UCSF

Steroid receptors are regulated by a diverse array of cellular effectors. Interactions with chaperone proteins, steroid hormones, kinase cascades, motor proteins, co-regulator proteins, and DNA determine the functional output of a receptor. Co-regulator proteins directly bind SRs at a surface site on the C-terminal ligand binding domain, apart from the buried ligand binding pocket, and are capable of activating and repressing transcriptional activity through the receptor. Most SRs prefer to bind co-regulators bearing the NR box motif LXXLL, where X is any amino acid. The sequences surrounding the Leu residues and the amino acids that comprise the receptor's activation function-2 surface determine the specificity of the interaction. The recruitment patterns of co-regulator proteins to SRs is key in determining cell and tissue-specific responses to natural and synthetic therapeutic steroids.

Androgen receptor is unlike the other SRs in that it preferentially binds co-regulators with aromatic residues comprising the NR box. To investigate this unusual preference, we solved X-ray structures of several aromatic-containing peptides bound to AR's AF-2 surface, and determined binding constants for the interactions by surface plasmon resonance (Chapter 2). Several AR residues were shown to be involved in an induced-fit mechanism that allows the receptor to recognize a wider variety of motifs.

Co-regulator recruitment to GR's AF-2 surface was investigated biochemically, revealing that binding events at AF-2 and the ligand binding pocket are allosterically coupled (Chapter 1). We found that NR box binding to GR's AF-2 slows association and dissociation of a fluorescent GR agonist, using fluorescence polarization. The effect on steroid binding was NR box sequence dependent with 7 of 18 peptides assayed showing the ability to reduce steroid binding rates significantly. Dissociation constants were determined for 8 of 18 peptides, revealing a more complete consensus sequence for NR box binding to GR. Additionally, mutation of a residue conserved in SRs shown to effect chaperone, steroid, and co-regulator binding to GR in cells was confirmed as a key component of the network connecting steroid and co-regulator binding sites.