Sequences in the ligand-binding domains of the human androgen and progesterone receptors which determine their distinct ligand identities.
The natural ligands of the progesterone (PR) and androgen (AR) receptors, progesterone and testosterone, differ only by their 17 beta-substitution. To identify within the AR and PR ligand-binding domains (LBDs) the sequences responsible for the differential recognition of these ligands, chimeric LBDs assembled from five homologous AR/PR 'cassettes' linked to the GAL4-DNA binding domain were constructed, and their ligand binding and transactivation characteristics were determined. Replacing the central cassette 3 of PR by that of AR generated a progesterone- and testosterone-responsive PR LBD with the AR residues 788-RHLS-791 being specifically involved in testosterone recognition, while the introduction of the C-terminal PR cassette 5 into AR conferred progestin responsiveness onto the AR LBD. These results suggest that residues within AR 788-RHLS-791 interact with the testosterone 17 beta-OH, while PR cassette 5 apparently contains the amino acid(s) specifically involved in the recognition of the progesterone 17 beta-acetyl group. However, ligand binding and transactivation by these chimeras were significantly decreased compared with those of the parental LBDs, indicating that residues located outside of these cassettes contribute to the proper positioning of the steroids in the AR and PR ligand-binding pockets (LBPs). Indeed, certain AR/PR chimeras acquired efficient ligand binding, but were unable to transactivate, indicating that the ligand was improperly bound in the chimeric. LBP and could not induce the conformational changes leading to a transcriptionally competent activation function (AF-2) within the LBD. The properties of the various LBD chimeras are discussed in view of the recently solved three-dimensional structures of the retinoid X receptor alpha apo- and retinoic acid receptor gamma holo-LBDs.
Vivat
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V
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, Illkirch, CU de Strasbourg, France.
Gofflo
D
D
Garcia
T
T
Wurtz
J M
JM
Bourguet
W
W
Philibert
D
D
Gronemeyer
H
H
D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism / Q000494:pharmacology; D004274:DNA, Recombinant; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011372:Progestins / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation / Q000187:drug effects
D000728:Androgens; D004274:DNA, Recombinant; D008024:Ligands; D011372:Progestins; D011944:Receptors, Androgen; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins