FIG. 5.

Transactivation and dominant negative activity of hGRα/β hybrids. (A) The amino acids 728 to 742 of hGRα were replaced with the corresponding amino acids of hGRβ in a sequential fashion. The residues from 728 through 742 were numbered 1 to 15, shown in the center. Note that three residues, Val729 (position 2), Leu732 (position 5), and Leu741 (position 14), are conserved in both hGRα and hGRβ (∗). Beginning with the mutation of Val728 to Asp, hGRα(β1), each additional mutant included the one before it, until a complete hGRα/β hybrid was generated. − indicates that no change from wt hGRα was made. Only the first 12 sequential mutants are shown. Dex-induced transactivation was measured as for Fig. 2A with the data expressed as percent conversion of [¹⁴C]chloramphenicol to the acetylated form. The top panel shows the sequential mutants, where each residue in hGRα was changed with a corresponding hGRβ residue in succession. The bottom panel shows data from single and double point mutants described in the text. For each data set the wt hGRα is shown as the top sequence and the hGRβ sequence is on the bottom. Note that each hybrid contains the H12 carboxy-terminal extension (→), except hGRβ, which terminates following Ile742. The shaded region through positions 6 and 7 (amino acids 733 and 734) indicates the two amino acid mutations sufficient to completely block transactivation. As in Fig. 2, Western blots indicated similar expression levels for each receptor tested (data not shown). (B) The hybrid receptors from panel A that were completely deficient in Dex-induced transactivation were tested for dominant negative activity following the experimental protocol described for Fig. 2A. The top two bars, hGRα (+ pCMV5) and hGRα + hGRβ, are the controls. The dashed line indicates the level of dominant negative activity observed with hGRβ. All receptors tested for dominant negative activity were transfected at 10-fold-higher levels relative to levels for hGRα. The middle bars are the sequential mutants that lack transactivation, and the double mutant, hGRα(β6-7), is shown at the bottom.