N-terminal mutations of the human estrogen receptor gene alter expression and function of its protein product

摘要

A novel recombinant expression system is described for production of full-length, highly-active receptor proteins of the steroid/thyroid hormone superfamily of genes. Systematic site-directed mutation of the amino-terminus sequence of the human estrogen receptor gene fused to the ubiquitin gene generated three classes of mutants. One group, composed of mutant receptors containing either asparagine, aspartic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, tryptophan or tyrosine, generally exhibited characteristics similar to those of wild-type receptor. Mutations with valine, threonine, lysine or arginine at the N-terminus, essentially eliminated expression and characteristic hER activities. However, a new class of hER-mutant molecules were generated in high quantities with superior properties for binding both (~3H)17beta-estradiol and the vitellogenin A2 ERE, as well as transactivation of a model gene in a yeast cell-based bioassay. Hyper-active mutant hER contained either alanine, cysteine, glutamic acid, glutamine, or serine at the amino-terminus. The striking efficiency of recombinant mutant hER produced in simple yeast expression systems suggests reagent quantities of these labile regulatory proteins may be generated for detailed structural and functional analyses.