Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor.
The trafficking of the androgen receptor (AR) in transfected cells was studied using a green fluorescent protein (GFP)-AR chimera. The reporter molecule GFP enabled the localization of AR in living cells with a good spatial and temporal resolution. After the construction of GFP-AR and verification of the size of the fusion protein produced, we demonstrated that GFP-AR conserves the functional properties of the AR: GFP-AR had the same androgen-binding affinity as AR, and GFP-AR efficiently transactivated an androgen-responsive gene in response to synthetic androgen at 30 degrees C. The fusion protein was first detected throughout the cytoplasm without hormone, fluorescence becoming nuclear rapidly after androgen incubation. This hormone dependence of AR trafficking was confirmed by the use of the mutant GFP-AR-del4, which lacked the androgen-binding function. The mutant was localized in the cytoplasm in the absence of hormone, but the distribution was not modified by androgen incubation. An ACAS 570 scanning laser cytometer was used to quantify fluorescence in a single living cell, first without and then with hormone. Different hormones and antihormones were tested to determine the dynamics of GFP-AR translocation into the nucleus. All the drugs used were able to induce nuclear translocation, and steady state level was rapidly attained within 1 h. The ratio of receptors in cytoplasmic and nuclear compartments was related to both affinity and concentration of ligand. The data from this follow-up study demonstrated for the first time the intracellular dynamics of the hormone-dependent trafficking of AR in a single living cell.
Georget
V
V
Institut National de la Santé et de la Recherche Médicale, INSERM U439, Pathologie Moleculaire des Récepteurs Nucléaires, Montpellier, France.
Lobaccaro
J M
JM
Terouanne
B
B
Mangeat
P
P
Nicolas
J C
JC
Sultan
C
C
D000728:Androgens / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D000728:Androgens; D008164:Luminescent Proteins; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins