Mineralocorticoid and Glucocorticoid Receptor Interactive Complexes and their Cellular Localisation

Abstract

Adrenal glucocorticoids (GCs) released into circulation exhibit circadian and ultradian profiles, which are important in regulating homeostasis and physiological pathways. GCs induce gene expression changes in targeted cells via ligand activated transcription factors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), which produce both physiological and adaptive responses to stress. MR and GR are highly abundant in the hippocampus, an area vital for learning and memory, and effective responses to stressful events involve modification of cognitive processes in the brain to improve coping mechanisms to future stressors. MR and GR regulate transcription of GC targets, often as homodimers, but the existence and significance of MR/GR heteromers in gene regulation remains unclear. Using a proximity ligation assay (PLA) for the first time, MR and GR have been shown to be in very close proximity and are consequently likely to be interacting together. MR:GR complexes were detected both in cultured cells (3617 and Neuro-2A) transfected with MR/GR expression vectors and in rat hippocampal tissue where endogenous MR and GR are co-expressed. Nuclear MR:GR complexes were localised to heterochromatic regions, in proximity to the nuclear lamina. The interaction appears to require neither the DNA binding domain, nor the dimerization interface, indicating another mode of MR:GR interaction. In the absence of ligand, MR:GR complexes were unexpectedly detected in the cytoplasm, predominantly at the nuclear envelope. Using RT-qPCR, the transcriptional consequences of MR/GR alone or together with ‘physiological’ levels of CORT suggested cooperative regulation of some GC target genes. An optimised protocol for in situ PLA and the modular image analysis (MIA) pipeline are presented, for the detection and analysis of MR:GR complexes in Neuro-2A cells and hippocampal tissue, which are applicable to other receptor-receptor interaction studies in order to reveal dynamic spatio-temporal protein-protein interactions.

Date of Award	12 May 2020
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stafford L Lightman (Supervisor) & Caroline A Rivers (Supervisor)