3 AMINO-ACID SUBSTITUTIONS SELECTIVELY DISRUPT THE ACTIVATION BUT NOTTHE REPRESSION FUNCTION OF THE GLUCOCORTICOID RECEPTOR N-TERMINUS

A 210-amino acid region, termed enh2, near the N terminus of the rat g lucocorticoid receptor, is necessary for both transcriptional activati on and repression. The mechanism(s) of transcriptional regulation conf erred by this region, however, are poorly understood. We screened in S accharomyces cerevisiae a library of random mutants in the enh2 region of a constitutive glucocorticoid receptor derivative and isolated a s eries of multiply substituted receptors that are specifically defectiv e in transcriptional activation. Although many substitutions in this a rea were tolerated, three amino acid substitutions (E219K, F220L, and W234R) within a 16-amino acid region were sufficient to disrupt the en h2 transcriptional activation function both in yeast and in mammalian cells. Although this region is rich in acidic residues, the conserved tryptophan at position 234 appears to be a more critical feature for e nh2 activity; hydrophobic but not charged residues were tolerated at t his position, Notably, the mutants uncoupled the activation and repres sion functions of enh2, as the activation defective isolates remained competent for repression of AP-1 at the composite response element plf G.