MUTATIONS FLANKING THE POLYGLUTAMINE REPEAT IN THE MODULATORY DOMAIN OF RAT GLUCOCORTICOID RECEPTOR LEAD TO AN INCREASE IN AFFINITY FOR HORMONE

Abstract

A polyglutamine repeat in the N-terminus of the rat glucocorticoid receptor shows polymorphism, with variants of Q₂RQ₅, Q₂RQ_15–21. We investigated whether these natural polymorphisms affect receptor function, and whether alleles with polyglutamine repeats shorter than Q₂RQ₅, between Q₂RQ_6–14, or longer than Q₂RQ₂₁ are not found naturally because they encode a dysfunctional receptor. Ligand binding and transactivation properties of sets of natural (Q₂RQ₅–Q₂RQ₂₁) and artificial (Q₄–Q₈₀) alleles were compared following expression in CV-1 cells. The sequence of artificial alleles at sites flanking the repeat region was altered slightly to facilitate cloning. Western blotting showed that all constructs expressed GR protein in CV-1 cells. When co-expressed with an MMTV-lacZ reporter plasmid, all GR proteins were shown to be transcriptionally active in the presence of hormone. Scatchard analysis of ligand binding curves showed that affinities for dexamethasone and corticosterone were not affected by variation in the polyglutamine repeat in either the natural or artificial sets of alleles. However, affinities were greater for the artificial compared with the natural alleles (2–3-fold for dexamethasone, p<0.001; and 4-fold for corticosterone, p<0.001). These differences provide evidence of a direct or indirect interaction within GR between the ligand binding domain and residues flanking the polyglutamine repeat of the N-terminal domain.

ACKNOWLEDGMENTS

This work was funded by the Medical Research Council, UK. The authors are grateful to Dr E. Stocklin and Professor B. Groner for confirmation of the transcriptional activity of the GR mutants.