Transcriptional activation and nuclear targeting signals of the human androgen receptor.
The androgen receptor (AR) is a signal-transducing protein required for sexual differentiation, development, and expression of the male phenotype. A series of human AR deletion mutants were created either by site-directed mutagenesis using restriction enzyme digestion, the polymerase chain reaction, or, for a series of unidirectional NH2-terminal deletions, exonuclease III digestion. Receptor mutants were expressed in monkey kidney COS cells as truncated AR proteins between 20 and 107 kDa as revealed on immunoblots, where wild type AR was a doublet of 114 and 108 kDa. Subcellular localization by immunocytochemical staining demonstrated androgen-dependent nuclear uptake of AR from a perinuclear region of the cytoplasm. A nuclear targeting signal similar in sequence and position to the glucocorticoid receptor and homologous to the SV40 large T antigen was required for androgen-induced nuclear uptake of wild type AR. AR mutants lacking the NH2-terminal and/or steroid binding domains were constitutively nuclear with reduced transcriptional activity. Transcriptional activation by wild type AR was androgen-dependent in cotransfection studies of CV1 cells using the chloramphenicol acetyltransferase reporter gene linked to the mouse mammary tumor virus promoter. Deletion mutagenesis revealed within the NH2-terminal region a domain required for full transcriptional activity and within the steroid binding domain, an inhibitory function, deletion of which yielded a constitutively active receptor. Inhibition of wild type AR by coexpression with an inactive NH2-terminal fragment suggested competition for nuclear factors required for transcriptional regulation. These studies demonstrate a concerted interplay among the domains of the AR protein in regulating gene transcription.
Simental
J A
JA
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
Sar
M
M
Lane
M V
MV
French
F S
FS
Wilson
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002872:Chromosome Deletion; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011944:Receptors, Androgen / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
D011944:Receptors, Androgen