Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and modulate the receptor transactivation capacity

To localize regions conferring ligand binding specificity of the human gluc ocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, l inked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hP R creates a chimeric protein GP3, which binds the progestin RU 27987 with h igh affinity, and results in a concomitant loss of glucocorticoid binding [ dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 279 87-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five r esidues that confer progestin responsiveness to GR or modulate ligand bindi ng and transcriptional activation. Notably, the simultaneous presence of re sidues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is speci fically involved in RU 27987 binding. The absence of residues Asp641, Gln64 2, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activ ation in response to DEX and RU 27987, respectively. DEX-dependent transact ivation is further enhanced by the presence of Leu647.