Identification and characterisation of an androgen receptor from zebrafish Danio rerio.
Androgens play key roles in vertebrate sex differentiation, gonadal differentiation and sexual behaviour. The action of androgens is primarily mediated through androgen receptors (ARs). The present study describes the isolation, sequencing and initial characterisation of an androgen receptor from zebrafish Danio rerio. The predicted protein of 868 residues has an estimated calculated molecular weight of 96 kDa. The amino acid sequence of the zebrafish AR (zfRA) shows high homology with other vertebrate ARs. The highest overall similarity was 82% to ARs from fathead minnow (Pimephales promelas) and goldfish (Carassius auratus). Binding assays with zfAR demonstrated high affinity, saturable, single class binding site, with the characteristics of an androgen receptor. Saturation experiments along with subsequent Scatchard analysis determined that the Kd of the zfAR for 3H-testosterone was 2 nM. Androgen binding affinities in competition with 3H-testosterone were: 5alpha-dihydrotestosterone>11-ketotestosterone>testosterone>androstenedione. The deletion of 12 amino acids (zfARd12) in the ligand binding domain of zfAR resulted in impaired binding to the receptor. Therefore, it was not possible to determine Kd for the zfARd12. The characterisation of this zfAR provides a new perspective for understanding the mechanisms underlying androgen actions in a model vertebrate species commonly used for studies investigating potential endocrine disrupters.
Jørgensen
Anne
A
Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. annejoer@ruc.dk
Andersen
Ole
O
Bjerregaard
Poul
P
Rasmussen
Lene Juel
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003530:Cyprinidae / Q000502:physiology; D006054:Goldfish / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013739:Testosterone / Q000378:metabolism; D015027:Zebrafish / Q000502:physiology*; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011944:Receptors, Androgen; D029961:Zebrafish Proteins; D013739:Testosterone