Characterization of the DNA-independent dimerization interface of the rat glucocorticoid receptor.

The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of transcription factors, binds DNA as a ligand induced homodimer. Within the DNA binding domain, a short sequence of amino acids called the D-box, has been shown by mutagenesis and structural studies to function as a DNA-dependent dimerization interface. The importance of the D-box for DNA-dependent dimerization by GR is well known. A second dimerization activity necessary for dimerization in solution in the absence of DNA has been proposed for GP, based on homology with other steroid receptors. While the DNA-dependent dimerization, activity has been extensively studied, very little is known about the DNA-independent dimerization activity in GR. The subject of this thesis is the characterization of the solution dimerization activity in GR. Three independent assays demonstrated solution dimerization of GR: co-immunoprecipitation, GST pull-down and two-hybrid assay in yeast. The GR, in its steroid free state, is packaged into a heat shock protein containing multiprotein complex. My results showed that solution dimerization of GR required dissociation of the heat shock protein complex. However, dimerization did not appear to have a strict ligand requirement as efficient dimerization occurred with both salt- and antagonist-treated receptors. Further, by all three assays, I show that amino acids 505 to 547 are required for GR solution dimerization. Interestingly, these amino acids overlap with the nuclear localization signal-1 in GR, the binding site for the NLS receptor importin alpha. Additionally, my data also provided evidence for protein interaction surfaces in the amino and carboxy terminal domains of GR, results that are consistent with the idea that multiple regions of GR contribute, either directly or indirectly, to homodimerization. Co-transfection experiments with wild type GR and a GR mutant, which had a mutation in one of its nuclear localization signals such that it was unable to efficiently transfer to the nucleus in response to hormone, demonstrated an interaction between full-length GR monomers in vivo. Data from the co-transfection assay also suggested that GR homodimerizes in the cytoplasm prior to nuclear import. Finally, I show for the first time that GR has the potential to heterodimerize with several members of the nuclear receptor superfamily in solution. These results provide the first detailed characterization of the GR solution dimerization activity