Receptor-protein interactions in transformed and untransformed glucocorticoid receptor complexes.

Abstract

The glucocorticoid receptor has a functional cycle which begins when the cytoplasmic receptor is transformed (activated) by hormone, translocates to the nucleus and induces gene transcription. This nuclear receptor is ultimately cycled back to the cytoplasm in its original, untransformed state. My thesis examines receptor-protein interactions which may occur during receptor translocation to the nucleus and recycling to the cytoplasm. I recently proposed that movement of glucocorticoid receptors into and within the nucleus may involve translocation along cytoskeletal protein scaffolds. In this work I describe a cytosolic system in which glucocorticoid receptors bind to a protein particulate composed largely of cytoskeletal proteins. Incubation of mouse L cell cytosol in a glutamate-containing buffer at 37$\sp\circ$C converts the receptor to a form that pellets with a large amount of tubulin, actin and vimentin (three cytoskeletal proteins) when centrifuged at 150,000 $\times$ g. Addition of an ATP-regenerating system increases the rate of receptor pelleting. Examination of the behavior of glucocorticoid receptor mutants demonstrates that residues 445-795 of the rat receptor (DNA-binding and hormone-binding domains) contain the features required for binding to the cytoskeletal complex. Although it is the transformed receptor that associates tightly with the complex, DNA-binding activity is not required for association with the cytoskeletal particulate. I believe this system may provide an in vitro biochemical model for receptor interactions which occur during nuclear transport. After receptor-induced transcriptional activation terminates, the receptor must reassociate with hsp90 and cycle back to the cytoplasm. I describe a rabbit reticulocyte lysate system that allows reconstitution of the monomeric receptor with hsp90 to form a functional, untransformed receptor. This same system can also release receptor from DNA in a functional, untransformed state in a steroid-inhibited manner and I speculate that this kind of process may be involved in termination of transcription. Finally, I describe a partially purified lysate fraction which has receptor reconstitution activity as well as an activity that facilitates protein import into mitochondria. Both of these processes probably require protein folding and I suggest that hsp70, in complex with hsp90, unfolds the receptor and allows hsp90 to reassociate.