EXCLUSIVE HOMODIMERIZATION OF THE ORPHAN RECEPTOR HEPATOCYTE NUCLEAR FACTOR-4 DEFINES A NEW SUBCLASS OF NUCLEAR RECEPTORS

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and five r-specific gene expression, is very similar to another superfamily mem ber, retinoid X receptor alpha (RXR alpha), in overall amino acid sequ ence and DNA binding specificity. Since RXR alpha is known to heterodi merize with many other nuclear receptors, the formation of heterodimer s between HNF-4 and RXR alpha was examined. With the electrophoretic m obility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not f orm heterodimeric complexes on DNA with a number of other receptors, i ncluding RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack o f heterodimerization between HNF-4 and RXR alpha is due to a strong ho modimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to Show that HNF-4 is in fact a stable homo dimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cel ls, which express endogenous HNF-4, and transfected COS cells, which o verexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nuc leus and which bind DNA and regulate transcription as homodimers.