Steroid hormone receptors and their regulation by phosphorylation.Recent studies have highlighted the importance of phosphorylation in receptor function. Although most of the phosphorylation sites are serine ligand binding domain of steroid hormone receptors threonine residues, a few of the family members are also phosphorylated on tyrosine. Those steroid receptor family members that are bound to heat-shock proteins in the absence of ligand typically are liganr phosphorylated and exhibit increases in phosphorylation upon steorid binding. Most of these sites contain Ser-Pro motifs, and there is evidence that cyclin-dependent kinases and Anabolic steroid drugs kinases mitogen-activated protein kinases ligamd subsets of these sites. In contrast, phosphorylation sites identified thus far in members of the family that bind to DNA in the absence of hormone typically do not contain Ser-Pro motifs and are frequently casein kinase II or protein kinase A sites. Phosphorylation has been implicated in DNA binding, transcriptional activation and stability of the receptors.
Visualizing the action of steroid hormone receptors in living cells
Steroid hormones exert a wide variety of effects on growth, development, and differentiation, including important regulatory and behavioral functions within the reproductive, central nervous system, and adrenal axis.
These hormones act through binding to specific intracellular receptor proteins that function as both signal transducers and transcription factors to modulate expression of target genes. Sequence comparison has revealed that steroid hormone receptors belong to a diverse family of ligand-activated gene regulators that share a highly conserved structure and common mechanisms affecting gene transcription.
The steroid receptors are considered class I members of the nuclear receptor superfamily while the other receptors are class II receptors.
Members of the steroid hormone receptor gene superfamily in mammalian tissues Receptors with known ligand s: In order to understand how steroid hormone receptors regulate gene function, it is important to know the structure of the receptor proteins as well as the identity and cellular function of the genes that they regulate.
Members of the steroid receptor superfamily share direct amino acid homology and a common structure Fig. Receptors in this superfamily contain several key structural elements which enable them to bind to their respective ligands with high affinity and specificity, recognize and bind to discrete response elements within the DNA sequence of target genes with high affinity and specificity, and regulate gene transcription.
Variability between members of the steroid hormone receptor family is due primarily to differences in the length and amino acid sequence of the amino N -terminal domain. Adapted from Wahli W, Martinez E. Superfamily of steroid nuclear receptors: Positive and negative regulators of gene expression. Molecular cloning of the complementary DNA cDNA for each of the major steroid receptors has greatly enhanced our understanding of the structure—function relationships for these molecules.
The receptor proteins have five or six domains called A—F from N- to C-terminus, encoded by 8—9 exons. The receptors contain three major functional domains that have been shown experimentally to operate as independent "cassettes", 13 unrestricted as to position within the molecule. The three major functional domains Fig. The horizontal lines indicate the domains of the receptor.
The F domain is thought to play a role in distinguishing estrogen agonists from antagonists, perhaps through interaction with cell-specific factors. In rat GR, the AF-1 region is called tau 1 or enh2 and constitutes aa — Tau 1 is necessary for transcriptional activation and repression. Some steroid receptors exist as isoforms, encoded by the same gene, but differing in their N-terminus. DNA-binding is achieved through the tetrahedral coordination of zinc Zn by four cysteine residues in each of two extensions, that form two structurally distinct "Zn fingers" Fig.
The more C-terminal part of the C2 Zn finger and amino acids in the hinge region are involved in receptor dimerization in coordination with amino acids in the hinge region and the LBD. Zinc fingers are common features of many transcription factors, allowing proteins to bind to DNA. Each circle represents one amino acid. Annu Rev Biochem ; Transcription activation by estrogen and progesterone receptors.
Annu Rev Genet ; The hinge region or D domain is a 40—50 amino acid sequence separating the DNA-binding and ligand-binding domains that contains sequences for receptor dimerization and ligand-dependent and independent nuclear localization sequences NLSs. The carboxy C -terminal or ligand-binding domain LBD is poorly conserved, ranging in size from to amino acids and is hydrophobic.
This region contains the ligand-binding site and dictates hormone binding specificity. Helix 12 is indispensable for AF-2 function. The C-terminal AF-2 transactivation domain is highly conserved within the nuclear receptor superfamily 36 and is recognized by various transcriptional coregulators, formerly referred to as coactivators or corepressors.
Additionally, the N- and C- terminals of the receptor interact with each other to increase transcriptional activation. All genes share a common basic design Fig. Several key elements in the regulatory region of the target gene must be activated before mRNA synthesis can occur. Located further upstream are one or more hormone response elements HREs , the specific DNA-binding sites to which steroid receptors bind, conferring hormone sensitivity to the gene.
Hormone response elements HREs are consensus 13—bp DNA sequences derived from alignment of genes responsive to a particular steroid hormone. HREs have two "half-sites" that each bind the C1 zinc finger of one receptor monomer. Steroid hormone receptors bind DNA as homodimers or heterodimers, e. Response elements for the class II nuclear receptors are direct repeats DR , inverted repeats IR , or everted repeats EvR of the indicated half-site with the letter following the DR or IR indicating the number of nucleotides separating the half-sites, e.
One of the major advances in the field of transcriptional regulation by ER has been the development of ChIP and the using of tiling arrays to identify ER binding sites throughout the human genome.
The response elements for the various class II NR, e. Steroid hormones are small hydrophobic and lipid-soluble molecules derived from cholesterol. When circulating levels of steroid hormones exceed the binding capacity of their respective binding proteins, they can then bind nonspecifically, and with low affinity, to albumin, from which they can readily dissociate and enter target cells. Extracellular binding proteins may modulate hormone response by regulating the amount of steroid available to the cell.
The current model is that the steroid hormone diffuses freely in the cytoplasm. Early attempts to isolate steroid hormone receptors led to a major controversy regarding their intracellular localization in the unliganded state.
Reports published before suggested that prior to hormone treatment, steroid receptors in target tissues were located predominantly in the cytosolic fraction as large protein complexes of — kDa. The exceptions are the glucocorticoid and mineralocorticoid receptors, which in the unliganded state reside in the cytoplasm in association with hsp90, hsp70, and a variety of receptor-associated proteins. All class II nuclear receptors are nuclear in the absence of ligand.
The newly synthesized unliganded receptor is highly unstable and either moves into the nucleus, targeted by its nuclear localization signals, soon after synthesis, or associates with the hsp90 complex of cytoplasmic proteins. In addition to hsp90, unliganded steroid receptors extracted from animal tissues or mammalian cells showed that GR and PR are complexed with a number of other proteins including hsp70, FKB59, p60, p48 Hip , and p A target cell contains the steroid hormone receptor s required to respond to a given steroid hormone.
Most steroid hormones that enter the cell travel in the bloodstream either in free state or loosely bound to serum albumins. Estrogens and androgens are bound to steroid hormone binding globulin SHBG with high affinity. The cell membrane contains a SHBG receptor linked to adenylate cyclase.
The exception is the glucocorticoid receptor GR which is complexed with the hsp90 chaperonin complex in the cytoplasm. Hsp90 and its accompanying proteins are thought to stabilize the receptor until hormone-binding occurs.
Although receptors for progesterone, estrogens, and androgens have also been found in complexes with the primarily cytosolic hsps, the function of these associations is unclear. In the case of estrogens, progestins, and androgens, the hormones freely enter the nucleus and bind to their cognate receptor.
This allows the receptors to form homodimers and bind to specific hormone response elements HREs, see Table 2 for specific sequences in the DNA in the regulatory region of the target gene.
Once bound to the HRE, the liganded steroid hormone receptor induces a DNA bend and, in the presence of agonist ligand, recruits coactivator proteins. Certain coactivator proteins have histone acetyltransferase HAT activity that acetylates lysine residues in histones H3 and H4. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D. The role of the hsp90 complex in ER function is not yet clear. Steroid hormone receptors within living cells are dynamic.
The hormone receptor enters the nucleus by two processes: The ligand-binding domain of the receptor may act as a repressor of receptor function since deletion of the LBD from the glucocorticoid and progesterone receptors causes constitutive gene activation. Binding of the hormone to the receptor may be only one of several factors that activates or transforms the receptor, enabling it to bind as a dimer to specific hormone response elements located adjacent to or sometimes at a distance from the transcription start site of the regulated gene.
It is important to note that Type II nuclear receptors, e. These receptors are bound to DNA in the absence of ligand and are associated with corepressor proteins, e. Hormone-dependent phosphorylation of steroid hormone receptors may play an important role in binding of the receptor to its specific response element on the gene and subsequent activation of transcription. Although steroid and nuclear receptors are classically activated by ligand binding and are subsequently phosphorylated, a second mode of activation in the absence of ligand has been detected for certain receptors.
Likewise activation of the protein kinase A PKA pathway stimulated transcription by the MR in a ligand-independent manner. DNA bending appears to be important in cellular processes mediated by multiprotein complexes, including transcription in both prokaryotes, and eukaryotes.
Initiation of transcription is a complex event occurring through the cooperative interaction of multiple factors at the target gene promoter Fig.
When bound to the specific HRE on the DNA, the hormone-receptor complex interacts with basal transcription factors and with other proteins to stabilize basal transcription factor binding and promote the assembly of the transcription initiation complex. It is clear that steroid receptors can interact with a number of different coactivators. Some of these coactivators, i. Steroid receptors show different affinities for the various coactivators and use different amino acids to contact the coactivators.
It is important to note that not all genes are affected by histone acetylation, and steroid and nuclear receptors show different affinities of interaction with coactivators. Examples of coactivator proteins that interact with steroid hormone receptors. These proteins have been identified in yeast and mammalian two hybrid screening, during purification, in immunoprecipitation assays, and by cross-linking studies. Some, but not all, have been shown to stimulate transcription in cell systems , , II holoenzyme to the promoter.
Tamoxifen agonism at AP-1 sites is cell type specific, i. A likely interpretation is that coactivators are required. One mechanism by which a steroid receptor represses gene transcription is by binding to the same, or an overlapping, DNA binding site as that used by a different activator protein, thus competitively blocking activator binding to DNA.
Unlike coactivator complexes that have endogenous HAT activity, corepressors do not appear to have enzymatic activity. A variety of mechanisms is likely to be involved in hormone antagonist action. First, the antagonist activity of an antihormone may depend on the cell or tissue type.
While most of the effects of steroid hormones are mediated through their interaction with their cognate receptors and subsequent effects on target gene transcription, certain rapid effects of steroid hormones are incompatible with a transcriptional mechanism. Specific binding sites for androgen, estrogen, progesterone, glucocorticoid, and vitamin D receptors have been reported in the plasma membrane of various target cell types.
GR can bind to cytoskeletal structures and glucocorticoids stimulate the rapid-onset of polymerization of actin in a non-genomic manner that involves decreased intracellular cAMP. There is clearly a need for further research to elucidate the roles of membrane PRs in mediating progestin activity in various tissues. Additionally, binding sites for estrogen with different biochemical properties from the classical nuclear receptor have been reported in the endoplasmic reticulum of uterine tissues.
Estradiol appears to have both genomic and nongenomic effects in the brain. Diversity of tissue responses to steroid hormone action, despite conservation of structure and function, is achieved through a variety of mechanisms.