L-764406 Is a Partial Agonist of Human Peroxisome Proliferator-activated Receptor γ

Abstract

Insulin-sensitizing thiazolidinedione (TZD) compounds are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) γ. A scintillation proximity assay for measurement of3H-radiolabeled TZD binding to human PPARγ under homogeneous conditions was developed. Using this approach, a novel non-TZD compound (L-764406) was shown to be a potent (apparent binding IC50 of 70 nm) PPARγ ligand. Preincubation of PPARγ with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addition, structurally related analogues ofL-764406, which would be predicted not to interact with PPARγ in a covalent fashion, did not displace [3H]TZD binding to PPARγ. Covalent binding of L-764406 was proven by an observed molecular weight shift of a tryptic PPARγ ligand binding domain (LBD) peptide by mass spectrometric analysis. A specific cysteine residue (Cys313 in helix 3 of hPPARγ2) was identified as the attachment site for this compound. In protease protection experiments, the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor containing the PPARγ LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast,L-764406 did not exhibit activity in cells transfected with chimeric receptors containing PPARα or PPARδ LBDs. The partial agonist properties of L-764406 were also evident in a co-activator association assay, indicating that the increased transcription in cells was co-activator mediated. Thus,L-764406 is a novel non-TZD ligand for PPARγ and is also the first known partial agonist for this receptor. The results suggest a critical functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes. Insulin-sensitizing thiazolidinedione (TZD) compounds are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) γ. A scintillation proximity assay for measurement of3H-radiolabeled TZD binding to human PPARγ under homogeneous conditions was developed. Using this approach, a novel non-TZD compound (L-764406) was shown to be a potent (apparent binding IC50 of 70 nm) PPARγ ligand. Preincubation of PPARγ with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addition, structurally related analogues ofL-764406, which would be predicted not to interact with PPARγ in a covalent fashion, did not displace [3H]TZD binding to PPARγ. Covalent binding of L-764406 was proven by an observed molecular weight shift of a tryptic PPARγ ligand binding domain (LBD) peptide by mass spectrometric analysis. A specific cysteine residue (Cys313 in helix 3 of hPPARγ2) was identified as the attachment site for this compound. In protease protection experiments, the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor containing the PPARγ LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast,L-764406 did not exhibit activity in cells transfected with chimeric receptors containing PPARα or PPARδ LBDs. The partial agonist properties of L-764406 were also evident in a co-activator association assay, indicating that the increased transcription in cells was co-activator mediated. Thus,L-764406 is a novel non-TZD ligand for PPARγ and is also the first known partial agonist for this receptor. The results suggest a critical functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes. peroxisome proliferator-activated receptor ligand binding domain scintillation proximity assay glutathioneS-transferase upstream activator sequence liquid chromatography-electrospray ionization-mass spectrometry homogeneous time-resolved fluorescence assay CREB-binding protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid retinoic acid receptor estrogen receptor Peroxisome proliferator-activated receptors (PPARs)1are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar, 2Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). They are currently divided into three subtypes: PPARα, PPARδ (also known as hNUC1 or PPARβ), and PPARγ, with each being encoded by a distinct gene. The presence of at least two promoters in the 5′-flanking region of PPARγ results in the production of two isoforms, γ1 and γ2 (3Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Sambasiva R.M. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (598) Google Scholar), where γ2 possesses an additional 30 residues at its amino terminus (4Elbrecht A. Chen Y. Cullinan C.A. Hayes N. Leibowitz M.D. Moller D.E. Berger J. Bichem. Biophys. Res. Commun. 1996; 224: 431-437Crossref PubMed Scopus (352) Google Scholar). Like other members of this superfamily, the PPARs exert their effects by regulating gene transcription and all three members bind to DR1 response elements (reviewed in Ref. 1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar).Although the three subtypes have been grouped together based on sequence homology, it is clear that they have distinct functions. PPARα is expressed predominantly in the liver and is involved in peroxisome proliferation and regulation of fatty acid catabolism (5Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar). PPARγ plays a pivotal role in fat cell differentiation and lipid storage (6Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3092) Google Scholar), while PPARδ is expressed in most cell types but its role remains unclear (7Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar). Consistent with their distinct physiological roles, each receptor has been shown to bind a discrete set of ligands, although, in general, these receptors seem to be regulated by fatty acids and eicosanoids (8Kliewer S.A. Lehmann J.M. et al.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1870) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google Scholar, 10Krey G. Wahli W. et al.Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar). Thus, for PPARα, selected fatty acids serve as potential natural ligands; in addition, the fibrate class of hypolipidemic drugs and a group of structurally diverse peroxisome proliferators can also function as PPARα ligands and/or activators. The synthetic thiazolidinedione insulin sensitizers (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. S.A. J. 1995; Full Text Full Text PDF PubMed Scopus Google and the bind and PPARγ S.A. J.M. I. Lehmann J.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar, B.M. P. Chen J. Spiegelman B.M. Evans R.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar), while fatty acids and eicosanoids can serve to PPARδ (7Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google receptors a role in lipid where can to and to PPARs have been to be for Ref. Scopus Google Scholar), and insulin Ref. B. J. N. J. 1994; PubMed Scopus Google Scholar). Thus, has been in and specific ligands for these receptors Smith-Oliver T.A. D.J. S.A. J.M. 1997; Full Text PDF PubMed Scopus Google Scholar, S. P. J. Res. Full Text Full Text PDF PubMed Google Scholar). clear that of the of natural or synthetic which can function as ligands and is an a of the residues in the ligand binding which with agonist ligands would the subsequent of and of these a thiazolidinedione and human PPARγ to a scintillation proximity assay in to ligands that bind this receptor. Using this approach, a novel non-TZD was shown to be a high affinity PPARγ ligand. were to that this compound as a specific agonist for PPARγ. was also shown to bind to PPARγ. was in to that the compound to in helix 3 of the LBD of human The of as the attachment site an role for this residue and for helix 3 in ligand binding and of a novel binding assay for the nuclear PPARγ. is a assay that not of and Using this was shown to function as a potent ligand for the of this that it bind to the PPARγ was also by the that three related compounds the were in the PPARγ binding in of this the that of PPARγ with this compound the of a potent TZD ligand to displace L-764406 covalent binding of L-764406 to PPARγ was proven by mass of PPARγ LBD with this a molecular mass in peptide was which to the molecular weight ofL-764406, for the of L-764406 [3H]TZD at it was of partial agonist activity at these its to gene transcription was was chimeric receptors in cells or in 3T3-L1 where receptors are and can serve to aP2 In of the activity with a TZD was partial agonist is for a PPARγ other known ligands thiazolidinedione insulin sensitizers (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. S.A. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. C. Cullinan C.A. Hayes R. Leibowitz M.D. 1996; PubMed Scopus Google and S.A. J.M. I. Lehmann J.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar, B.M. P. Chen J. Spiegelman B.M. Evans R.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google function as in transfected cells and the molecular for partial agonist activity is not it be to the production of a receptor with a affinity for Mol. Endocrinol. 1995; PubMed Google have that the estrogen receptor can distinct of the receptor to ligands with agonist also that these are on and it is as a agonist under In protease protection a that was the thiazolidinedione agonist estrogen receptor are known to a distinct protease protection that of J. 1992; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1995; PubMed Google Scholar), that conformational for the partial agonist activity of L-764406 be for by the protease protection agonist activity of L-764406 is specific for PPARγ, activity was with chimeric receptors containing PPARα or PPARδ LBDs. A sequence of the for and that the cysteine in PPARγ is in the other two PPARγ has cysteine in the the PPARα LBD two additional cysteine residues the with of these additional also being in Thus, the of potential attachment in the of and not these receptors in the LBD of PPARγ is a predicted domain predicted the LBD were the sequence that to helix 3 as in the of the N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google and M. Nature. 1997; PubMed Scopus Google LBDs. residues helix 3 of the LBD have been shown to interact with the of K. J. Full Text Full Text PDF PubMed Scopus Google Scholar), as as the M. Nature. 1997; PubMed Scopus Google Scholar). The specific residues of the LBD that interact with and are not in a cysteine has not been identified in the estrogen a cysteine to is in the of human and but not in and In to with its helix 3 the LBD of with helix and be involved in the of the domain N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). a ligand at helix 3 are into with the have shown that the partial a interaction with this interaction not be by subsequent of a potent TZD it that the of the the ligand binding to by that is not an for TZD binding the is in this at a these suggest that the activity of L-764406 as a partial agonist is by a interaction with it be that L-764406 as a partial agonist in the and and in the in co-activator association assay it is that this compound function as a agonist in not a cysteine residue to the in the LBD of PPARγ, have a residue at this In the of for is the the of and and has been shown to for their ligand N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). that the residue at this in helix 3 is involved in the of ligand for other nuclear the of et S. R. Nature. PubMed Scopus Google their results where was to the PPARγ LBD that is helix 3 and that helix 3 an of the ligand binding by the are two and which of a by the of the TZD in S. R. Nature. PubMed Scopus Google Scholar). is to of a by the of S. R. Nature. PubMed Scopus Google Scholar). results a novel covalent ligand an of which the of helix 3, and in Cys313, in critical of the PPARγ ligand binding L-764406 was shown to agonist activity in cells and an LBD protease protection that was to that by a known it is that the interaction of ligands with helix 3 is for the of conformational which co-activator and of is also known PPARγ ligands in that it as a partial agonist co-activator and the that PPARγ ligands, which exhibit or and or Peroxisome proliferator-activated receptors (PPARs)1are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar, 2Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar). They are currently divided into three subtypes: PPARα, PPARδ (also known as hNUC1 or PPARβ), and PPARγ, with each being encoded by a distinct gene. The presence of at least two promoters in the 5′-flanking region of PPARγ results in the production of two isoforms, γ1 and γ2 (3Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Sambasiva R.M. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (598) Google Scholar), where γ2 possesses an additional 30 residues at its amino terminus (4Elbrecht A. Chen Y. Cullinan C.A. Hayes N. Leibowitz M.D. Moller D.E. Berger J. Bichem. Biophys. Res. Commun. 1996; 224: 431-437Crossref PubMed Scopus (352) Google Scholar). Like other members of this superfamily, the PPARs exert their effects by regulating gene transcription and all three members bind to DR1 response elements (reviewed in Ref. 1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar). the three subtypes have been grouped together based on sequence homology, it is clear that they have distinct functions. PPARα is expressed predominantly in the liver and is involved in peroxisome proliferation and regulation of fatty acid catabolism (5Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar). PPARγ plays a pivotal role in fat cell differentiation and lipid storage (6Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3092) Google Scholar), while PPARδ is expressed in most cell types but its role remains unclear (7Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar). Consistent with their distinct physiological roles, each receptor has been shown to bind a discrete set of ligands, although, in general, these receptors seem to be regulated by fatty acids and eicosanoids (8Kliewer S.A. Lehmann J.M. et al.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1870) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google Scholar, 10Krey G. Wahli W. et al.Mol. Endocrinol. 1997; 11: 779-791Crossref PubMed Scopus (908) Google Scholar). Thus, for PPARα, selected fatty acids serve as potential natural ligands; in addition, the fibrate class of hypolipidemic drugs and a group of structurally diverse peroxisome proliferators can also function as PPARα ligands and/or activators. The synthetic thiazolidinedione insulin sensitizers (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. S.A. J. 1995; Full Text Full Text PDF PubMed Scopus Google and the bind and PPARγ S.A. J.M. I. Lehmann J.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar, B.M. P. Chen J. Spiegelman B.M. Evans R.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar), while fatty acids and eicosanoids can serve to PPARδ (7Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar, 9Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1850) Google Scholar). receptors a role in lipid where can to and to PPARs have been to be for Ref. Scopus Google Scholar), and insulin Ref. B. J. N. J. 1994; PubMed Scopus Google Scholar). Thus, has been in and specific ligands for these receptors Smith-Oliver T.A. D.J. S.A. J.M. 1997; Full Text PDF PubMed Scopus Google Scholar, S. P. J. Res. Full Text Full Text PDF PubMed Google Scholar). clear that of the of natural or synthetic which can function as ligands and is an a of the residues in the ligand binding which with agonist ligands would the subsequent of and of these a thiazolidinedione and human PPARγ to a scintillation proximity assay in to ligands that bind this receptor. Using this approach, a novel non-TZD was shown to be a high affinity PPARγ ligand. were to that this compound as a specific agonist for PPARγ. was also shown to bind to PPARγ. was in to that the compound to in helix 3 of the LBD of human The of as the attachment site an role for this residue and for helix 3 in ligand binding and of PPARγ. a novel binding assay for the nuclear PPARγ. is a assay that not of and Using this was shown to function as a potent ligand for the of this that it bind to the PPARγ was also by the that three related compounds the were in the PPARγ binding in of this the that of PPARγ with this compound the of a potent TZD ligand to displace L-764406 covalent binding of L-764406 to PPARγ was proven by mass of PPARγ LBD with this a molecular mass in peptide was which to the molecular weight ofL-764406, for the of L-764406 [3H]TZD at it was of partial agonist activity at these its to gene transcription was was chimeric receptors in cells or in 3T3-L1 where receptors are and can serve to aP2 In of the activity with a TZD was partial agonist is for a PPARγ other known ligands thiazolidinedione insulin sensitizers (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. S.A. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. C. Cullinan C.A. Hayes R. Leibowitz M.D. 1996; PubMed Scopus Google and S.A. J.M. I. Lehmann J.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar, B.M. P. Chen J. Spiegelman B.M. Evans R.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google function as in transfected cells and the molecular for partial agonist activity is not it be to the production of a receptor with a affinity for Mol. Endocrinol. 1995; PubMed Google have that the estrogen receptor can distinct of the receptor to ligands with agonist also that these are on and it is as a agonist under In protease protection a that was the thiazolidinedione agonist estrogen receptor are known to a distinct protease protection that of J. 1992; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1995; PubMed Google Scholar), that conformational for the partial agonist activity of L-764406 be for by the protease protection agonist activity of L-764406 is specific for PPARγ, activity was with chimeric receptors containing PPARα or PPARδ LBDs. A sequence of the for and that the cysteine in PPARγ is in the other two PPARγ has cysteine in the the PPARα LBD two additional cysteine residues the with of these additional also being in Thus, the of potential attachment in the of and not these receptors in the LBD of PPARγ is a predicted domain predicted the LBD were the sequence that to helix 3 as in the of the N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google and M. Nature. 1997; PubMed Scopus Google LBDs. residues helix 3 of the LBD have been shown to interact with the of K. J. Full Text Full Text PDF PubMed Scopus Google Scholar), as as the M. Nature. 1997; PubMed Scopus Google Scholar). The specific residues of the LBD that interact with and are not in a cysteine has not been identified in the estrogen a cysteine to is in the of human and but not in and In to with its helix 3 the LBD of with helix and be involved in the of the domain N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). a ligand at helix 3 are into with the have shown that the partial a interaction with this interaction not be by subsequent of a potent TZD it that the of the the ligand binding to by that is not an for TZD binding the is in this at a these suggest that the activity of L-764406 as a partial agonist is by a interaction with it be that L-764406 as a partial agonist in the and and in the in co-activator association assay it is that this compound function as a agonist in not a cysteine residue to the in the LBD of PPARγ, have a residue at this In the of for is the the of and and has been shown to for their ligand N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). that the residue at this in helix 3 is involved in the of ligand for other nuclear the of et S. R. Nature. PubMed Scopus Google their results where was to the PPARγ LBD that is helix 3 and that helix 3 an of the ligand binding by the are two and which of a by the of the TZD in S. R. Nature. PubMed Scopus Google Scholar). is to of a by the of S. R. Nature. PubMed Scopus Google Scholar). results a novel covalent ligand an of which the of helix 3, and in Cys313, in critical of the PPARγ ligand binding L-764406 was shown to agonist activity in cells and an LBD protease protection that was to that by a known it is that the interaction of ligands with helix 3 is for the of conformational which co-activator and of is also known PPARγ ligands in that it as a partial agonist co-activator and the that PPARγ ligands, which exhibit or and or a novel binding assay for the nuclear PPARγ. is a assay that not of and Using this was shown to function as a potent ligand for the of this that it bind to the PPARγ was also by the that three related compounds the were in the PPARγ binding in of this the that of PPARγ with this compound the of a potent TZD ligand to displace L-764406 covalent binding of L-764406 to PPARγ was proven by mass of PPARγ LBD with this a molecular mass in peptide was which to the molecular weight ofL-764406, for the of L-764406 [3H]TZD at it was of partial agonist activity at these its to gene transcription was was chimeric receptors in cells or in 3T3-L1 where receptors are and can serve to aP2 In of the activity with a TZD was partial agonist is for a PPARγ other known ligands thiazolidinedione insulin sensitizers (11Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. S.A. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. P. C. Cullinan C.A. Hayes R. Leibowitz M.D. 1996; PubMed Scopus Google and S.A. J.M. I. Lehmann J.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google Scholar, B.M. P. Chen J. Spiegelman B.M. Evans R.M. Cell. 1995; 83: Full Text PDF PubMed Scopus Google function as in transfected cells and the molecular for partial agonist activity is not it be to the production of a receptor with a affinity for Mol. Endocrinol. 1995; PubMed Google have that the estrogen receptor can distinct of the receptor to ligands with agonist also that these are on and it is as a agonist under In protease protection a that was the thiazolidinedione agonist estrogen receptor are known to a distinct protease protection that of J. 1992; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1995; PubMed Google Scholar), that conformational for the partial agonist activity of L-764406 be for by the protease protection The agonist activity of L-764406 is specific for PPARγ, activity was with chimeric receptors containing PPARα or PPARδ LBDs. A sequence of the for and that the cysteine in PPARγ is in the other two PPARγ has cysteine in the the PPARα LBD two additional cysteine residues the with of these additional also being in Thus, the of potential attachment in the of and not these receptors in the LBD of PPARγ is a predicted domain predicted the LBD were the sequence that to helix 3 as in the of the N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google and M. Nature. 1997; PubMed Scopus Google LBDs. residues helix 3 of the LBD have been shown to interact with the of K. J. Full Text Full Text PDF PubMed Scopus Google Scholar), as as the M. Nature. 1997; PubMed Scopus Google Scholar). The specific residues of the LBD that interact with and are not in a cysteine has not been identified in the estrogen a cysteine to is in the of human and but not in and In to with its helix 3 the LBD of with helix and be involved in the of the domain N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). a ligand at helix 3 are into with the have shown that the partial a interaction with this interaction not be by subsequent of a potent TZD it that the of the the ligand binding to by that is not an for TZD binding the is in this at a these suggest that the activity of L-764406 as a partial agonist is by a interaction with it be that L-764406 as a partial agonist in the and and in the in co-activator association assay it is that this compound function as a agonist in not a cysteine residue to the in the LBD of PPARγ, have a residue at this In the of for is the the of and and has been shown to for their ligand N. M. Chambon P. D. Nature. 1995; PubMed Scopus Google Scholar). that the residue at this in helix 3 is involved in the of ligand for other nuclear the of et S. R. Nature. PubMed Scopus Google their results where was to the PPARγ LBD that is helix 3 and that helix 3 an of the ligand binding by the are two and which of a by the of the TZD in S. R. Nature. PubMed Scopus Google Scholar). is to of a by the of S. R. Nature. PubMed Scopus Google Scholar). results a novel covalent ligand an of which the of helix 3, and in Cys313, in critical of the PPARγ ligand binding In L-764406 was shown to agonist activity in cells and an LBD protease protection that was to that by a known it is that the interaction of ligands with helix 3 is for the of conformational which co-activator and of is also known PPARγ ligands in that it as a partial agonist co-activator and the that PPARγ ligands, which exhibit or and or are to the for additional in addition, was by Mark and