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Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1)., (Copyright 2000 Academic Press.)

We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors. In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system. The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS). The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues. Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample. DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS. Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations. Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well.