In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein.
Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.
Kuiper
G G
GG
Department of Endocrinology and Reproduction, Medical Faculty, Erasmus University Rotterdam, The Netherlands.
de Ruiter
P E
PE
Trapman
J
J
Jenster
G
G
Brinkmann
A O
AO
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D015741:Metribolone / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D018075:RNA, Complementary / Q000378:metabolism*; D011817:Rabbits; D011944:Receptors, Androgen / Q000096:biosynthesis* / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic; D014316:Tritium
D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D018075:RNA, Complementary; D011944:Receptors, Androgen; D014316:Tritium; D015741:Metribolone; D000255:Adenosine Triphosphate; D008715:Methionine