Structural and functional analysis of N-terminal point mutants of the human estrogen receptor.
Twenty N-terminal point mutations of the human estrogen receptor (hER) were constructed as ubiquitin fusion products and expressed under the control of the copper regulated promoter CUP1 in Saccharomyces cerevisiae. The objective of these studies was to overexpress hER in yeast and also to evaluate the functional properties of the N-terminal variants of hER. Fusion of the C-terminus of ubiquitin to the N-terminus of other proteins has been shown to increase the level of protein expression in yeast. Ubiquitin C-terminal hydrolases (UCHs) in yeast efficiently and precisely cleave at the junction with ubiquitin and render free hER with desired amino termini. The variant hER proteins, that were generated by mutating the N-terminus of hER, showed enormous differences in receptor protein levels and transactivation potential. All variant hER proteins were synthesized as 66 kDa species as identified by Western blotting with the exception of the proline-containing variant (Pro-ER). The UB-Pro-ER variant was cleaved inefficiently by UCHs in yeast. The UB-Pro-hER [correction of UB-Pro-hEr] variant also exhibited a different DNA band-shift profile compared to those of the other receptor variants and the wild-type. Val-, Thr-, and Lys-ER did not express, as measured by enzyme-immunoassay and Western blotting; nor did they transactivate a beta-galactosidase reporter gene in yeast. However, the Glu-ER was 50% more active in transactivation as compared to the wild-type. The results of the receptor content, DNA binding properties and transactivation analysis in yeast demonstrate that the N-terminal residue plays an important role in the structure and function of hER.
Graumann
K
K
Institute for Applied Microbiology, University of Agriculture, Forestry and Biotechnology, Vienna, Austria.
Wittliff
J L
JL
Raffelsberger
W
W
Miles
L
L
Jungbauer
A
A
Butt
T R
TR
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014452:Ubiquitins / Q000737:chemistry
D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D011994:Recombinant Proteins; D014452:Ubiquitins