MODULAR STRUCTURE OF GLUCOCORTICOID RECEPTOR DOMAINS IS NOT EQUIVALENT TO FUNCTIONAL INDEPENDENCE - STABILITY AND ACTIVITY OF THE STEROID-BINDING DOMAIN ARE CONTROLLED BY SEQUENCES IN SEPARATE DOMAINS

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolat ion, For this reason, the precise boundaries of the steroid binding do main have not been de fined. These questions have How been systematica lly examined with a variety of receptor deletion constructs, Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not y ield stable proteins, while the fusion of receptor or non-receptor seq uences upstream of 537-673 afforded stable proteins that did not bind steroid, Wild type steroid binding affinity could be obtained, however , when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 531-795, Studies of a seri es of dhfr/receptor constructs with deletions at the amino- and carbox yl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795, The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domai n was consistent with improperly folded receptor sequences. This concl usion was supported by analyses of the proteolysis and thermal stabili ty of the mutant receptors, Thus, three independent regions appear to be required for the generation of the steroid binding form of receptor s: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required far the co rrect folding of the steroid binding domain, and 3) amino-terminal seq uences (550-673), which may be sufficient for steroid binding after th e entire steroid binding domain is properly folded. These results esta blish that the steroid binding domain of glucocorticoid receptors is n ot independently functional and illustrate the importance of both prot ein stability and protein folding when constructing mutant proteins.