The putative androgen receptor-A form results from in vitro proteolysis

Activation domains in the 114kDa androgen receptor (AR) NH2- and carboxyl-t erminal regions are thought to contribute to different extents to AR-mediat ed transactivation. We investigated using anti-peptide antibodies whether s maller AR forms that migrate like the previously described 87 kDa AR-A occu r in vivo resulting in constitutive or increased gene activation. Immunoblo ts of prostate cancer and fibroblast cell Culture extracts revealed 114 and 84 kDa AR forms. Antibody mapping indicated the 84 kDa AR lacked the ligan d-binding domain and comigrated with the constitutively active AR fragment AR1-660. AR expressed in COS cells was 114 and 92 kDa. Migration of the 92 kDa AR was slightly slower than that of a 90 kDa expressed fragment that wa s designed to initiate at the second methionine (residue 189) and lacked th e NH2-terminal FxxLF interaction sequence. The 92 kDa AR did not result fro m alternative initiation since it was observed when the second methionine w as changed to alanine. Optimization of extraction conditions indicated that both 84 and 92 kDa forms resulted from in vitro proteolytic cleavage and t hat cleavage by caspase-3 could account for the 92 kDa form. The results su ggest that AR forms with gel mobility similar to that of the previously des cribed 87 kDa AR-A result from in vitro proteolytic cleavage of NH2- or car boxyl-terminal regions during cell extraction and storage and that smaller forms with increased transcriptional activity do not occur in vivo.