title: Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: association with altered ligand affinity.
abstract: Although LXXLL motifs in coactivators mediate binding to liganded nuclear receptors, the roles of comparable motifs within nuclear receptors are less understood. We investigated the role of the LXXLL motifs in the human glucocorticoid receptor both in transcriptional activation and repression of gene expression. The two conserved LXXLL motifs within the ligand binding domain of the receptor, amino acids 532-536 (helix 1) and 718-722 (helix 10), were characterized by evaluating LXXLL mutant receptors as well as comparable mutants in other helices of the ligand binding domain. All mutant receptors were expressed at comparable levels to wild type in COS-1 cells. Mutation of 532-536 LXXLL to LXXAA completely disrupted dexamethasone induced transcription, whereas the 718-722 LXXAA mutant fully activated reporter genes at high ligand concentrations. Ligand binding analysis demonstrated that both LXXLL motif mutations resulted in disruption of ligand binding capacity without altering their association with hsp90. Proteolytic cleavage studies suggested that mutations of the LXXLL motifs introduced changes in the receptor conformation. Interestingly, the 532-536 LXXAA mutant was not able to transrepress NF-kappaB activity, whereas the 718-722 LXXAA mutant did so in the presence of ligand. These data suggest that although LXXLL motifs in helices 1 and 10 appear to lie outside the predicted ligand binding pocket they may contribute to receptor ligand binding affinity.
authors.affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, United States.
authors.forename: Diane D
authors.initials: DD
authors.lastname: Dong
authors.forename: Christine M
authors.initials: CM
authors.lastname: Jewell
authors.forename: Rachelle J
authors.initials: RJ
authors.lastname: Bienstock
authors.forename: John A
authors.initials: JA
authors.lastname: Cidlowski
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D015870:Gene Expression; D005938:Glucocorticoids / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection; D014222:Triamcinolone Acetonide / Q000494:pharmacology
chemical_list: D005938:Glucocorticoids; D018841:HSP90 Heat-Shock Proteins; D008024:Ligands; D016328:NF-kappa B; D011965:Receptors, Glucocorticoid; D049452:Green Fluorescent Proteins; D014222:Triamcinolone Acetonide