The putative androgen receptor-A form results from in vitro proteolysis.
Activation domains in the 114 kDa androgen receptor (AR) NH(2)- and carboxyl-terminal regions are thought to contribute to different extents to AR-mediated transactivation. We investigated using anti-peptide antibodies whether smaller AR forms that migrate like the previously described 87 kDa AR-A occur in vivo resulting in constitutive or increased gene activation. Immunoblots of prostate cancer and fibroblast cell culture extracts revealed 114 and 84 kDa AR forms. Antibody mapping indicated the 84 kDa AR lacked the ligand-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 was designed to initiate at the second methionine (residue 189) and lacked the NH(2)-terminal FxxLF interaction sequence. The 92 kDa AR did not result from alternative initiation since it was observed when the second methionine was changed to alanine. Optimization of extraction conditions indicated that both 84 and 92 kDa forms resulted from in vitro proteolytic cleavage and that cleavage by caspase-3 could account for the 92 kDa form. The results suggest that AR forms with gel mobility similar to that of the previously described 87 kDa AR-A result from in vitro proteolytic cleavage of NH(2)- or carboxyl-terminal regions during cell extraction and storage and that smaller forms with increased transcriptional activity do not occur in vivo.
Gregory
C W
CW
Laboratories for Reproductive Biology, Department of Pediatrics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA.
He
B
B
Wilson
E M
EM
D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008970:Molecular Weight; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011944:Receptors, Androgen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D020033:Protein Isoforms; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D010450:Endopeptidases