SAT271 Ligand Induced GR Heterodimerization With MR In Living Cells

Disclosure: T. Sueyoshi: None. L. Perera: None. J.A. Cidlowski: None. We have established methods for monitoring heterodimer formation between GR and MR in living cells upon the addition of ligands. Two protein fragments derived from NanoBit luciferase (Small Bit peptide composed of 11 amino acids and Large Bit peptide sized 17.6 kDa) can form functional enzyme when protein-protein interaction brings these fragments close proximity. Utilizing the enzyme fragments for fusion protein partners for the hormone receptors, we have constructed expression plasmids for every combination of receptor and enzyme fragments comprising N-terminal or C-terminal fusion proteins. GR and MR fusion proteins with the luciferase fragments were expressed in HEK293 cells and the enzyme activities were determined in time course before and after the addition of ligands (dexamethasone and aldosterone). We observed robust increases of NanoBit luciferase activity in GR and MR transfected cells less than 20 min after the addition of ligands suggesting the heterodimer was formed in the time period. Similar assays detected these receptors formed homodimer after the addition of ligand for each receptor suggesting homodimer and heterodimer co-exists when appropriate ligands are in this assay system. DNA binding null mutations of GR DNA binding domain effectively abolished GR homodimers but not GR - MR heterodimers suggesting mechanistical differences between the homodimer and heterodimer formation. Based on the results only C-terminal NanoBit fusion receptors showed positive heterodimerization-induced enzyme activity, we proposed ligand binding domain 3D structure heterodimer models and found amino acid residues in helix 9, helix 10, and near C-terminus are involved in the heterodimer interaction interface. Thus, this method will be a powerful tool for analyzing molecular mechanisms of GR - MR heterodimer formation in living cells. Presentation: Saturday, June 17, 2023