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Abstract
Based on the finding that some transcription factors contain multiple transcriptional regulatory activities, we constructed a panel of rat androgen receptor (AR) mutants containing small internal deletions and point mutations within the amino-terminal region of the receptor. Trans-activation assays in CV-1 cells using AR-responsive reporter genes were performed and led to the identification of two noncontiguous trans-activation regions in the AR amino terminus. One of these regions, termed activator function 1a (AF-1a) is a highly-conserved 14-amino acid segment that is predicted to form a beta-turn followed by an acidic amphipathic alpha-helix. Point mutagenesis within AF-1a revealed that two adjacent hydrophobic residues were required for full AR trans-activation function, as arginine substitutions resulted in a 60% reduction in transcriptional activity. A second amino-terminal region was also identified and has been designated AF-1b. Deletion of the 65-amino acid AF-1b segment, which contains numerous glutamate and aspartate residues, caused a 55% decrease in trans-activation function. An AF-1a/AF-1b double mutant retains less than 10% trans-activation function compared with wild-type AR, suggesting that AF-1a and AF-1b may each contribute separately to maximal AR activity. To determine whether AF-1a and AF-1b play a role in AR-mediated trans-repression of AP-1 function, we tested single and double AF-1a/AF-1b mutants in a transient trans-repression assay. Our results showed that neither AF-1a nor AF-1b was required for AP-1 trans-repression, demonstrating that AR-mediated trans-repression and trans-activation are discrete functions.
Highlights
The steroid hormones testosterone and dihydrotestosterone (DHT)1 exert their physiological effects through the intracellular androgen receptor (AR)
Simental et al [4] previously showed that deletion of amino acids 1–141 of human AR had no effect on trans-activation function, whereas deletion of residues 1–338 resulted in a complete loss in trans-activation without altering hormone binding or nuclear localization functions
We demonstrate in this study that the amino-terminal activation domain of the rat AR is similar to the activation domains of other eukaryotic transcription factors in that it contains multiple subregions that together are required for full trans-activation function
Summary
Construction of AR Amino-terminal Mutants—All AR derivatives were cloned into the expression vectors p6R [9] and pCMX [10] for chloramphenicol acetyltransferase (CAT) and ligand binding assays, respectively. For trans-activation assays using the AR deletion mutants, 1 ϫ 106 CV-1 cells were transfected by the calcium phosphate method [11] using equimolar receptor expression plasmid (10 g of for p6RAR-AB), 2 g of pMM-CAT reporter plasmid, 5 g of the -galactosidase (-gal) expression plasmid pEQ176 [9], and carrier DNA (sheared calf thymus DNA or pSKϩ plasmid DNA) at up to 25 g/10-cm plate. For each ligand binding assay and Western blot sample, 15-cm plates containing 1.8 ϫ 106 COS-7 cells in Dulbecco’s modified Eagle’s medium plus 5% charcoal-stripped calf bovine serum were transfected as above, except that ϳ30 g of each pCMX receptor expression plasmid and 1 g of pRSCAT [14] were used per plate, and the DNA precipitates were left on the cells for 16 h. The membrane was incubated with the peroxidase substrates hydrogen peroxide and ImmunoPure 3-amino-9-ethylcarbazole (Pierce) to visualize the bands
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