Abstract
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.
Highlights
Nuclear receptors are, in general, defined as ligand-activated transcription factors
To confirm a previous finding that cytoplasmic CAR retention protein (CCRP) binds to ligand binding domain (LBD) of glucocorticoid receptor (GR),[17] a co-IP analysis was conducted using COS-1 cells overexpressing GR deletion mutants (Figure 1a)
GR∆LBD did not interact with CCRP while full length GR and GR LBD were co-immunoprecipitated with CCRP (Figure 1b)
Summary
In general, defined as ligand-activated transcription factors. The interaction is mediated by the binding of the LBD to a specific FXXLF sequence in the NTD and serves to modulate protein-protein interactions of AR.[1,2,3,4] The N/C interaction regulates AR functions including protein stability, dimerization, and gene activation.[5,6,7,8] The N/C interaction-defective mouse model (deletion of FXXLF) showed a clear delay of neurodegeneration induced by aggregation of AR with an expanded glutamine repeat, further confirming a physiological importance of this interdomain interactions for nuclear receptor regulation.[5] Whereas the N/C interactions were demonstrated in various other nuclear receptors, progesterone receptor (PR), estrogen receptor alpha (ERα), and mineralocorticoid receptor (MR), this concept has not yet been observed in glucocorticoid receptor (GR).[9,10,11] Whereas the N/C interactions of AR, PR, and MR are induced by ligand binding, the ERα N/C interaction does not require ligands and is suggested to be modified by cell-specific factors such as co-chaperone proteins.[3,9,10,11] It brought us a hypothesis that GR utilizes a co-chaperone protein to form a stable N/C interaction. We have investigated whether GR requires co-chaperone CCRP (cytoplasmic constitutive active/androstane receptor retention protein) for the N/C interaction and what the biological significance is
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