Cortisol resistance and the guinea pig glucocorticoid receptor

Abstract

The guinea pig has been employed as a model to study the structure/function relationships of the glucocorticoid receptor (GR), and to determine the regions of the receptor important for binding hormone and antihormone. Guinea pigs have high levels of circulating cortisol and GR with a ∼ 20-fold lower affinity for dexamethasone than mouse GR. Cloning and sequencing of guinea pig GR has identified 24 amino acid changes in the ligand-binding domain (LBD) compared to the human GR. By substituting the guinea pig GR LBD for the human LBD in a human GR expression vector we have shown in cotransfection studies that guinea pig GR LBD confers glucocorticoid resistance as observed in vivo. In initial studies guinea pig GR LBD appeared constitutively active; in subsequent studies to determine which of the 24 amino acid changes present in the guinea pig GR LBD conferred resistance, it became apparent that the guinea pig LBD (LBDΔ), amplifed by PCR for subcloning into the human GR expression vector, contained a single adenine deletion in the hinge region within ten bases of the PCR primer. This single base deletion resulted in a frameshift bringing a stop codon into frame one codon after the deletion. While this now clearly accounts for the observed constitutive activity, since it is known that C-terminally truncated steroid receptors exhibit constitutive activation such a truncation is more difficult to reconcile with the repeatedly demonstrable hormone dose-response curves obtained with this guinea pig GR LBDΔ. Studies with the correct guinea pig GR LBD show hormone resistance, with half-maximal activity at 3 nM dexamethasone (compared to 0.8 nM with the human GR), but not constitutive activity. Secondly, it has recently been shown that the protein kinase A activator, 8-bromo-cAMP, is able to enhance the response of the mouse GR to dexamethasone, and in addition elicits an agonist response from the glucocorticoid antagonist RU 486. We have examined whether this also holds for the human GR, and whether changes in the LBD alter the response for guinea pig GR. For guinea pig GR LBDΔ constitutive activity is enhanced 3-fold in the presence of 8-bromo-cAMP alone; in contrast with the results for mouse GR, RU 486 uniquely antagonizes the action of 8-bromo-cAMP. Both wild-type guinea pig GR LBD and human GR respond similarly to mouse GR in this context. Third, guinea pig GR have tryptophan at residue 632, which in all other species examined to date is the cysteine, shown to be the site of covalent attachment for the affinity label dexamethasone 21-mesylate. Mutation of cysteine to tryptophan in human GR had no effect on the dose response curve, and the reciprocal mutation of tryptophan to cysteine in the guinea pig GR LBD was also without effect. Despite the predicted importance of this residue, it would thus appear not to be critical for receptor response to steroid.