ausblenden:
Schlagwörter:
Amino Acids/chemistry
Animals
Arginine/chemistry
Biotin/chemistry
COS Cells
Cell Line
Chloramphenicol O-Acetyltransferase/metabolism
DNA-Binding Proteins/chemistry/*physiology
Detergents/pharmacology
Dimerization
Gene Deletion
Genetic Vectors
Glutathione Transferase/metabolism
Hepatocyte Nuclear Factor 4
Humans
Isoleucine/chemistry
Leucine/chemistry
Ligands
Methionine/chemistry
Models, Biological
Mutation
Phosphoproteins/chemistry/*physiology
Plasmids/metabolism
Point Mutation
Protein Binding
Protein Structure, Tertiary
Serine/chemistry
Transcription Factors/chemistry/*physiology
Transcription, Genetic
Transfection
Zusammenfassung:
Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.
