1A. of basal and stress-related homeostasis and essential for life. They modulate the expression of 10% of human genes and function in every organ and in a wide variety of physiologic, cellular, and molecular networks (1). The biological action of glucocorticoids is usually mediated primarily through the activation of the cytoplasmic glucocorticoid receptor (GR), a member of the nuclear receptor family of ligand-dependent transcription factors. Like other members of the family, GR possesses a modular structure consisting of three major domains (2): an N-terminal, a central DNA binding domain name, and a C-terminal ligand binding domain name. There are numerous aspects of GC action that we do not yet know or are just beginning to appreciate. Glucocorticoid secretion is determined through a complex series of feedback regulation. Endogenous cortisol binds to GR in the hypothalamus, acting as a potent negative regulator of the hypothalamic-pituitary-adrenal (HPA) axis. Alterations in the number or sensitivity of GR to glucocorticoids can significantly influence HPA axis activity and, in particular, can regulate hormone levels by mediating the strength of cortisol feedback inhibition (3,4). Abnormalities in glucocorticoid sensitivity can be divided into two major groups: resistance (CR) and hypersensitivity (CH). To our knowledge, only one AVL-292 AVL-292 case of CH has been reported, in a patient with Cushingoid manifestations despite persistent hypocortisolemia (5); the molecular basis of cortisol hypersensitivity was not elucidated. Polymorphisms in GR may also change glucocorticoid sensitivity, as has been suggested for the N363S AVL-292 polymorphism, which has been linked with extra adiposity in European populations (6). CR was first described as an inherited disorder, characterized by hypercortisolism without Cushingoid features (7). Diminished GC sensitivity in the hypothalamus results in higher cortisol secretion by the adrenal glands, thus maintaining relative glucocorticoid balance in tissues also bearing diminished GC sensitivity. CR is caused by loss-of-function mutations in the GC receptor (GR) (811). Patients homozygous for such mutations exhibit severe hypertension and hypokalemia due to hypercortisolemia and secondary activation of the mineralocorticoid receptor. Heterozygotes, on the other hand, generally exhibit relatively moderate symptomatology, although virilization and hirsuitism has been reported in female patients due to shunting of cortisol precursors into the androgen biosynthetic pathway (12). We previously described the construction of a mutant human glucocorticoid receptor with increased activity in the presence of GCs (13). The mutation, a substitution of leucine for methionine at residue 604 in the hormone-binding domain name of the receptor, increases the affinity of the receptor for GCs by creating a novel helix 3-helix 5 contact. The receptor has Rabbit Polyclonal to MSK2 increased sensitivity, being activated by 510-fold lower corticosterone concentration that GRWTin vitrowithout any alteration in specificity. This is one of a small group of gain of function mutations that have been described in the steroid hormone receptor family (14,15). Whether such point mutations can be responsible forin vivoglucocorticoid hypersensitivity remains to be shown. The availability of a GR with increased activity suggests an intriguing reagent for the study of tissue-specific glucocorticoid effect. To gain new insights into this gain of function of GR function and more directly examine glucocorticoid sensitivityin vivo, we aimed to take advantage of this mutation receptor with increased steroid affinity to study physiological aspects of glucocorticoid biology relevant to the genetics of endocrine activity via creation of a knock-in mouse bearing this gain of function receptor. == MATERIALS AND METHODS == Animal CareAll experiments were performed according to an Institutional Animal Care and Use Committee-approved.