Glucocorticoid receptor structure as probed by endogenous proteases

Area of Science:

• Molecular biology
• Cellular signaling
• Biochemistry

Background:

• The glucocorticoid receptor (GR) mediates cellular responses to glucocorticoids.
• Transformation of the GR complex involves conformational changes affecting its function.
• Proteolytic processing plays a role in regulating receptor activity and turnover.

Purpose of the Study:

• To investigate the proteolytic processing of the glucocorticoid receptor during transformation.
• To identify cleavage sites and their impact on receptor structure and function.
• To understand the role of calcium and inhibitors in GR processing.

Main Methods:

• Incubation of rat thymus and brain cytosol with calcium and heating.
• Covalent labeling of the GR with [3H]dexamethasone mesylate.
• Analysis of molecular weight and Stokes' radii of receptor complexes.
• Proteolytic digestion and fragment analysis.

Main Results:

• Heating cytosol induced formation of truncated GR complexes (DI, DIIc).
• Leupeptin inhibited DIIc formation, favoring intact DIIa.
• DI and DIIc share Mr (48 kDa) but differ in shape, suggesting cleavage at the same site.
• Proteolysis identified specific cleavage sites in non-transformed and transformed GR, with some sites protected in the transformed state.
• Receptor fragments were detected in thymocyte nuclei, indicating involvement in nuclear translocation and turnover.

Conclusions:

• Glucocorticoid receptor transformation involves specific proteolytic cleavages.
• Calcium and inhibitors modulate these cleavages, affecting receptor processing.
• Proteolytic susceptibility changes in the transformed receptor may relate to unmasking of enhancer-activating and DNA-binding functions.