UBC Theses and Dissertations
Identification of proteins that interact with the N-terminal domain of the androgen receptor Quayle, Alandra Nola
The androgen receptor (AR) plays an essential role in the proliferation of prostate cells, so the progression of prostate cancer to the incurable androgen independent (AI) state likely involves alterations in the regulation of AR-mediated gene transcription. One possible route to AI progression is through changes in the activity of coregulator proteins, which interact with AR to contribute further control and specificity to the process of transcriptional regulation. A variety of affinity assays was used to try and identify AR-specific coregulator proteins through their ability to bind to unique sequences in the AR N-terminal domain. Detection of calreticulin (a known AR-binding protein) following GST-pulldown with AR-GST fusion proteins from LNCaP cells (a human prostate cancer cell line), proved that this method was able to selectively pull down human proteins that interact with AR. GST-pulldown with the N-terminal domain of AR identified two LNCaP nuclear proteins of approximately 40 kDa and 70 kDa and a yeast protein of about 70 kDa that specifically interacted with the N-terminal domain, relative to other regions of AR. Yeast proteins of about 32 kDa, 45 kDa, and 55 kDa that appeared to interact uniquely with the N-terminal domain of AR were co-immunoprecipitated with fragments of the AR N-terminal domain expressed in yeast cells. Further characterization of these protein bands was complicated by high levels of non-specific background associated with these techniques. The yeast 2-hybrid system was used to screen a human prostate cDNA library, utilizing the first 232 amino acids of the N-terminal domain of human AR as bait. A positive clone, ARBP1, was identified with very strong sequence homology to a known human protein, RanBPM, that was previously shown to be involved in microtubule nucleation. Hybridization of an ARBP1 cDNA probe to multiple tissue expression blots showed that the expression of this gene was much higher in testis tissue than in other human tissues. In the future, the interaction between AR and the approximately 70 kDa protein expressed by the ARBP1 clone can be confirmed using GST-pulldown or co-immunoprecipitation. Elucidating the role of ARBP1 in AR function will provide an excellent focus for future experiments.
Item Citations and Data