Distinct amino acid residues may be involved in coactivator and ligand interactions in hepatocyte nuclear factor-4alpha.
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.
Iordanidou
Panagiota
P
Department of Genetics, Development and Molecular Biology, Laboratory of Developmental Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece 54124.
Aggelidou
Eleni
E
Demetriades
Constantinos
C
Hadzopoulou-Cladaras
Margarita
M
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019556:COS Cells; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D017353:Gene Deletion; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D000596:Amino Acids; D004268:DNA-Binding Proteins; D003902:Detergents; C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D008024:Ligands; D010750:Phosphoproteins; D014157:Transcription Factors; D007532:Isoleucine; D012694:Serine; D001710:Biotin; D001120:Arginine; D008715:Methionine; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase; D007930:Leucine