GR Utilizes a Co-Chaperone Cytoplasmic CAR Retention Protein to Form an N/C Interaction.

The N-terminal domain (NTD) of nuclear receptor superfamily members has been recently reported to regulate functions of the receptor through the interaction between the NTD and the C-terminal ligand binding domain (LBD), so-called an N/C interaction. Although this N/C interaction has been demonstrated in various nuclear receptors, eg, androgen receptor, this concept has not been observed in glucocorticoid receptor (GR). We hypothesized that GR requires its co-chaperone CCRP (cytoplasmic constitutive active/androstane receptor retention protein) to form a stable N/C interaction. This hypothesis was examined by co-immunoprecipitation assays using GR fragments overexpressing COS-1 cell lysate. Here, we demonstrated that GR undergoes the N/C interaction between the 26VMDFY30 motif in the NTD and the LBD. More importantly, co-chaperone CCRP is now found to induce this interaction. By the fact that a negative charge at Y30 disrupts this interaction, this residue, a potential phosphorylation site, was indicated to regulate the GR N/C interaction critically. Utilizing Y30F and Y30E mutants as N/C interacting and noninteracting forms of GR, respectively, a 2-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to examine whether or not the N/C interaction regulated formation of GR complexes. A cDNA microarray analysis was performed with COS-1 cells expressing Y30F or Y30E. We will present experimental data to demonstrate that CCRP is essential for GR to form the N/C interaction and will discuss its implications in GR functions.