Genetic studies have shown that mutations in the gene encoding hepatocyte nuclear factor (HNF)-4alpha, a member of the steroid/thyroid hormone receptor superfamily, give rise to early-onset type 2 diabetes (MODY1). The functional properties of mutant HNF-4alpha proteins and the molecular mechanisms by which they impair insulin secretion are largely unknown. In the present study, we have investigated transcriptional activation, DNA binding properties, and protein dimerization activity of three HNF-4alpha missense mutations--HNF4(R127W), HNF4(V255M), and HNF4(E276Q)--that have been associated with type 2 diabetes. We demonstrate that HNF4(E276Q) has lost its ability to bind to HNF-4 consensus binding sites and activate transcription. HNF4(E276Q) had no effect on the functional activity of wild-type HNF-4alpha in the pancreatic beta-cell line HIT-T15, but it exhibited weak dominant-negative activity in other cell types. Analysis of HNF4(E276Q) protein showed that it exists in two forms: a full length 54-kDa protein and a 40-kDa COOH-terminal protein lacking the NH2-terminal transactivation domain and the DNA binding domain. Immunoprecipitation experiments indicate that this truncated protein can bind to wild-type HNF-4alpha and may be responsible for the weak dominant-negative effects seen in these cells. In addition, we show that the transcriptional transactivation of HNF4(R127W) and HNF4(V255M) is indistinguishable from that of wild-type HNF-4alpha, suggesting that they are sequence polymorphisms. Our results demonstrate that HNF4(E276Q) is a loss-of-function mutation and that it identifies glutamic acid 276 in alpha-helix 8 of the ligand-binding domain of HNF-4alpha protein as a critical residue for DNA binding, transcriptional activation, and protein stability in vivo.

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