Expression of recombinant androgen receptor in cultured mammalian cells.
Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.
Quarmby
V E
VE
Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill 27599.
Kemppainen
J A
JA
Sar
M
M
Lubahn
D B
DB
French
F S
FS
Wilson
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D000882:Haplorhini; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007668:Kidney; D008297:Male; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D011944:Receptors, Androgen; D011994:Recombinant Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase