Steroid receptor mediated gene activation is crucial for cellular processes involved in cell proliferation, differentiation and development. Transcriptional activation domains in steroid receptors include activation function 1 in the NH2-terminal region and activation function 2 (AF2) in the ligand binding domain. For most steroid receptors, AF2 functions as a binding site for the LXXLL motifs of p160 coactivators. Androgen receptor (AR) AF2 functions primarily as the interaction site for the androgen dependent and specific NH 2- and carboxyl-terminal (N/Q interaction. Two LXXLL-like motifs were identified in the AR NH2-terminal region: FXXLF with sequence 23FQNLF27 and WXXLF with sequence 433WHTLF437, both of which bind AF2 in the presence of androgen. The N/C interaction stabilizes AR by protecting the ligand in the binding pocket and may be required for transactivation in vivo. Inhibition of p160 coactivator recruitment to AF2 by the androgen dependent N/C interaction raises questions regarding the role of AF2 in AR mediated transactivation.
In AIM 1 we will determine the sequence and structural requirements for peptide and protein interactions with AF2. The sequence requirements flanking the FXXLF and LXXLL motifs for binding AF2 will be established. We will determine the crystal structure of the AR ligand binding domain bound to FXXLF and WXXLF peptides and investigate whether the FXXLF or WXXLF motifs serve as binding sites for regulatory proteins or transcription factors. We will investigate the interaction of hsp90 peptides and protein with AF2 and identify peptide inhibitors of AR function.
In AIM 2 we will determine the characteristics of naturally occurring enhancer/promoter elements that require the AR N/C interaction and the role of the N/C interaction in AR mediated transactivation of these androgen regulated genes. Androgen response region spacing sequence, and binding of other factors will be investigated together with the effect of response element DNA on AR conformation and the recruitment of coactivators.
In AIM 3 we will determine the functional importance of the AR N/C interaction by analyzing mice expressing a mutant AR that lacks the N/C interaction. Mice carrying the FXXLF/WXXLF AR mutation will be created by homologous recombination.
In AIM 4 we will characterize the structural arrangement of the AR N/C interaction using electron microscopy of biotinylated purified AR to determine the orientation of the AR dimer bound to androgen response element DNA. We will scale up purification of the androgen-bound full-length baculovirus expressed AR for co-crystallization with androgen response element DNA. The studies will establish the properties and functional importance of the N/C interaction in AR mediated gene activation that could lead to the development of selective therapeutic inhibitors of AR activity.
Showing the most recent 10 out of 18 publications
