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Another type of relatively simple, though much slower, signaling is seen in pathways in which the signals are steroid hormones, like estrogen or testosterone, pictured below. Steroid hormones, as you are aware, are related to cholesterol, and as hydrophobic molecules, they are able to cross the cell membrane by themselves. This is unusual, as most signals coming to cells are incapable of crossing the plasma membrane, and thus, must have cell surface receptors.
By contrast, steroid hormones have receptors inside the cell (intracellular receptors). Steroid hormone receptors are proteins that belong in a family known as the nuclear receptors. Nuclear hormone receptors are proteins with a double life: they are actually dormant transcription regulators. In the absence of signal, these receptors are in the cytoplasm, complexed with other proteins (HSP in Figure 8.3.2) and inactive. When a steroid hormone enters the cell, the nuclear hormone receptor binds the hormone and dissociates from the HSP. The receptors, then, with the hormone bound, translocate into the nucleus.
In the nucleus, Nuclear hormone receptors regulate the transcription of target genes by binding to their regulatory sequences (labeled HRE for hormone- response elements). The binding of the hormone-receptor complex to the regulatory elements of hormone-responsive genes modulates their expression. Because these responses involve gene expression, they are relatively slow. Most other signaling pathways, besides the two we have just discussed, involve multiple steps in which the original signal is passed on and amplified through a number of intermediate steps, before the cell responds to the signal.
We will now consider two signaling pathways, each mediated by a major class of cell surface receptor- the G-protein coupled receptors (GPCRs) and the receptor tyrosine kinases (RTKs). While the specific details of the signaling pathways that follow the binding of signals to each of these receptor types are different, it is easier to learn them when you can see what the pathways have in common, namely, interaction of the signal with a receptor, followed by relaying the signal through a variable number of intermediate molecules, with the last of these molecules interacting with target protein(s) to modify their activity in the cell.
Nuclear Hormone Receptor
Nuclear hormone receptors are implicated in a variety of disorders such as asthma (glucocorticoid receptor), hyperlipidemia and diabetes mellitus (PPARs), rickets (vitamin D), gender disorders (progesterone, androgen and estrogen receptors), congestive heart failure (mineralocorticoid receptor), stress (glucocorticoid receptor), breast cancer (estrogen receptor), integumental disorders (retinoid receptors), and contraception (estrogen and progesterone receptors).
Hormone Receptors: Meaning and Types
A hormone receptor is a receptor protein on the surface of a cell or in its interior that binds to a specific hormone. The hormone causes many changes that take place in the cell. Binding of hormones to hormone receptors often trigger the start of a biophysical signal that can lead to further signal transduction pathways, or trigger the activation or inhibition of genes.
Types of Hormone Receptors:
Peptide Hormone Receptors:
Are often trans membrane proteins. They are also called G-protein- coupled receptors, sensory receptors or ionotropic receptors. These receptors generally function via intracellular second messengers, including cyclic AMP (cAMP), inositol 1, 4, 5-triphosphate (IP3) and the calcium (Ca 2+ )—calmodulin system.
Steroid Hormone Receptors and Related Receptors:
Are generally soluble proteins that function through gene activation. Their response elements are DNA sequences (promoters) that are bound by the complex of the steroid bound to its receptor. The receptors themselves are zinc-finger proteins. These receptors include those for glucocorticoids, estrogens, androgens, thyroid hormone (T3), calcitriol (the active form of vitamin D), and the retinoids (vitamin A).
Receptors for Peptide Hormones:
With the exception of the thyroid hormone receptor, the receptors for amino acid derived and peptide hormones are located in the plasma membrane. Receptor structure is varied.
Some receptors consist of a single polypeptide chain with a domain on either side of the membrane, connected by a membrane-spanning domain. Some receptors are comprised of a single polypeptide chain that is passed back and forth in serpentine fashion across the membrane, giving multiple intracellular, trans membrane, and extracellular domains. Other receptors are composed of multiple polypeptides. Ex. The insulin receptor is a disulfide linked tetramer with the β-subunits spanning the membrane and the α-subunits located on the exterior surface.
Subsequent to hormone binding, a signal is transduced to the interior of the cell, where second messengers and phosphorylated proteins generate appropriate metabolic responses. The main second messengers are cAMP, Ca 2+ , inositol triphosphate (IP3), and diacylglycerol (DAG).
Proteins are phospho­rylated on serine and threonine by cAMP-dependent protein kinase (PKA) and DAG-activated protein kinase C (PKC). Additionally a series of membrane-associated and intracellular tyrosine kinases phosphorylate specific tyrosine residues on target enzymes and other regulatory proteins.
The hormone-binding signal of most, but not all, plasma membrane receptors is transduced to the interior of cells by the binding of receptor-ligand complexes to a series of membrane-localized GDP/GTP binding proteins known as G-proteins. The classic interactions between receptors, G-protein transducer, and membrane-localized adenylate cyclase are illustrated using the pancreatic hormone glucagon as an example.
When G-proteins bind to receptors, GTP exchanges with GDP bound to the α-subunit of the G-protein. The Ga-GTP complex binds adenylate cyclase, activating the enzyme. The activation of ade­nylate cyclase leads to cAMP production in the cytosol and to the activation of PKA, followed by regulatory phosphorylation of numerous enzymes. Stimulatory G-proteins are designated Gs, inhibitory G-proteins are designated Gi.
A second class of peptide hormones induces the transduction of 2 second messengers, DAG and IP3. Hormone binding is followed by interaction with a stimulatory G-protein which is followed in turn by G-protein activation of membrane-localized phospholipase C-y, (PLC-y). PLC-y hydrolyzes phosphatidylinositol bisphosphate to produce 2 messengers viz. IP3, which is soluble in the cytosol, and DAG, which remains in the membrane phase.
Cytosolic IP3 binds to sites on the endoplasmic reticulum, opening Ca 2+ channels and allowing stored Ca 2+ to flood the cytosol. There it activates numerous enzymes, many by activating their calmodulin or calmodulin-like subunits.
DAG has 2 roles-it binds and activates PKC, and it opens Ca 2+ channels in the plasma membrane, reinforcing the effect of IP3. Like PKA, PKC phosphorylates serine and threonine residues of many proteins, thus modulating their catalytic activity.
Is a trans membrane receptor that is activated by insulin. It belongs to the large class of tyrosine kinase receptors. Two alpha subunits and two beta subunits make up the insulin receptor. The beta subunits pass through the cellular membrane and are linked by disulfide bonds. The alpha and beta subunits are encoded by a single gene (INSR). The insulin receptor has been designated as CD220 (cluster of differen­tiation 220).
Function of insulin receptor-effect of insulin on glucose uptake and metabolism:
Insulin binds to its receptor which in turn starts many protein activation cascades.
i. Translocation of Glut-4 transporter to the plasma membrane and influx of glucose
iii. Glycolysis and fatty acid synthesis
Insulin receptors (a family of tyrosine kinase receptors), mediate their activity by causing the addition of a phosphate group to particular tyrosine’s on certain proteins within a cell. The ‘substrate’ proteins which are phosphorylated by the insulin receptor include a protein called ‘IRS-1’ for ‘Insulin Receptor Substrate-1’.
IRS-1 binding and phosphorylation eventually leads to an increase in the high affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue, and therefore to an increase in the uptake of glucose from blood into these tissues. Briefly, the glucose transporter (Glut4) is transported from cellular vesicles to the cell surface, where it then can mediate the transport of glucose into the cell. Glycogen synthesis is also stimulated by the insulin receptor via IRS-1.
Pathology of insulin receptors:
The main activity of activation of the insulin receptor is inducing glucose uptake. For this reason ‘insulin insensitivity’, or a decrease in insulin receptor signaling, leads to diabetes mellitus type 2 – the cells are unable to take up glucose, and the result is hyperglycemia (an increase in circulating glucose), and all the sequelae which result from diabetes. Patients with insulin resistance may display acanthosis nigricans.
A few patients with homozygous mutations in the INSR gene have been described, which causes Donohue syndrome or Leprechauns. This autosomal recessive disorder results in a totally non-functional insulin receptor. These patients have low set, often protruberant ears, flared nostrils, thickened lips, and severe growth retardation.
In most cases, the outlook for these patients is extremely poor with death occurring within the first year of life. Other mutations of the same gene cause the less severe Rabson-Mendenhall syndrome, in which patients have characteristically abnor­mal teeth, hypertrophic gingiva (gums) and enlargement of the pineal gland. Both diseases present with fluctuations of the glucose level—after a meal the glucose is initially very high, and then falls rapidly to abnormally low levels.
Degradation of insulin and its receptors:
Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment or it may be degraded by the cell. Degradation normally involves endocytosis of the insulin-receptor complex followed by the action of insulin degrading enzyme. Most insulin molecules are degraded by liver cells. It has been estimated that a typical insulin molecule is finally degraded about 71 minutes after its initial release into circulation.
It is a 62 kDa peptide that is activated by glucagon and is a member of the G- protein coupled family of receptors, coupled to Gs. Stimulation of the receptor results in activation of adenylate cyclase and increased levels of intracellular cAMP. Glucagon receptors are mainly expressed in liver and in kidney with lesser amounts found in heart, adipose tissue, spleen, thymus, adrenal glands, pancreas, cerebral cortex, and G.I. tract.
Steroid Hormone Receptors:
Are proteins that have a binding site for a particular steroid molecule. Their response elements are DNA sequences that are bound by the complex of the steroid bound to its receptor. The response element is part of the promoter of a gene. Binding by the receptor activates or represses, as the case may be, the gene controlled by that promoter. It is through this mechanism that steroid hormones turn genes on (or off).
The DNA sequence of the glucocorticoid (a protein homodimer) response element is:
where n represents any nucleotide (a palindromic sequence)
The glucocorticoid receptor, like all steroid hormone receptors, is a zinc-finger transcription factor there are four zinc atoms each attached to four cysteine’s.
For a steroid hormone to turn gene transcription on, its receptor must:
(ii) Bind to a second copy of itself to form a homodimer
(iii) Be in the nucleus, moving from the cytosol if necessary
(iv) Bind to its response element
(v) Activate other transcription factors to start transcription
Each of these functions depends upon a particular region of the protein (Ex. The zinc fingers for binding DNA). Mutations in any one region may upset the function of that region without necessarily interfering with other functions of the receptor.
Nuclear Receptor Superfamily:
The zinc-finger proteins that serve as receptors for glucocorticoids and progesterone are members of a large family of similar proteins that serve as receptors for a variety of small, hydrophobic molecules. These include other steroid hormones like the mineralocorticoid-aldoster- one, estrogens, the thyroid hormone (T3), calcitriol (the active form of vitamin D), rednoids—vitamin A (retinol) and its relatives-retinal/retinoic acid, bile acids and fatty acids.
These bind members of the superfamily called Peroxisome Proliferator Activated Receptors (PPARs). They got their name from their initial discovery as the receptors for drugs that increase the number and size of peroxisomes in cells.
In every case, the receptors consists of at least three functional modules or domains from N-terminal to C-terminal, these are:
i. A domain needed for the receptor to activate the promoters of the genes being controlled
ii. The zinc-finger domain needed for DNA binding (to the response element)
iii. The domain responsible for binding the particular hormone as well as the second unit of the dimer
Receptors for Thyroid Hormones:
Are members of a large family of nuclear receptors that include those of the steroid hormones. They function as hormone-activated transcription factors and thereby act by modulating gene expression.
Thyroid hormone receptors bind DNA in absence of hormone:
Usually leading to transcriptional repression. Hormone binding is associated with a conformational change in the receptor that causes it to function as a transcriptional activator.
Mammalian thyroid hormone receptors are encoded by two genes, designated alpha and beta. Further, the primary transcript for each gene can be alternatively spliced, generating different alpha and beta receptor isoforms. Currently, four different thyroid hormone receptors are recognized as-(i) α-1 (ii) α-2 (iii) β-1 and (iv) β-2.
Like other members of the nuclear receptor superfamily, thyroid hormone receptors encapsulate three functional domains:
i. A transactivation domain at the amino terminus that interacts with other transcription factors to form complexes that repress or activate transcription. There is considerable divergence in sequence of the transactivation domains of alpha and beta isoforms and between the two beta isoforms of the receptor.
ii. A DNA-binding domain that binds to sequences of promoter DNA known as hormone response elements.
iii. A ligand-binding and dimerization domain at the carboxy-terminus.
Disorders of thyroid hormone receptors:
A number of humans with a syndrome of thyroid hormone resistance have been identified, and found to have mutations in the receptor beta gene which abolish ligand binding. Clinically, such individuals show a type of hypothyroidism characterized by goiter, elevated serum concentrations of T3 and thyroxine and normal or elevated serum concentrations of TSH.
More than half of affected children show attention-deficit disorder, which is intriguing considering the role of thyroid hormones in brain development. In most affected families, this disorder is transmitted as a dominant trait, which suggests that the mutant receptors act in a dominant negative manner.
Adrenergic Receptors (or Adrenoceptors):
Are a class of G-protein coupled receptors that are targets of the catecholamine’s. Adrenergic receptors specifically bind their endogenous ligands, the catecholamine’s adrenaline and noradrenalin (called epinephrine and norepinephrine), and are activated by these.
Many cells possess these receptors, and the binding of an agonist will generally cause a sympathetic response (i.e. the fight-or-flight response) viz. the heart rate will increase and the pupils will dilate, energy will be mobilized, and blood flow diverted from other, non-essential, organs to skeletal muscle. There are several types of adrenergic receptors, but there are two main groups viz. a-adrenergic and P-adrenergic.
These receptors bind noradrenalin (norepinephrine) and adrenaline (epineph­rine). Phenylephrine is a selective agonist of the a-receptor. They exist as α1-adrenergic receptors and α2-adrenergic receptors.
These receptors are linked to Gs proteins, which in turn are linked to adenyl cyclase. Agonist binding thus causes a rise in the intracellular concentration of the second messenger cAMP. Downstream effectors of cAMP include cAMP-dependent protein kinase (PKA), which mediates some of the intracellular events following hormone binding.
Epinephrine reacts with both α and β-adrenoreceptors, causing vasoconstricdon and vasodilation, respectively. Although receptors are less sensitive to epinephrine, when activated, they override the vasodilation mediated by β-adrenoreceptors. The result is that high levels of circulating epinephrine cause vasoconstriction. Lower levels of epinephrine dominates β-adrenoreceptor stimulation, producing an overall vasodilation.
The mechanism of adrenergic receptors:
Adrenaline or noradrenalin is receptor ligands to either α1, α2 or β-adrenergic receptors, a, couples to Gq, which results in increased intracellular Ca 2+ which results in smooth muscle contraction. α2 on the other hand, couples to Gi, which causes a decrease of cAMP activity, resulting in smooth muscle contraction. β receptors couple to Gs, and increase intracellular cAMP activity, resulting in heart muscle contraction, smooth muscle relaxation and glycogenolysis.
Functions of α-receptors:
α-Receptors have several functions in common. They are:
(i) Vasoconstriction of arteries to heart (coronary artery)
(ii) Vasoconstriction of veins
(iii) Decrease motility of smooth muscle in gastrointestinal tract
Alpha-1 adrenergic receptor:
Alpha-1 -adrenergic receptors are members of the G protein-coupled re­ceptor superfamily. Upon activation, a heterotrimeric G-protein, Gq, activates phospholipase C (PLC), which causes an increase in IP3 and calcium. This triggers all other effects. Specific actions of the β1 receptor mainly involve smooth muscle contraction.
It causes vasoconstriction in many blood vessels including those of the skin & gastrointestinal system and to kidney (renal artery) and brain. Other areas of smooth muscle contraction are for instance – ureter, vas deferens, hairs (arrector pili muscles), uterus (when pregnant), urethral sphincter, bronchioles (although minor to the relaxing effect of β2 receptor on bronchioles). Further effects include glycogenolysis and gluconeogenesis from adipose tissue and liver, as well as secretion from sweat glands and Na reabsorption from kidney.
Alpha-2 adrenergic receptor:
There are 3 highly homologous subtypes of α2 receptors viz. α2A, α2B, and α2C. Specific actions of the a2-receptor include:
i. Inhibition of insulin release in pancreas
ii. Induction of glucagon release from pancreas
iii. Contraction of sphincters of the gastrointestinal tract
Beta-1 adrenergic receptor:
Specific actions of the β1 receptor include:
i. Increase cardiac output, both by raising heart rate and increasing the volume expelled with each beat (increased ejection fraction)
ii. Renin release from juxtaglomerular cells
iii. Lipolysis in adipose tissue
Beta-2 adrenergic receptor:
Specific actions of the β2 receptor include:
i. Smooth muscle relaxation, e.g. in bronchi
ii. Relaxes urinary sphincter and pregnant uterus
iii. Relaxes detrusor urinary muscle of bladder wall
iv. Dilates arteries to skeletal muscle
v. Glycogenolysis and gluconeogenesis
vi. Contract sphincters of GI tract
vii. Thickened secretions from salivary glands
viii. Inhibit histamine-release from mast cells
ix. Increase renin secretion from kidney
5 Properties of Hormone Receptor Interactions | Biology
The following points highlight the top five properties of hormone receptor interactions. The properties are: 1. Hormone Receptor Interaction is Rapid and Reversible 2. Receptor Specificity 3. Receptor Affinity 4. Saturation 5. Agonist and Antagonist.
Property # 1. Hormone Receptor Interaction is Rapid and Reversible:
Most hormones bind to their receptors rapidly and reversibly. The interaction also terminates very rapidly. The number of receptors for a given hormone on any cell type varies from zero to more than 1 million. Moreover, non-target cells may also possess receptors. For example, TSH recep­tors are not only found on thyroid cells but also on adipocytes.
Property # 2. Receptor Specificity:
The most characteristic and essential feature of hor­mone receptor interaction is the specificity of binding. At the first level, specificity means that each hormone binds to its specific recep­tor. For example, glucagon binds only to glucagon receptors, insulin to insulin recep­tors. At a more subtle level, specificity implies that a hormone and its derivatives bind to their receptors with an affinity that is directly related to its bioactivity.
Human insulin is about 50 times as potent in stimu­lating the metabolism of isolated fat cells as human pro-insulin. It binds to insulin recep­tor with 50 times the affinity of pro-insulin. Insulin-like growth factor-I has an even lower affinity for the insulin receptors and even lower insulin-like activity.
Property # 3. Receptor Affinity:
Hormones bind to their receptors with high affinity and specificity, because most peptide hormones are present in the circulation in pico-molar to Nano molar concentrations. Receptors must have approximately high affinities to achieve significant binding at physiological levels. Receptor affinity may be calculated from the kinetics of association and dissociation.
The affinity of a hormone for its receptor results from non-covalent binding, primarily in the form of hydrophobic interactions that provide the driving force for the binding reaction, and from electrostatic interactions.
Property # 4. Saturation:
The binding of hormone to receptor is a saturable process there are a finite number of receptors on a target cell. In addition, the binding of hormone to receptor must either precede or accompany the bio­logical response, and the magnitude of the biological response must be associated, in some manner, with receptor occupancy.
Property # 5. Agonist and Antagonist:
Hormones or analogues that bind to receptors and elicit the same biological response as the naturally occurring hormone are termed as agonists. Molecules that bind to receptors but fail to elicit the normal biological response are termed competitive antagonists, as they occupy the receptors and prevent the binding of the biologically active molecules.
Molecules that bind to receptors but are less biologically active than the native hor­mones are termed partial agonists.
Nuclear Receptors (With Diagram) | Biology
Steroid hormones exert their action on target cells after binding to specific receptors which are ligand-inducible transcription factors localized primarily within the nucleus. The steroid-receptor complex regulates the synthesis of specific proteins primarily by altering the rate of transcription of specific genes.
The steroid hormone receptors belong to a super-family of so-called nuclear receptors that also includes the receptors for 1, 25- di-hydroxy-vitamin D3, several tissue-specific thyroid receptors and retinoic acid. By phylogenetic analysis, the superfamily can be divided into three subfamilies:
(1) Type I receptors include glucocorti­coid receptor (GR), mineralocorticoid recep­tor (MR), androgen receptor (AR) and estro­gen receptor (ER).
(2) Type II receptors include thyroid receptor (TR), all-trans retinoic acid receptor (RAR), 9-cis retinoic acid receptor (RXR) and 1, 25-di-hydroxy-vitamin D3 receptor (VDR).
(3) Type III recep­tors include the orphan receptors that have no known ligands. All these receptors have specific domains that are highly conserved structurally and functionally among all members of this superfamily (Fig. 7.9).
All these receptors have a DNA-binding domain (DAB), which is composed of 66-68 amino acids and contain nine perfectly con­served cysteine residues. This domain consists of two repeated units, each of which is folded into a finger-like structure containing four cysteines that coordinate one zinc ion. These DNA binding “fingers” have the capacity to insert into a half-turn of DNA.
Near the carboxy-terminal end of the receptor molecules are two conserved regions (HBD1 and HBD2) of 42 and 22 amino acids respectively, which com­prise the hormone binding domain. The HBD also contains a transcription activation do­main, termed as activation function 2 (AF-2), which is essential for ligand dependent acti­vation of transcription by nuclear receptors.
(1) Activation of the receptors:
“Free” glucocorticoid, mineralocorticoid, androgen, and estrogen receptors are known to exist in the cell as monomers associated with a com­plex of proteins that include phosphoproteins (hsp) and other proeins. In this conformation, the receptor is incapable of binding DNA or regulating gene transcription.
The binding of hormone to receptors results in the dissocia­tion of receptor from this complex, which forms a homodimer with another receptor molecule. This receptor homodimer has a greatly increased affinity for binding to HRE (Hormone receptor elements in DNA. Upon binding to DNA, the receptor homodimer recruits proteins termed as co-activators which facilitate transcription initiation.
On the other hand, “free” receptors for thyroid hormone, retinoic acid and 1, 25- di-hydroxy-vitamin D3 are not bound to hsps. Rather, they are bound to their HREs either as homodimers or as heterodimers. In their free state, receptors in this subgroup recruit proteins termed as co-repressors that inhibit transcription of their respective target genes.
(2) Regulation of gene expression:
As discussed above, the GR, MR, AR and ER upon binding to DNA, the receptor homo- dimer recruits proteins called co-activators that have histone acetyltransferase acti­vities.
Such co-activators then destabilize the nucleosome structure and cause a local open­ing of chromatin into a transcriptionally permessive state. Subsequently other transcrip­tion factors and RNA polymerase II are recruited and gene transcription is initiated.
The “free” dimeric TR, RAR and VDR interact with a co-repressor complex contai­ning histone deacetylase, which keeps chro­matin in a ‘closed’ conformation and silence transcription. The binding of T3 to T3R stabi­lizes formation of a heterodimer with RXR that causes dissociation of the co-repressor complex and recruitment of a co-activator complex containing histone acetyl-transferases.
Histone acetylation then causes de-stabilisation of the nucleosomal structure, resulting in an opening of the local chromatin structure, into a transcriptionally permissive state that allows assembly of the pre-initiation complex and subsequent recruitment of transcription factors and Pol II. As a result transcription of thyroid hormone-responsive genes is initia­ted.
With the fall in hormone concentration in the target cells, the hormone receptor com­plex gets dislodged from the HRE of the DNA and dissociates into the hormone and the receptor. The receptors are immediately inactivated by processes like conformational or ionisation changes, oligomerisation or phosphorylation.
B. Hormone Response Elements
Nuclear receptors regulate transcription by binding to specific DNA sequences in target genes known as hormone response elements or HREs. These elements are located in regulatory sequences normally present in the 5′-flanking region of the target gene. Although often the HREs are found relatively close to the core promoter, in some cases they are present in enhancer regions several kilobases upstream of the transcriptional initiation site. The analysis of a large number of naturally occurring as well as synthetic HREs revealed that a sequence of 6 bp constitutes the core recognition motif. Two consensus motifs have been identified: the sequence AGAACA is preferentially recognized by steroid class III receptors, whereas AGG/TTCA serves as recognition motif for the remaining receptors of the superfamily (17). It should be noted that these motifs represent consensus idealized sequences and that naturally occurring HREs can show significant variation from the consensus. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. For dimeric HREs, the half-sites can be configured as palindromes (Pal), inverted palindromes (IPs), or direct repeats (DRs).
Steroid hormone receptors typically bind to palindromes of the AGAACA sequence separated by three nucleotides, with the exception of the ERs that recognize the consensus AGGTCA motif with the same configuration. On the basis of the analysis of glucocorticoid receptor/ER chimeras, the first zinc finger has been identified as the one responsible for the discrimination of the DNA motif (91). Further studies have shown that mutation of three residues in the P box, which are identical in the glucocorticoid, progesterone, androgen, and mineralocorticoid receptors that recognize the same HRE, was sufficient to switch the sequence recognized by glucocorticoid receptors and ERs. Furthermore, cocrystal structures of receptor DBDs with DNA have shown that P box residues, which are contained within the recognition helix 1 of the DBD, were indeed involved in interaction with specific bases of the recognition motifs (for a detailed review on the interaction of receptors with the HREs, see Ref. 87).
In contrast to steroid receptors that almost exclusively recognize palindromic elements, nonsteroidal receptors can bind to HREs with different configurations (Fig. 5). In this case, the arrangement as well as the spacing between the motifs are determinant to confer selectivity and specificity. Some of these response elements are capable of mediating transcriptional responses to more than one ligand. This is the case of the palindromic element AGGTCATGACCT that confers regulation by both thyroid hormones and retinoic acid (271). As a consequence, both ligands can control overlapping gene networks as demonstrated by the regulation of the rat growth hormone gene by the two hormones via a common HRE (19). Similarly, IPs can also mediate transcriptional responses to both ligands as well as to vitamin D. However, a careful analysis of natural and synthetic HREs has shown that the most potent HREs for nonsteroid receptors are configured as DRs. Analysis of variably spaced DRs suggested that the length of the spacer region was an important determinant of the specificity of hormonal responses. Thus DRs separated by 3, 4, and 5 bp (i.e., DR3, DR4, and DR5) mediate preferential regulation by vitamin D, thyroid hormone, and retinoic acid, respectively (183, 272). The subsequent demonstration that DR1 serves as the preferred HRE for the RXR or for the PPAR and that RARs can also activate transcription through a DR2, expanded the model from a 3-to-5 rule to a 1-to-5 rule (reviewed in Ref. 163). Furthermore, a DR0 sequence can also act as a receptor binding site, and widely spaced DRs can act as promiscuous response elements for different nonsteroid receptors and even for ERs (132). The configuration of the preferred HREs for different classical and orphan receptors has been included in Table 1.
More recent results have shown that in addition to spacing, small differences in the half-site sequence and the sequence of the flanking extension of the response elements also appear to be important parameters in determining receptor binding efficiency (162).
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8.3: Nuclear Hormone Receptors - Biology
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Hormones can also bind to intracellular receptors, protein receptors found inside the target cell, to alter cellular functions.
First, lipid-soluble hormones like the steroid testosterone easily diffuse across the cell membrane of the endocrine cell of the testes. Once outside in the extracellular fluid, these hormones attach to transport proteins to remain soluble, which is particularly important in the aqueous blood stream.
At the target cell, the lipid-soluble hormone detaches from the transport protein and then diffuses through the cell membrane. Now inside the cell, it binds to its intracellular hormone receptors, in this case, androgen receptors. This complex can enter the nucleus and then bind to a specific DNA sequence called a hormone response element, which triggers gene transcription and translation, thus regulating the production of mRNA and gene expression.
21.3: Intracellular Hormone Receptors
Lipid-soluble hormones diffuse across the plasma and nuclear membrane of target cells to bind to their specific intracellular receptors. These receptors act as transcription factors that regulate gene expression and protein synthesis in the target cell
Based on their mode of action, intracellular hormone receptors are classified as Type I or Type II receptors. Type I receptors, including steroid hormone receptors such as the androgen receptor, are present in the cytoplasm. Hormone binding transports the hormone-receptor complex to the nucleus, where it binds to regulatory DNA sequences called hormone response elements and activates gene transcription.
Type II receptors, such as the thyroid hormone receptor, are bound to their DNA response elements within the nucleus even in the absence of hormone. In this state, the receptor acts as an active repressor of transcription. However, upon hormone binding, the receptor-hormone complex activates transcription of thyroid hormone-inducible genes.
Sever, Richard, and Christopher K. Glass. &ldquoSignaling by Nuclear Receptors.&rdquo Cold Spring Harbor Perspectives in Biology 5, no. 3 (March 2013). [Source]
B. Transcriptional Antagonism and “Cross-Talk” With Other Signaling Pathways
Although as described in section ii C, the orientation and spacing of the half-sites can determine selective transcriptional responses to nuclear receptors, specificity is not total, and some HREs can bind different heterodimers with high affinity. However, only a subset of receptor DNA binding elements function as response elements. As described above, the heterodimer RAR/RXR binds to a DR1 in a transcriptionally inactive form and antagonizes the response mediated by the active RXR homodimers (140). Equally, VDR/RXR can bind retinoic acid and thyroid hormone response elements in a transcriptionally inactive form, and under these circumstances vitamin D can inhibit the response to those ligands (81, 120). However, although competition for DNA binding by transcriptionally inactive VDR/RXR heterodimers may contribute to this inhibitory response, mutants lacking the A/B domain and the DNA-binding domain also display a dominant negative activity, suggesting that titration of coactivators could be involved in the inhibitory effect of vitamin D (121). Similarly, mutant or truncated transcriptionally inactive receptors in some syndromes of hormone resistance can compete binding of wild-type receptors to DNA, presenting a dominant-negative activity and reducing hormone-mediated transcriptional responses.
In the case of heterodimeric receptors, competition for limiting concentrations of RXR may also represent a mechanism for modulating transcriptional responses to several partner receptors (12). Thus COUPs can act as transcriptional repressors antagonizing activation mediated by different receptors, and this antagonism may involve competition for DNA binding sites, competition for RXR, and formation of inactive complexes with other receptors (269). An unusual receptor, the small heterodimer partner (SHP), lacks a typical DBD and can heterodimerize with different nuclear receptors leading to inhibition of binding to DNA and transcriptional inactivation (122, 240, 241).
Nuclear receptors can also modulate gene expression by mechanisms independent of binding to an HRE. Thus they can alter expression of genes that do not contain an HRE through positive or negative interference with the activity of other transcription factors, a mechanism generally referred to as “transcriptional cross-talk” (90). The ERs utilize protein-protein interactions to enhance transcription of genes that contain AP-1 sites (83). The AP-1 complex that is composed of dimers of Jun family proteins and preferently of Jun/Fos heterodimers plays an important role in cell proliferation. ERα and ERβ have been shown to signal in opposite ways at AP-1 sites. ERα activates transcription in the presence of estradiol, whereas with ERβ estradiol inhibits AP-1-dependent transcription. Furthermore, antiestrogens can act as agonists of ER action at AP-1 sites. This is particularly evident in the case of ERβ, which enhances AP-1-dependent transcription in the presence of antiestrogens but not estrogens (194).
One of the best known examples of the cross-talk between nuclear receptors and AP-1 complexes is the finding that several receptors, such as TR, RAR, or GR, can act as ligand-dependent transrepressors of AP-1 (Jun/Fos) activity, and reciprocally, that AP1 can inhibit transactivation by nuclear receptors (203). It is believed that many of the antiproliferative effects of ligands of nuclear receptors could be mediated by their anti-AP-1 activity. Similarly, some nuclear receptors, specifically GR, can also mutually interfere with NF-κB activity, which could be involved in the anti-inflammatory and immunosuppressive effects of glucocorticoids.
In some cases the cross-talk between the receptors and AP-1 can involve binding to a “composite element” that can bind both the receptors and the AP-1 complex, and depending on the composition of the AP-1 complexes they can either cooperate or antagonize transcription by nuclear receptors (for a review see Ref. 203). However, the receptors can negatively regulate target gene promoters that carry AP-1, NF-κB, or CREB binding sites, without binding to these DNA elements themselves. It was originally proposed that the receptors directly contact the basic leucine zipper region of c-Jun or therel homology domain of the p65 subunit of NF-κB and that this interaction inhibits binding to their corresponding cognate sites (291). However, more recent evidence suggested that competition for common transcriptional mediators could be involved in the antagonism observed (90 see also sect. v C). Additional mechanisms have been suggested, including an induction of the Ik-Bα factor that sequesters NF-κB in the cytoplasm (5), or an inhibition of the Jun-NH2-terminal kinase (JNK) activity by the receptors that would prevent phosphorylation of c-Jun (35).
A most interesting finding is that receptor-mediated transactivation and transrepression can be separated: mutations that impair transactivation, retain their ability to antagonize AP-1 or NF-κB activity. Interestingly, it has been possible to generate synthetic ligands of GR and retinoid receptors that dissociate transactivation from transrepression (217). These ligands are largely devoid of the ability to activate target genes containing HREs, but they retain in vivo anti-inflammatory or antiproliferative activity (53, 159, 275). These “dissociated” ligands have a large potential as pharmacological tools in the treatment of a variety of diseases including cancer and inflammatory diseases.
That transrepression plays a very important role in vivo has been demonstrated in a “knock-in” mouse in which the wild-type glucocorticoid receptor has been replaced by a mutant receptor containing a substitution in the DBD that results in a dimerization-defective receptor (GR dim ). This mutation allows transrepression, but the mutant receptor no longer binds with high affinity to the glucocorticoid response element. Whereas GR “knock-out” mice die at birth as a result of a failure in lung maturation, the GR dim survives despite impairment of several physiological functions of glucocorticoids (214).
The cross-talk between nuclear receptors and other signaling pathways is not restricted to the transcriptional antagonism described above (198). Phosphorylation of nuclear receptors provides an important link between signaling pathways. As already stated in section ii A, multiple kinases activated by extracellular signals that bind to surface receptors, including for instance MAPKs, cell cycle-dependent kinases (CDKs), casein kinase, and protein kinase A, affect receptor activity through phosphorylation events (243). Depending on the receptor and in the residue involved, in some cases phosphorylation can inhibit ligand-dependent activation by nuclear receptors due to a reduction in ligand binding or in DNA binding affinity. However, in other cases, the receptors can be activated in the absence of its cognate ligand by phosphorylation through signals originated in membrane receptors.
Contrary to the antiproliferative effects of some nuclear receptor ligands, ovarian hormones stimulate growth of breast cancer cells. It has been reported that estrogens activate the Src/Ras/MAPK signal transduction pathway and that this cross-talk could be crucial for their growth-promoting effect in these cells. MAPK activation occurs very rapidly and is receptor mediated, but appears to represent a nongenomic action of the steroid (172). A direct interaction of ER with c-Src could be involved in this phenomenon, and the progesterone receptor (PR) that does not interacts with c-Src can activate this pathway by association with ER (173).
A novel mechanism of cross-talk between nuclear receptors, specifically VDR, and transforming growth factor-β (TGF-β) has been recently reported (300). Smad3, one of the proteins downstream in the TGF-β signaling pathway, was found to act as a coactivator for VDR by forming a complex with a nuclear receptor coactivator. These interactions are potentially important in the control of cell proliferation and differentiation by vitamin D and the growth factor.
Plant Nuclear Hormone Receptors: A Role for Small Molecules in Protein-Protein Interactions
Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.