CONSTITUTIVELY ACTIVE HUMAN ESTROGEN-RECEPTORS CONTAINING AMINO-ACID SUBSTITUTIONS FOR TYROSINE-537 IN THE RECEPTOR PROTEIN

To better understand structure-activity relationships in the human est rogen receptor (ER), we examined the role of tyrosine 537 in the trans criptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino a cid has been proposed to be a tyrosine kinase phosphorylation site imp ortant in the activity of the ER. We substituted five amino acids at t his position (alanine, phenylalanine, glutamic acid, lysine, or serine ) and screened these mutants for their biological activities in the pr esence and absence of estradiol. Two of the ER mutants, Y537A and Y537 S, displayed estrogen-independent constitutive activity that was appro ximately 20% or 100%, respectively, of the activity of the wild type r eceptor with estradiol, when assessed in two different cell background s using three different estrogen-responsive promoters. In some circums tances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as we ll as their activity in the presence of E(2), was fully suppressed by antiestrogen. The extent of interaction of the constitutively active E Rs with the steroid receptor coactivator-1 (SRC-1) closely parallel th e magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen , Y537A and Y537S ER showed moderate or full interaction in the absenc e of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon est radiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformationa l state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional act ivity and that the presence of certain amino acids at this position ca n shift ER into a conformation that is active even without ligand. How ever, tyrosine is not required at this site for estrogen binding or tr anscriptional response to estrogen in the systems investigated. Our fi ndings, interpreted in light of the recently published x-ray crystal s tructure of the ligand-binding domains of three related receptors of t he nuclear receptor superfamily, suggest that some of the amino acid s ubstitutions introduced at position 537 may facilitate the shift of he lix 12 of the ER into an active conformation and/or allow for differen tial stabilization of the receptor in its active form.