MODY1 MUTATION Q268X IN HEPATOCYTE NUCLEAR FACTOR 4-ALPHA ALLOWS FOR DIMERIZATION IN SOLUTION BUT CAUSES ABNORMAL SUBCELLULAR-LOCALIZATION

Recent studies have shown that mutations in the hepatocyte nuclear fac tor (HNF)-4 alpha gene give rise to maturity-onset diabetes of the you ng, type 1 (MODY1). HNF-4, an orphan member of the nuclear receptor su perfamily, contains a DNA-binding domain (DBD) and a putative ligand-b inding domain (LBD) that can act independently of each other. The firs t MODY1 mutation identified creates a stop codon at amino acid 268 in the LED of HNF-4 (Q268X) that leaves the DBD intact, suggesting that t he mutant protein may retain some of the properties of the wild-type p rotein. To determine the functional properties of this mutant, we cons tructed HNF4.Q268X and tested it in vitro and in vivo for DNA binding, protein dimerization, and transactivation activity. Results of an ele ctrophoretic mobility shift assay showed that HNF4.Q268X neither binds DNA alone nor binds it as a dimer with wild-type HNF-4 (HNF4.wt). In contrast, a co-immunoprecipitation assay showed that HNF4.Q268X is cap able of dimerizing in solution with HNF4.wt. Transient transfection as says, however, indicated that HNF4.Q268X does not affect transactivati on by HNF4.wt in vivo, supporting the argument against a dominant nega tive effect. Additional results suggest that the lack of a dominant ne gative effect could be due to a striking differential subcellular loca lization of the HNF4.Q268X protein: HNF4.Q268X could be extracted from transfected cells only when treated with SDS. Taken together, our res ults suggest that the MODY1 phenotype is due to a loss of functional H NF-4 protein that is aggravated in tissues that express relatively low amounts of HNF-4, such as pancreas.