this activity is due to T3 generated from T4 in the cell, these results suggest that T4 may act as a TR agonist. Normal concentrations of plasma-free T4 are about 4-6-fold higher than those of T3 (19 pmol/liter of T4 versus 4.3 pmol/liter T3) and intracellular T4 and T3 levels can differ because of variations in uptake and T4 to T3 conversion (3Braverman L.E. Utiger R.D. 8th Ed. Werner's and Ingbar's The Thyroid: A Fundamental and Clinical Text. Lippincott Williams & Wilkins, Philadelphia, PA2000Google Scholar); thus, it is conceivable that intracellular T4 in some context could occupy a significant fraction of nuclear TRs. If T4 does behave as an agonist, then it should bind to TR in a similar way to T3 and induce conformational changes in the TR similar to those induced by T3 (13Weatherman R.V. Fletterick R.J. Scanlan T.S. Annu. Rev. Biochem. 1999; 68: 559-581Crossref PubMed Scopus (286) Google Scholar, 14Ribeiro R.C. Apriletti J.W. Wagner R.L. Feng W. Kushner P.J. Nilsson S. Scanlan T.S. West B.L. Fletterick R.J. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 1998; 65: 133-141Crossref PubMed Scopus (59) Google Scholar). T3 interacts with the TR ligand binding domain (LBD), located in the receptor C terminus. The x-ray crystal structure of TRα or TRβ complexed with T3 reveals that hormone is completely enclosed in a ligand binding pocket within the core of the LBD. It is thought that the enclosure is due to ligand-induced packing of the LBD C-terminal helix 12 (H12) against the LBD; a rearrangement that also disrupts the corepressor binding surface and the coactivator binding to of and on in 2000; PubMed Google Scholar). T3, T4 a bulky iodine at the of the crystal structures have determined for complexed with several high affinity T3, and the and R.L. Apriletti J.W. West B.L. Baxter J.D. Fletterick R.J. 1995; PubMed Scopus Google Scholar, Wagner R.L. Apriletti J.W. Kushner P.J. Baxter J.D. Fletterick R.J. K.R. 1998; PubMed Scopus Google Scholar, R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). of these the agonist contains a 5′ group that to the surface of We therefore predicted that with bulky side the of against the of the LBD and this to TR on the that 5′ appropriate packing and coactivator binding J. and of Scholar, Apriletti J.W. Wagner R.L. West B.L. Feng W. Kushner P.J. Nilsson S. Scanlan T.S. Fletterick R.J. F. Baxter J.D. 1998; Google Scholar, Apriletti J.W. R.C. West B.L. K. Nilsson S. Kushner P.J. Fletterick R.J. Scanlan T.S. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 2002; PubMed Scopus Google Scholar, J.D. Apriletti J.W. West B.L. Feng W. K. Fletterick R.J. Wagner R.L. Kushner P.J. Scanlan T.S. Nilsson S. 2002; PubMed Scopus Google Scholar, Apriletti J.W. Baxter J.D. Scanlan T.S. Chem. 2002; PubMed Scopus Google Scholar, Apriletti J.W. Baxter J.D. Scanlan T.S. J. Chem. 2002; PubMed Scopus Google Scholar). of a 5′ similar in to an iodine to the agonist the TR J.D. Apriletti J.W. West B.L. Feng W. K. Fletterick R.J. Wagner R.L. Kushner P.J. Scanlan T.S. Nilsson S. 2002; PubMed Scopus Google Scholar). it is conceivable that T4, with a 5′ iodine could behave as an in some of the way that the TRs adapt to the 5′ iodine group be therefore important for T4 and of design. this we T4 with TR, the way that T4 TR activity in vitro and in in culture and determined the x-ray crystal structure of TR in complex with We that the TR-T4 complex is than the TR-T3 and that TRs properties that are similar to TRs in solution. Nonetheless, T4 behaves as an agonist in and The x-ray structure of the TR complex reveals that a niche in the ligand binding pocket to the of the pocket in the to the 5′ to pack against the LBD surface in the of the the enclosed TR hormone binding pocket accommodates T4 complete of results suggest that T4 act as a TR agonist present at high concentrations in the Thyroid hormone binding and as J.W. Baxter J.D. Lau K.H. West B.L. Protein Expr. 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The binding at the a that for at the at for at a and J.D. Apriletti J.W. West B.L. Feng W. K. Fletterick R.J. Wagner R.L. Kushner P.J. Scanlan T.S. Nilsson S. 2002; PubMed Scopus Google Scholar), and a with a into the of the hormone of and TRβ LBD for affinity and hydrophobic interaction and then with hormone as R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). of the T3 complex obtained by the with a and of and by in of and with 3, and to a through a with in of the T4 complex obtained with a of and of and with LBD of the to be to then as for the T3 with a of and at the the T3 of with for the T4 of with in and in of the the structure with hormone as a The structure then to of by and in in for then of the the of to the structure and in with in and by and of the and a of the T4 to in and and and in as for the T3 of the higher of the for the T3 the T3 structure then least in to the of the T4 A of from the T3 complex and ligand from the T4 complex and used for against the T4 complex against this to and in this as with of the that the structure of the T4 complex is to the T3 complex used to the and as for the T3 The the and a in of TRs with T3 and T4 We determined that T3 an affinity for TRs about higher than T4 J.W. Eberhardt N.L. Latham K.R. Baxter J.D. J. Biol. Chem. 1981; 256: 12094-12101Abstract Full Text PDF PubMed Google Scholar). T4 also from TRs than T3 and Whereas it and for the T3 to from in vitro of TRα and TRβ at it about and for of the T4 to from TRα and TRs form a complex with T4 that is than the TR-T3 We examined of TRs from hydrophobic interaction an that conformational alterations in and a of J.W. Baxter J.D. Lau K.H. West B.L. Protein Expr. Purif. 1995; 6: 363-370Crossref PubMed Scopus (35) Google Scholar). with results J.W. Baxter J.D. Lau K.H. West B.L. Protein Expr. Purif. 1995; 6: 363-370Crossref PubMed Scopus (35) Google Scholar), of TRs Moreover, TR in complex with several T3, and together, with of ligand for TR the complex to results also obtained with the TRβ LBD; the complex from the the complex and TRβ It is that the of the TR-T4 complex is related to the affinity of T4 for the complex at a similar to TR agonist complexes binds TRα with an affinity times lower than T4 for versus for It is also that the is related to rapid T4 over dissociation of T4 to a and not a as the with the complex may T4 dissociation Nonetheless, we examined migration of to TRα on T4 at the as the T4 not from the in these that TR to T4 as it over the of the TR-T4 complex an conformation that hydrophobic surface than TRs in complex with T3 or of of determined whether T4 as an agonist that T4 and T3 levels of TR binding to nuclear receptor of the and T4 and T3 also activity in binding of and to TR from of the of the T4 and T3 behave as in TR complete dissociation of TR not from DNA as or R.C. Kushner P.J. Apriletti J.W. West B.L. Baxter J.D. Mol. Endocrinol. 6: Google Scholar, R.C. Apriletti J.W. P.M. Baxter J.D. PubMed Scopus Google Scholar). T4 and T3 TRα from an and of TR with the of TH binding in the results also obtained and with TRβ and Nonetheless, TR migration in the of T4 than T3 for the T4 T3 in of of DNA binding TR-T4 and TR-T3 complexes the that TR-T4 complexes adopt a structure from TR-T3 complexes in (see as a TR in examined the of T4 in cell T4 a similar to T3 in that with a and a that TRα cells, or TRβ cells, J.D. Apriletti J.W. West B.L. Feng W. K. Fletterick R.J. Wagner R.L. Kushner P.J. Scanlan T.S. Nilsson S. 2002; PubMed Scopus Google Scholar, K. K. K. J. J. J. Chem. PubMed Scopus Google Scholar). T4 a that is about that of T3. It is that T4 to T3 conversion for the activity of T4 in these for several of with of the acid or not the T3 or T4 and we not significant T4 to T3 conversion by high of cell Moreover, T4 with T3 for of TH on a a in to T4 that T4 to T3 conversion of can be in this T4 behaves as a agonist in in in which conversion to T3 is to contribute to agonist activity. The of the the LBD to the of the how TRs T4 within the ligand binding pocket and the as an agonist the of the bulky 5′ iodine we obtained structures of the in complex with T3 and and are in A of the TRβ structure over the TRβ structure is in The complexes are similar in to and to determined structures of TR to Moreover, T4 is completely buried within the core of the LBD, The structure a pocket of the structure a pocket of as determined by K.A. PubMed Scopus Google Scholar). The and structures the group and crystal in pocket these two structures are to the in the of the and in a of the TR-T4 and TR-T3 complexes LBD structures in in distinct The the loop and the of the ligand binding pocket and to the 5′ iodine group The of the DNA binding domain C-terminal helix which is in this of the and and the loop and and the of of these of TR in that to of the R.L. Apriletti J.W. West B.L. Baxter J.D. Fletterick R.J. 1995; PubMed Scopus Google Scholar, Wagner R.L. Apriletti J.W. Kushner P.J. Baxter J.D. Fletterick R.J. K.R. 1998; PubMed Scopus Google Scholar, R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). alterations in these are to be significant for ligand than those of The loop is by about in the TR-T4 structure to the TR-T3 structure the C of is the pocket, a also present in the T3 structure and TR LBD and the C-terminal of is in the of these that the coactivator binding surface and the of and adopt a structure with to those in the TR-T3 and side is with the that T4 coactivator binding in and agonist activity in Nonetheless, in that with the of the LBD in the T3 complex than in the T4 that against the LBD. Moreover, the for higher for TR-T4 than for TR-T3 and the TR-T4 structure lower than the TR-T3 structure the complex a of than the TR-T3 The conformational alterations that within the hormone binding pocket the T4 5′ iodine group are in in and the 5′ iodine into a in the of the pocket is to similar that the iodine in the T4 and T3 structures and is of two distinct an that of from several helices that the pocket of the LBD on and on and a lower of on and and on niche permits TR to T4 completely within the enclosed pocket the of the 5′ iodine The niche is also present within the it is smaller of the TR-T4 and TR-T3 complexes reveals this of the pocket in the of T4 T4 with is a of a of amino acid side The which the 5′ iodine in the TR-T4 If the receptor in the in a and the the ligand the of the receptor to the of ligand in the complex in the 5′ iodine group and side chains of two on the in results from a the 5′ iodine and (see for alterations enlarge the niche that accommodates the 5′ iodine and to pack against the LBD and complete the coactivator binding alterations in the ligand binding pocket that TRβ to of the side chains and of T4 and of in a The studies T4 with TRs. in the T3 is thought to be the major active form of TH and binds TRs with the affinity of T4 J.W. Eberhardt N.L. Latham K.R. Baxter J.D. J. Biol. Chem. 1981; 256: 12094-12101Abstract Full Text PDF PubMed Google Scholar). T4 levels about to times that of T3 and intracellular can it is conceivable that T4 could occupy a significant fraction of nuclear TRs in some The lower affinity of T4 for TRs is related to the bulky on our structures of in complex with T3 and related should not fit into the hormone binding pocket R.C. Apriletti J.W. Wagner R.L. Feng W. Kushner P.J. Nilsson S. Scanlan T.S. West B.L. Fletterick R.J. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 1998; 65: 133-141Crossref PubMed Scopus (59) Google Scholar, R.L. Apriletti J.W. West B.L. Baxter J.D. Fletterick R.J. 1995; PubMed Scopus Google Scholar, Wagner R.L. Apriletti J.W. Kushner P.J. Baxter J.D. Fletterick R.J. K.R. 1998; PubMed Scopus Google Scholar, R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). of some bulky 5′ on high affinity TR can Apriletti J.W. R.C. West B.L. K. Nilsson S. Kushner P.J. Fletterick R.J. Scanlan T.S. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 2002; PubMed Scopus Google Scholar). we how T4 interacts with TR, whether it behaves as an agonist or and how it can fit into the TR ligand binding We examined properties of TR-T4 We that T4 to TR than T3, and that T4 from TRs than T3, Moreover, the TR complex with T4 is than that with T3, as by migration of TR-T4 complexes to TRs than to complexes on A and and in with DNA The is not a of lower affinity of TR for T4, because TR complexes with a 5′ of the affinity of T4 for at a similar to TR-T3 complexes It is also to rapid dissociation of T4 on the because TRs in complex with T4 also at a similar to TRs in complex with T4 the of the TR-T4 of T4 as as those of T3 in of and of and and dissociation of TR from DNA Moreover, of T4 as as those of T3 in activity of a in that TRα or and in in which it is that agonist activity of T4 from T3 or intracellular conversion of T4 to T3. it is that T4 and T3 similar conformational within the in these and that into the active conformation in the of The crystal structure of the complex the that T4 a TR conformation similar to that with higher affinity TR this T4 into the ligand binding pocket the of the 5′ iodine The pocket accommodates the bulky 5′ iodine in the of several amino acid side chains in the pocket to in the TR-T3 changes enlarge a niche that to the of the and the and of the 5′ iodine and The for these alterations for of T4 to T3 the affinity of the TRs for T4 to T3. the niche permits to over the bulky iodine group and complete the coactivator binding surface. the of an niche in the TR ligand binding pocket T4 to behave as agonist, the the active conformation in the of T4, our crystal structures that the loop is and against the LBD in the of T4 than in the of T3 and suggest for the the of TR-T4 and TR-T3 A of to that and the of the of the pocket and T4 to packing of also of the hydrophobic of the of the TR-T4 complex in and While T4 behaves as a agonist in our it is conceivable that the packing of induced by T4 versus T3 could the TR to that the to the in with high corepressor coactivator be into the conformation and T4 could agonist, or activity. We not have a similar structure of TRα in complex with T4, are the TR in of LBD R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar), as activity in the of T4 and T3 and that TRα adapt to the 5′ iodine extension in a similar way to T4 from TRβ than from TRα this TRβ to in the of the than structures of TRα in complex with the ligand by R.L. Scanlan T.S. Apriletti J.W. Baxter J.D. West B.L. Fletterick R.J. Mol. Endocrinol. 2001; PubMed Scopus Google Scholar). in within the TR may the dissociation of We that TR with bulky 5′ side chains should and act as in Ref. Apriletti J.W. R.C. West B.L. K. Nilsson S. Kushner P.J. Fletterick R.J. Scanlan T.S. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 2002; PubMed Scopus Google Scholar). the extension J. and of Scholar, Apriletti J.W. Wagner R.L. West B.L. Feng W. Kushner P.J. Nilsson S. Scanlan T.S. Fletterick R.J. F. Baxter J.D. 1998; Google Scholar), by our of TR on these J.D. 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Fletterick R.J. Scanlan T.S. Baxter J.D. J. Steroid. Biochem. Mol. Biol. 2002; PubMed Scopus Google and studies to an which that in a of to hormone and to act as The TR-T4 crystal structure reveals that the pocket can to the 5′ iodine with a of the structure to T3. of is for the pocket to and to smaller P.R. J. Mol. Biol. PubMed Scopus Google Scholar, P.R. PubMed Scopus Google Scholar, Williams 2001; PubMed Scopus Google Scholar). this packing does not to in the we have versus TRs. we that TR accommodates a ligand that binds TRβ with about the affinity of and a extension and a 5′ S. M. Baxter J.D. Scanlan T.S. Fletterick R.J. S. PubMed Scopus Google Scholar), by a hydrophobic on the of and that is not of the It be to how TR accommodates with 5′ J.D. Apriletti J.W. West B.L. Feng W. K. Fletterick R.J. Wagner R.L. Kushner P.J. Scanlan T.S. Nilsson S. 2002; PubMed Scopus Google Scholar, Apriletti J.W. Baxter J.D. Scanlan T.S. Chem. 2002; PubMed Scopus Google Scholar, Apriletti J.W. Baxter J.D. Scanlan T.S. J. Chem. 2002; PubMed Scopus Google Scholar), or with 5′ as the group and the act as antagonists. studies not the question of whether T4 is a species of TH in in the concentrations in the it to the to which intracellular T4 in TR Nonetheless, the that TR can to a niche that accommodates the T4 5′ with the that the of T4 is about that of T3 in cell culture and that T4 concentrations are 4-6-fold those of T3, the distinct that T4 could significant agonist activity in