PMLRARm4 and RARm4.
A, PMLRARαm4: the Leu to Pro point mutation at codon 398 ofRARα was introduced into a human PMLRARα cDNA whose chromosome 15 breakpoint lies in breakpoint cluster 1.16The PML portion of the fusion is shown with hatching with selected structural domains labeled and shown in white. The B-F domains that encompass the RARα portion of the fusion are labeled and functional regions are noted. RARαm4: the Leu to Pro point mutation was introduced into a human RARα1 cDNA. B, Hormone binding by PMLRARα (“Wild-type,” WT) andPMLRARαm4 (m4 mutant). Radiolabeled proteins synthesized by in vitro transcription and translation were incubated without or with increasing amounts of trypsin (indicated above the panels) in the absence or presence of 1 μmol tRA. The protein products were resolved by denaturing PAGE and visualized by autoradiography. The arrows show the position of the full-length undigested proteins. Smaller bands represent partially degraded products. C, Dominant negative activity of the m4 mutant proteins. CV-1 cells were transiently transfected with pSG5 constructs containing no exogenous receptor, RARα (WT),RARαm4, PMLRARα (WT), and PMLRARαm4. Luciferase activity expressed from a cotransfected βRARE-luciferase reporter gene was normalized to β-galactosidase activity from a cotransfected pCH210-LacZ plasmid. D, Decreased hormone-induced dissociation of SMRT corepressor by the m4 mutant proteins. GST-SMRT fusion protein was synthesized in E coli and was immobilized on glutathione agarose. The different receptor proteins were synthesized by in vitro transcription and translation and were incubated with the immobilized GST-SMRT in the absence or presence of 1 μmol tRA, as indicated below the panels. Equivalent amounts of GST-SMRT and radiolabeled receptor protein were used for each panel. Nonrecombinant GST, immobilized on glutathione agarose, was used in parallel as a negative control. The radiolabeled receptors remaining bound to the GST or GST-SMRT matrix after washing were eluted, were resolved by denaturing PAGE, and were visualized and quantified by phosphorimager analysis. The amount of radiolabeled receptor bound to the GST or GST-SMRT matrix, relative to the amount of receptor input in each binding reaction, is displayed beneath each panel.
