AUUUA motifs in the 3'UTR of human glucocorticoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor protein expression

An association between a gene polymorphism of the human glucocorticoid receptor (hGR) gene and rheumatoid arthritis has recently been suggested. This polymorphism contains an A to G mutation in the 3'UTR of exon 9beta, which encodes the 3'UTR of the mRNA of the hGRbeta isoform. The hGRbeta isoform can act as a dominant negative inhibitor of hGRalpha, and therefore may contribute to glucocorticoid resistance. The A to G mutation is located in an AUUUA motif, which is known to destabilize mRNA. In the present study, the importance of the mutation in this AUUUA motif was further characterized and mutations in other AUUUA motifs in the 3'UTR of hGRbeta and hGRalpha mRNA were studied. hGRbeta and hGRalpha expression vectors, carrying mutations in one AUUUA motif or all AUUUA motifs were transiently transfected into COS-1 cells. Each transfected vector was analyzed for the mRNA expression level, the mRNA turnover rate and the protein expression level. The naturally occurring mutation in the 3'UTR of hGRbeta mRNA increased mRNA stability and protein expression. Mutation of two other AUUUA motifs in the 3'UTR of hGRbeta, or mutation of all four AUUUA motifs resulted in a similar effect. Mutation of the most 5' AUUUA motif did not alter hGRbeta mRNA expression or mRNA stability. Mutation of all 10 AUUUA motifs in the 3'UTR of hGRalpha mRNA increased hGRalpha mRNA expression and mRNA stability as well as expression of the receptor protein level. Thus, the naturally occurring mutation in an AUUUA motif in the 3'UTR of hGRbeta mRNA results not only in increased mRNA stability, but also in increased receptor protein expression, which may contribute to glucocorticoid resistance. A similar role is suggested for two other AUUUA motifs in the 3'UTR of hGRbeta mRNA and for the 10 AUUUA motifs that are present in the 3'UTR of hGRalpha.     

Molecular origins for the dominant negative function of human glucocorticoid receptor beta

This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRbeta, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRalpha mutants and sequential mutagenesis to generate a series of hGRalpha/beta hybrids, we find that the absence of helix 12 is neither necessary nor sufficient for the GR dominant negative phenotype. Moreover, we have localized the dominant negative activity of hGRbeta to two residues and found that nuclear localization, in addition to heterodimerization, is a critical feature of the dominant negative activity. Molecular modeling of wild-type and mutant hGRalpha and hGRbeta provides structural insight and a potential physical explanation for the lack of hormone binding and the dominant negative actions of hGRbeta.     

An antiestrogen-responsive estrogen receptor-alpha mutant (D351Y) shows weak AF-2 activity in the presence of tamoxifen

Antiestrogens, including tamoxifen and raloxifene, block estrogen receptor (ER) action by blocking the interactions of an estrogen-dependent activation function (AF-2) with p160 coactivators. Although tamoxifen does show some agonist activity in the presence of ERalpha, this stems from a distinct constitutive activation function (AF-1) that lies within the ERalpha N terminus. Previous studies identified a naturally occurring mutation (D351Y) that allows ERalpha to perceive tamoxifen and raloxifene as estrogens. Here, we examine the contributions of ERalpha activation functions to the D351Y phenotype. We find that the AF-2 function of ERalpha D351Y lacks detectable tamoxifen-dependent activity when tested in isolation but does synergize with AF-1 to allow enhanced tamoxifen response. Weak tamoxifen-dependent interactions between the ERalpha D351Y AF-2 function and GRIP1, a representative p160, can be detected in glutathione S-transferase binding assays and mammalian two-hybrid assays. Furthermore, tamoxifen-dependent AF-2 activity can be detected in the presence of ERalpha D351Y and high levels of overexpressed GRIP1. We therefore propose that the D351Y mutation allows weak tamoxifen-dependent AF-2 activity but that this activity is only detectable when AF-1 is strong, and AF-1 and AF-2 synergize, or when p160s are overexpressed. We discuss the possible structural basis of this effect.