Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain

Abstract

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652–795, was non-covalently associated with another, ∼17-kDa species that was determined to be amino acids 518–651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537–673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518–795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be α-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.