Functional Probing of the Human Glucocorticoid Receptor Steroid-interacting Surface by Site-directed Mutagenesis

Abstract

To elucidate which amino acids in the glucocorticoid receptor ligand-binding domain might be involved in determining steroid binding specificity by interaction with the D-ring of glucocorticoids, we have performed site-directed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-739 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or steroid-dependent transactivation. The results indicate that these residues are located in close proximity to the ligand and appear to play a role in steroid recognition and/or transactivating sensitivity, possibly by changes in the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked decrease in affinity for steroids containing a 17␣-OH group. This effect was alleviated by the presence of a 16␣-CH 3 group to a varying degree. Thr-739 appears to form a hydrogen bond with the 21-OH group of the steroid, as well as possibly forming hydrophobic interactions with the steroid. Met-560 and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be characterized in more detail at this point. The glucocorticoid receptor (GR)1 belongs to the superfamily of hormone-dependent nuclear receptors and consists of three structural and functional main domains: the N-terminal domain, which harbors the major transactivating function (AF-1); the central domain, which binds to DNA in glucocorticoid regulated genes; and the C-terminal domain, which binds the ligand (1-4).The ligand-binding domain (LBD) comprises approximately 250 amino acids and is in its unliganded state associated with a complex containing heat shock proteins and immunophilins (5). Upon ligand binding this complex dissociates and a cascade of events are triggered leading to induction or repression of target genes. Within the ligand-binding domain there are also a hormone-dependent nuclear localization signal (6) and hormone-dependent transactivation functions (AF-2) (7-10).The crystal structure of the GR LBD is not yet available, but the crystal structures of the LBDs of other members of the nuclear receptor superfamily including the peroxisome proliferator activated receptor, retinoic acid receptor, retinoid X receptor, thyroid hormone receptor, progesterone receptor (PR), estrogen receptor ␣ (ER␣), and estrogen receptor ␤ (ER␤) have been solved (11-17). Their structures contain 12 ␣-helices that are folded in a very similar way into a three-layered antiparallel ␣-helical sandwich that creates a hydrophobic pocket for the ligand. Upon ligand binding a conformational change occurs, mainly involving helix 12, which folds up against the protein body and creates a lid for the ligand binding pocket. This also leads to formation of the AF-2 interface, which has been shown to interact with transcriptional coactivators (18 -21).The mechanisms that determine the binding affi...

Human Glucocorticoid Receptor β Binds RU-486 and Is TranscriptionallyActive

“…Our dose-response results for the nuclear translocation of YFP-hGR␤ indicate that at least 100 nM RU-486 is necessary for translocation, which is consistent with the estimated K d of 138 nM for RU-486 binding to hGR␤. It is therefore likely that in our original experiment we did not observe binding of RU-486 to hGR␤ because the concentration of 3 H-ligand was too low (18). Because the RU-486 affinity for hGR␤ is low compared to the interaction of compounds such as dexamethasone or RU-486 with hGR␣, we were concerned that the effects of RU-486 on hGR␤ might instead be due to an unidentified contaminant in the RU-486 stock.…”

Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

“…(B) t 1/2 for wild-type YFP-hGR␣ and mutant Q642V in the presence of 1,000 nM corticosterone, dexamethasone, and triamcinolone acetonide. Mutation Q642V slightly increased the affinity for corticosterone (50% inhibitory concentration (IC 50 ) 2.3 times lower than wild type [34]), markedly decreased the affinity for dexamethasone (IC 50 11-fold increased compared to wild type in a competition binding assay), and slightly decreases the affinity for triamcinolone acetonide (K d 1.4 times higher than wild type in a binding assay). Consistent with the alterations in affinity, the mobility of the mutant receptor induced by these ligands was altered compared to the wild type.…”

“…Glutamine 642 supposedly interacts with the 17␣-OH group of glucocorticoid ligands (5), and therefore mutation of this residue markedly decreases the affinity for ligands with a 17␣-OH group like dexamethasone, whereas it alters the affinity for ligands that lack this group only slightly (the mutant has decreased affinity for triamcinolone acetonide and increased affinity for corticosterone [34]). We made this mutation in YFP-hGR␣ and performed FRAP analysis after addition of 1,000 nM triamcinolone acetonide, dexamethasone, or corticosterone (Fig.…”

A critical role of helix 3–helix 5 interaction in steroid hormone receptor function

“…Key residues within the LBDs of nuclear and steroid hormone receptors that interact with specific steroid functional groups have been identified, providing a structural basis for the steroid specificity of these receptors (2)(3)(4)(5)(6)(7)(8). For example, in the mineralocorticoid receptor (MR), it has been proposed that conserved residues in H3 and H5, Gln-776 and Arg-817, form hydrogen bonds with the 3-ketone group of steroid hormones to anchor aldosterone's A-ring, whereas conserved residues in H3 and helix 12, Asn-770 and Thr-945, mediate binding of the D-ring (2).…”