ENDOCRINE RECEPTORS: 2 SEPTEMBER 2010 Registrar: Dr. C Behrens Consultant: Dr. T Mofokeng Hormones: History: - About 100 years ago Starling coined the term “hormone” to describe secretin, a substance secreted by the small intestine into the bloodstream to stimulate pancreatic secretion. - Since the first connection between a viral oncogene, a mutated receptor tyrosine kinase and human cancer was made in 1984 (Ullrich, et al. 1984) it is well known that aberrant signalling by receptor tyrosine kinases is critically involved in human cancer and other hyper-proliferative diseases. The multiple activities of the cells, tissues and organs of the body are coordinated by the interplay of several types of communication systems, including 1. Neural: in which chemicals (neurotransmitters) are released at synaptic junctions and act locally to control cell function. 2. Endocrine, in which lands or specialized cells release into the circulating blood chemicals (hormones) that influence the function of cells at another location in the body. 3. Neuroendocrine, in which neurons secrete substances (Neuro hormones) that reach the circulating blood and influence the function of cells at another location in the body. 4. Paracrine, in which cells secrete substances that diffuse into the extracellular fluid and affect neighbouring cells 5. Autocrine, in which a cell secretes substances that affect the function of the same cell by binding to the cell surface receptors. 6. Tropic hormones are secreted by the hypothalamus and pituitary gland and activate specific pathways for hormone synthesis and release. Typical examples are luteinizing hormone, and follicle-stimulating hormone, thyroid-stimulating hormone and adrenocorticotrophic hormone (LH, FSH, TSH, ACTH) The major hormonal signalling programs are: (and all these are also used by paracrine factors) - G protein coupled receptors Tyrosine kinase receptors Serine/threonine kinase receptors Ion channels Cytokine receptors Nuclear receptors In contrast paracrine signalling are usually only used by paracrine factors and not by hormones. Receptors on the Cell Surface: Signalling pathways that begin with activation of receptors located on the surface of target cells. There are two essential functions that define hormone receptors: 1. The ability to bind the hormone 2. The ability to couple hormone binding to hormone action. The cell-surface receptors can be classified according to the molecular mechanisms by which they accomplish their signalling function: - G protein-coupled receptors (e.g., receptors for adrenergic agents, muscarine cholinergic agents, glycoprotein hormones, glucagon and parathyroid hormone - Ligand-gated ion channels (e.g., nicotinic acetylcholine receptors) - Receptor tyrosine kinases (e.g., receptors insulin and insulin-like growth factor I [IGF-I]) - Receptor serine/threonine kinases (e.g., atrial natriuretic factor receptor) - Cytokine receptors (e.g., receptors for growth hormone, prolactin and leptin) Scatchard plot, describes the affinity of the hormone for the receptor. It is usually a straight line, but several mechanisms contribute to a non-linearity: - More than one type of receptor that binds to hormone - More than one binding site - Cooperative interactions among binding sites Chemical structure and syntheses of hormones: There are three general classes of hormones: 1. Protein and polypeptides: including hormones secreted by the ant. and post. pituitary gland, the pancreas, the parathyroid gland, etc. 2. The steroids secreted by the adrenal cortex (cortisol and aldosterone), the ovaries, the testes and the placenta. - The chemical structure of steroid hormones is similar to cholesterol, in most instances they are synthesized from cholesterol itself. - Large stores of cholesterol esters in cytoplasm vacuoles can be rapidly mobilized for steroid synthesis after a stimulus. - Once they are synthesized, they simply diffuse across the cell membrane and enter the interstitial fluid and then the blood. 3. Derivatives of the amino acid tyrosine, secreted by the thyroid and the adrenal medullae. - The two groups of hormones derived from tyrosine are the thyroid hormones and adrenal medullary hormones. Hormone formation: Hormone formation may occur either in localized collection of specific cells, the endocrine glands or in cells that have additional roles. Many protein hormones, such as growth hormone, parathyroid hormone, prolactin, insulin and glucagon are produced in dedicated cells by standard protein synthetic mechanisms common to all cells. These secretory cells usually contain specialized secretory granules designed to store large amounts of hormone and to release the hormone in response to specific signals. Formation of small hormone molecules initiates with commonly found precursors, usually in specific glands such as the adrenals, gonads or thyroid. - Steroid hormone: The precursor is cholesterol, which is modified by various hydroxylation, methylations and demethylation to form glucocorticoid, androgens, estrogens and their biological derivatives. - In contrast the precursor of Vit D, 7-dehydrocholesterol, is produced in skin keratinocytes again from cholesterol, by a photochemical reaction. Leptin, which regulate appetite and energy expenditure, is formed in adipocytes, thus providing a specific signal reflecting the nutritional state to the central nervous system. Thyroid hormone synthesis occurs via a unique pathway. The thyroid cell synthesizes thyroglobulin, which is then iodinated at specific iodotyrosines. Certain of these couple to form the iodothyronine molecule within thyroglobulin, which is then stored in the lumen of the thyroid follicle. In order for this to occur, the thyroid cell must oxidize it via a specific peroxidase. Release of thyroxine (T4) from the thyroglobulin requires its phagocytosis and cathepsin-catalysed digestion by the same cells. - Endocrine diseases: Endocrine diseases fall into 4 broad categories: 1. Hormone overproduction 2. Hormone underproduction 3. Altered tissue responses to hormones Resistance to hormones can be causes by a variety of genetic disorders. Examples: Growth hormone receptor in Laron dwarfism Mutation in the Gsα gene in the hypoparathyroidism of pseudohypopararthyroidism type 1a. Insulin resistance in muscle and liver central to the aetiology of type 2 diabetes mellitus. 4. Tumours of the endocrine glands Hormone Receptors and Their Activation: Hormones must bind to specific receptors at the target cell. Receptors for some hormones are located on the target cell membrane, whereas other hormone receptors are located in the cytoplasm or the nucleus. When the hormone combines with its receptor, this usually initiates a cascade of reactions in the cell, with each stage becoming more powerfully activated so that even a small concentration of the hormone can have a large effect. Each receptor is usually highly specific for a single hormone – this determines the type of hormone that will act on a particular tissue. The location for the different types of hormone receptors is generally the following: In or on the surface of the cell membrane. Specific mostly for the protein peptide and catecholamine hormones In the cell cytoplasm Receptors for steroid hormones are found mostly in the cytoplasm In the cell nucleus (nuclear receptor superfamily) Receptors for the thyroid hormones are found in the nucleus These hormones bind to intracellular receptors that function in the nucleus of the target cell to regulate gene expression. Classical hormones that utilize intracellular receptors include thyroid and steroid hormones. In addition to above, lipophilic signalling molecules that utilize nuclear receptors include the following: (these are ligands for nuclear receptors) i. Derivatives of Vitamin A and D ii. Endogenous metabolites such as oxysterols and bile acids iii. Non-natural chemicals (xenobiotics) The number of Hormone Receptors is Regulated The receptor proteins themselves are often inactivated or destroyed during the course of their function, and at other times either they are reactivated or new ones are manufactured. Down Regulation: binding of a hormone with its target cell receptors often causes the number of active receptors to decrease, either because of inactivation of some of the receptor molecules or because of decreased production of the receptors. This decreases the response of the target tissue to the hormone Up Regulation: The stimulating hormone induces the formation of more receptor molecules than normal by the protein-manufacturing machinery of the target cell. Intracellular Signalling after Hormone Receptor Activation: Almost without exception, a hormone affects its target tissues by first forming a hormone-receptor complex. This alters the function of the receptor itself, and the activated receptor initiates the hormonal effects. Some Hormones change membrane permeability: Virtually al the neurotransmitter substances, such as acetylcholine and norepinephrine, combine with receptors in the postsynaptic membrane. This causes a change in the structure of the receptor, usually opening or closing a channel for one or more ions. It is the altered movement of these ions through the channels that causes the subsequent effects on the postsynaptic cells Membrane associated receptor proteins usually consist of extracellular sequences that recognize and bind ligand, transmembrane anchoring hydrophobic sequences, and intracellular signalling is mediated by soluble second messengers or by activation of intracellular signalling molecules. Receptor dependent activation of heterotrimeric G-proteins, comprising α, β and γ. Some hormones activate intracellular enzymes when they combine with their receptors: Hormones activate an enzyme immediately inside the cell membrane when binding with the membrane receptor. insulin binds with that portion of its membrane receptor that protrudes to the exterior of the cell that causes a structural change in the receptor molecule itself, causing the portion of the molecule that protrudes to the inside to become an activated kinase. The kinase then promotes phosphorylation of several different substances inside the cell. Both the number of receptors expressed per cell, as well as their responses, is also regulated, thus providing a further level of control for hormone action. Several mechanisms account for altered receptor function. o Receptor endocytosis causes internalization of cell-surface receptor o The hormone-receptor complex is subsequently dissociated, resulting in abrogation of the hormone signal. o Receptor trafficking may then result in recycling back to the cell-surface (e.g. insulin) or internalized receptor may undergo lysosomal degradation. Both these mechanisms may lead to impaired hormone signalling by down-regulation. o Receptor function may also be limited by action of specific phosphatases (e.g. SHP) or by intracellular negative regulation of the signalling cascade (e.g. SOCS proteins inhibiting JAK-STAT signalling) o Mutational changes in receptor structure can also determine hormone action. Constitutive receptor activation may be induced by activating mutations leading to endocrine organ hyper function, even in the absence of hormone. Some hormones activate Genes by binding with intracellular receptors: Several hormones, especially the steroid hormones and thyroid hormones, bind with the protein receptors inside the cell, not in the cell membrane. The activated hormone-receptor complex then binds with or activates specific portions of the DNA strands of the cell nucleus, which in turn initiates transcription of specific genes to form messenger RNA (mRNA). Newly formed proteins appear in the cell and become the controllers of new or increased cellular function. ------------------------------------------------------------------------------------------------------------------------------Generally, the second messengers activate serine/threonine kinases, which phosphorylate serine or threonine residues, or both on proteins, whereas the receptor kinases are tyrosine-specific kinases that phosphorylate tyrosine residues. Examples of receptor tyrosine kinases are growth factor receptors such as those for insulin, insulin-like growth factor (IGF), epidermal growth factor and platelet –derived growth factor. Receptors in the cytokine receptor family, which include leptin, growth hormone and prolactin, activate associated tyrosine kinases in a variation on the theme. Growth factor receptor tyrosine kinases activate transcription factors through cascades that involve both tyrosine phosphorylation and serine/threonine kinases such as mitogen-activated protein kinases, whereas the janus kinases (JAKs) activated by cytokine receptors directly tyrosine phosphorylate the signal transducer and activator of transcription (STAT) factors. Receptor Tyrosine Kinases: Receptor Tyrosine kinases have several structural mechanisms in common: an extracellular domain containing the ligand-binding site, a single transmembrane domain and an intracellular portion that includes the tyrosine kinase catalytic domain. There are approximately 100 receptor tyrosine kinases, and can be classified into 16 subfamilies, based on the differences in the structure of the extracellular domain. They mediate the biological action of a wide variety of ligands, including insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and vascular endothelial cell derived growth factor. The insuline receptor closely resembles the type 1 receptor for IGFs. This is the receptor that mediates the biologic actions of IGF-I and therefore also plays an important role in the physiology of growth hormone (GH) in vivo. Although the kinase domains of receptors for insulin and IGF-I closely resemble other receptor tyrosine kinases, at least two distinctive features set them apart. 1. The receptors are synthesized as proreceptors that undergo proteolytic cleavage into two subunits (β,α). The α subunit contains the ligand binding site and the β subunit includes the transmembrane and tyrosine kinase domains. 2. Both receptors exist as α2β2 heterotetramers that are stabilized by intersubunit disulfide bonds. In contrast to other receptor tyrosine kinases, which are thought to dimerize in response ligand binding, the insulin receptor exists as a dimer of αβ monomers even in the absence of ligand. Receptor Activation: Role of Receptor Dimerization: Dimerization plays a central role in the mechanism whereby most receptor tyrosine kinases are activated by their cognate ligands. Examples of mechanisms of receptor dimerization: Dimeric Ligand: PDGF and vascular endothelial cell-derived growth factor are such examples. Each subunit of ligand can bind one receptor molecule, simultaneous binding of two receptor molecules drives receptor dimerization. Direct support for this type of mechanism is provided by the crystal structure of vascular endothelial cell derived growth factor. Two Receptor binding sites on a Monomeric Ligand: One molecule of ligand can bind two molecules of receptor. In fact, there are two distinct receptor-binding sites on each GH molecule, and this enables the ligand to promote receptor dimerization. By binding to the receptors, the mutant ligand acquires the ability to inhibit the action of endogenous hormone. Such mutant GH molecules have been developed as therapeutic agents, for example, in conditions such as acromegaly. Pre-existing Receptor Dimer The insulin receptor represents a paradox. The insulin receptor exists as a dimer even in the absence of ligand. It actually is an α2β2 heterotetramer, which is a dimer of αβ monomers. It seems that the two halves of the insulin receptor are not orientated in an optimal way to permit receptor activation in the absence of ligand. Perhaps insulin binding triggers a conformational change that somehow mimics the effects of dimerization in other receptor tyrosine kinases. Receptor Activation: Conformational Changes in Kinase Domain When ligand binds to the extracellular domain, it stimulates the tyrosine kinase activity of the intracellular domain. This can be demonstrated in insulin. In the inactive form of insulin receptor kinase, TYR1162 is located in a position so that it blocks protein substrates from binding to the active site, and that does not accommodate magnesium adenosine triphosphate (ATP) thus tyrosine kinase is inactive because it cannot bind either of its substrates. Insulin binding triggers autophosporylation of three tyrosine residues (Tyr1158, Tyr1162,Tyr1163) in the activation loop. When the three tyrosine residues in the activation loop become phosphorylated, an important conformation change occurs and as a result the ability to bind both ATP and protein substrates. Receptor Tyrosine kinases Phosphorylate other intracellular proteins: Once activated, tyrosine kinases are capable of phosphorylating other protein substrates. There are factors that determine which proteins will be phosporylated: Amine acid sequence context of Tyr Residue’ Tyrosine kinase does not exhibit strict specificity with respect to amino acid sequence of the phosphorylation site, but most sites are located in the vicinity of acidic amino acid residues (i.e., Glu or Asp) Binding to Tyrosine Kinase Some protein substrates bind directly to the intracellular domain of the receptor. The binding interaction brings the substrate into close proximity to the kinase, thereby promoting phosphorylation of substrate. Subcellular Localization Because receptor tyrosine kinases are located in the plasma membrane, they are in close proximity to other plasma membrane proteins. This colocalization has the potential to promote phosphorylation. Functional significance of Tyrosine Phosphorylation: There are at least two distinct mechanisms whereby tyrosine phosphorylation regulates protein function. Tyrosine phosphorylation can induce a conformational change in a protein, thereby altering its function. As for example the three Tyr residues in the activation loop of the insulin receptor changes the conformation of the active site, thereby facilitating binding of substrates and activating the receptor tyrosine kinase. Downstream Signalling Pathways: Receptor tyrosine kinases mediate the action of a wide variety of ligands in a wide variety of cell types. For example, activated insulin receptor phosphorylates multiple substrates including IRS-1, IRS-2, IRS-3 and IRS-4. Each of the substrates contains multiple tyrosine phosphorylation sites. Thus IRS proteins serve as docking proteins that bind SH2 domain-containing proteins. Among these, PI3-kinase and Grb-2 are the most important. Binding of the SH2 domains triggers multiple downstream signalling pathways. - - Phosphatidylinositol 3-Kinase: The catalytic subunit of PI 3-kinase is bound to a regulatory subunit. All four IRS molecules contain multiple tyrosine phosphorylation sites. Therefore insulin-stimulated phosphorylation promotes binding of IRS proteins to the SH2 domains in the regulatory subunit of PI3-kinases, thereby increasing the enzymatic activity of the catalytic subunit. Activation of PI 3-kinase triggers activation of a cascade downstream kinases, beginning with phosphoinositide-dependent kinases 1 and 2. These phosphoinositide-dependent kinases phosphorylate and activate multiple downstream protein kinases including protein kinase B and atypical isoforms of protein kinase C. Grb-2 and the activation of RAS: Grb-2 is a short adaptor molecule that contains an SH2 domain capable of binding to pTyr residues in several signalling molecules for example IRS-1 and Shc. The SH2 domain of Grb-2 is flanked by two SH3 domains, which bind to proline containing sequences in mSos. mSos is capable of activating the Ras, a small G protein that plays an important role in intracellular signalling pathways. mSos activates Ras by catalysing the exchange of GTP to GDP in the guanine nucleotide-binding site of Ras. This in turn triggers the activation of a cascade of serine/threonine-specific protein kinases including Raf, mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mitogenactivated protein (MAP) kinase. These pathways downstream from Ras contribute to the ability of tyrosine kinases to promote cell growth and regulate the expression of various genes. Off Signals: Termination of Hormone action Just as there are complex biochemical mechanisms to mediate hormone action, there are also mechanisms to terminate the biological response. - Receptor –mediated endocytosis: - - Insulin binding to its receptor triggers endocytosis of the receptor. Although most of the internalized receptors are recycled to the plasma membrane, some receptors are transported to lysosomes, where they are degraded. As a result, insulin binding accelerates the rate of receptors degradation, thereby down-regulating the number of receptors on the cell surface. Protein Tyrosine Phosphatases: Tyrosine kinases catalyze the phosphorylation of tyrosine residues, but there are also protein tyrosine phosphatases (PTPases) to remove the phosphatases. Thus PTPases antagonize the action tyrosine kinases. Serine/Threonine Kinases: Most receptor tyrosine kinases, including the insulin receptor, are substrates for phosphorylation by Ser/Thr-specific protein kinases, and it’s phosphorylation inhibit the action of Tyrosine kinase. Receptor tyrosine kinase and disease patterns: In most cases gene amplification, overexpression or mutations are responsible for the acquired transforming potential of oncogenic RTK’s. Somatic and germline mutations, which are associated with distinct inherited and spontaneous human cancer syndromes, have been observed in at least ten different RTK families (Robertson et al. 2000) These alterations include deletions or mutation within the extracellular region or alterations of the catalytic domain, especially of the ATP-binding motif, which all result in a constitutive active RTK. In addition, mutations within the transmembrane domain have been shown to lead to ligandindependent kinase activation as reported for the RTK HER2/neu. Intervention Strategies: Increasing knowledge of the structure and activation mechanism of RTKs and signalling pathways controlled by tyrosine kinases provided the possibility for the development of target – specific drugs and new anti-cancer therapies (Plowman et al 1994). Development of selective components that target either the extracellular ligand-binding domain or the intracellular tyrosine kinase or substrate binding region. The most successful strategy to selectively kill tumour cells is the use of monoclonal antibodies (mAbs) that are directed against the extracellular domain of RTKs which are critically involved in cancer and are expressed at the surface of tumour cells (Fan & Mendelsohn 1998) Mechanistically, anti-RTKs mAbs might work by blocking the ligand-receptor interaction ant therefore inhibiting ligand-induced RTK signalling and increasing RTK downregulation and internalization. In addition by binding of mAbs to certain epitopes on the cancer cells they induce immune mediated responses such as opsonization and complement-mediated lysis and trigger antibody dependent cellular cytotoxicity by macrophages or natural killer cells. In contrast, bispecific antibodies can bridge selected surface molecules on a target cell with receptors on an effector cell triggering cytotoxic responses against the target cell that is thus killed. Another promising approach to inhibit aberrant RTK signalling are small molecule drugs that selectively interfere with the intrinsic tyrosine kinase activity and thereby blocking receptor autophosphorylation and activation of downstream signal transducers (Levitzki 1999). The tyrphostins, which belong to the quinazolines, are one important group of such inhibitors that compete with ATP for the ATP binding site at the receptor’s tyrosine kinase domain and some members have been shown to specifically inhibit the EGFR (Levitski & Gazit 1995) Recombinant immunotoxins provide another possibility of target-selective drug design. They are composed of a bacterial or plant toxin either fused or chemically conjugated to a specific ligand such as the variable domain of the heavy and light chains of mAbs or to a growth factor (Kreiman 1999). Immunotoxins either contain the bacterial toxins Pseudomonas exotixin A or diphtheria toxin or the plant toxins ricin A or clavin. The use of antisense oligonucleotides represent another strategy to inhibit the activation of RTKs. Antisense oligonucleotides are short pieces of synthetic DNA or RNA that are designed to interact with the mRNA to block the transcription and thus the expression of specifictarget proteins (Marcusson et al. 1999). Epidermal growth factor receptor family (EGFR) This family comprise of four members: 1. the archetypal EGFR/ErbB1, which was the first RTK to be molecularly cloned (Ulrich et al. 1984) 2. HER2/ErbB2 3. HER3/ErbB3 4. HER4/ErbB4 This four receptors share two extracellular cysteine-rich domains and an intracellular portion with a long C-terminal tail carrying most of the autophosphorylation sites. HER2: The RTK HER2 was originally identified at both the transforming gene in a chemically transformed rat neuroblastoma cell line and has been most frequently implicated in human neoplasias and cancer (King et al. 1985) Despite the fact that no ligand is known to bind with high affinity to HER2, it is well known that HER2 acts as a co-receptor for the other EGFR family members. These heteromolecular interactions are of pathophysiological relevance, because such receptor combinations show strong mitogenic signalling and tumorgenicity. - A large-scale population-based case control study in China, recently identified that there is an association of genetic HER2 polymorphism and an increased risk of breast cancer among young women (Xie et al. 2000) - And subsequently, it became evident that elevated levels of HER2, either with or without gene amplification, correlated with a more aggressive progression of disease and a reduced patient survival time (Paik et al. 1990) The role of HER2 in tumour growth: Recently it became evident that the transforming ability of HER2 is linked to cell survival and the ability of HER2 to directly affect components of the cell cycle machinery. Progression through the cell cycle is critically dependent on active complex formation of cyclin-dependent kinases (CDKs) with cyclin D1, which is a key regulator of G1/S phase transition of the cell cycle. It was shown that cyclin D1 expression is upregulated by activated HER2/neu and that blocking HER2 specifically inhibits the growth of tumour cells by arresting the cells in the G1 phase of the cell cycle. HER2 as a prognostic and predictive indicator: The relatively low expression level of HER2 in normal epithelial cells is significantly enhanced in several types of human cancers, including breast, ovarian, gastric, lung, bladder and kidney carcinomas. Clinical as well as laboratory data revealed that overexpression of HER2 increases the metastatic potential of human breast and lung cancer cells and correlates with the number of lymph node metasteses in node-positive breast cancer patients (Yu&Hung 2000). Elevated levels of a processed HER2 extracellular domain metastatic breast cancer patients seem to reduce the efficacy of certain chemotherapy combinations. HER2 overexpression correlates with a lack of response to anti-oestrogen hormonal therapy and confers resistance to tamoxifen, an antioestrogen that is administered as endocrine therapy in breast cancer patients. Herceptin – the first RTK-specific anti-oncogene drug Application of Herceptin in combination with paclitaxel or doxorubicin with cyclophosphamide signigicantly increased response duration, time to progression and survival in first line metastatic breast cancer patients. Therefore, it seems reasonable that Herceptin should be considered as one of the routine treatment options in metastatic breast cancer. EGFR The EGFR is frequently overexpressed in non-small cell lung, bladder, cervical, ovarian, kidney and pancreatic cancer and occurs with very high incidence in squamous cell carcinomas of the head and neck. The predominant mechanism leading to EGFR overexpression is EGFR gene amplification, with more than 15 copies per cell reported in certain tumours are associated with late stage of disease progression and often correlate with high metastatic rate and increased rate of tumour proliferation (Pavelic et al. 1993) A very potent mechanism of constitutive EGFR activation in a variety of human cancers is autocrine stimulation via growth factor loops. The most prominent ligand, which is involved in autocrine growth receptor activation, is TGFα and EGFR is frequently observed in glioblastomas and squamous cell carcinomas of the head and neck where it is correlated with poor prognosis (Grandis et al 1998) Since aberrant EGFR signalling is implicated in many cancers and seems to correlate with poor prognosis, it is an excellent target for anti-cancer therapy(Huang &Harari 1999). Currently the monoclonal antibody-based treatments using cetuximab (C225) represent the most successful antiEGFR therapies. C225 is directed against the extracellular domain of the EGFR and inhibits receptor activation and downstream signalling by blocking ligand binding to the EGFR and inducing receptor internalization. Insulin growth factor receptor (IGFR) Structurally quite different from the EGFR family are the insulin receptor (IR) and the insulin-like growth factor (IGF) receptor (IGF-IR). Both receptors consist of two extracellular α subunits, which are responsible for ligand binding, and two membrane spanning β subunits bearing the tyrosine kinase domain and the autophosphorylation site. The ligands for the two receptors include insulin, IGF-I and IGF-II. While insulin is mostly a metabolic hormone, IGF-I and IGF-II are crucial for normal development and carcinogenesis. In the last 10 years it has been demonstrated that IGF-IR and its ligands are involved in the pathogenesis of a variety of human tumours, particular in breast and prostate cancer (Perks & Holly 2000). IGF-I is synthesized by multiple cells, and therefore there are two major regulating mechanisms: 1. Growth hormone: IGF-I is synthesized in liver and secreted into blood. 2. Autocrine/Paracrine: IGF-I is synthesized in peripheral tissues, such as bone. It is still under the control of GH. The IGF-I receptor is present in multiple cell types. The receptor number is regulated by GH and thyroxine and range from 20 to 35,000 receptors per cell. Other growth factors such as plateletderived growth factor and fibroblast growth factor can also stimulate an increase in the number of receptors. Receptor activation happen when the ligand bind to the receptor’s alpha subunit. A conformational change in the receptor leads to auto-activation of the tyrosine kinase activity, followed by autophosphorylation of six tyrosines. Following phosphorylation, IRS-1 bind to receptor at tyrosine 950. Other kinases are then activated such as PI-3-kinase. After its tyrosine phosphorylation, IRS-1 binds to other signalling molecules through its Src homology 2 domain. Grb-2 forms a complex with the Ras activating protein son of sevenless (SOS) which leads to p21 Ras Activation. This activates the mitogen activated protein kinase (MAP) pathway which is very important for cell growth by IGF-I. IGF-I also play a role in prevention of apoptosis. The presence of IGF-I can rescue a cell from apoptosis by downstream signalling. Vascular endothelial growth factor receptor family (VEGFR) The vascular endothelial growth factor (VEGF) is one of the main inducers of endothelial cell proliferation and permeability of blood vessels. They are expressed on endothelial cells during embryonic development and are the key regulators for angiogensis, a process which leads to the formation of new blood vessels developing from pre-existing ones. Blood vessel formation is usually quiescent in the adult organism except for wound repair and the female menstrual cycle. However, expansion of solid tumours beyond a diameter of 1-2mm requires de novo formation of a vascular network that provides the growing tumour with oxygen and essential nutrients. Mechanistically, the tumour secretes VEGF that in turn activates VEGFR-2 and induces proliferation of stromal endothelial cells and sprouting of new blood vessels. Inhibition of angiogenesis has several advantages compared with other attempts in cancer therapy. This includes the fact that endothelial cells are easily accessible from the blood stream and are not likely to develop resistance to cancer therapy. Fibroblast growth factor receptor (FGFR): The fibroblast growth factors represent the largest family of growth factor ligands. More than 20 distinct members have been identified. The FGFs and their designated receptors appear to play critical roles no only in normal development but also in tumour formation and progression. Two classes of FGFRs were discovered in the past. The first class comprise the four high affinity FGFRs, whereas the second class is defined by low affinity FGF binding sites. The remarkable number of FGFs were identified as genes isolated from tumours due to their ability to induce proliferation or transformation of fibroblasts indicates their functional role in tumorigenesis. Since FGFR signalling shows a physiological profile of action that includes mitogenic and angiogenic activity and is frequently altered in human tumours, this system might be a useful target for anti-cancer therapy. Suramin, a compound that complexes heparin-binding growth factors, such as FGF-2, has been shown to inhibit cell migration and FGF-2 mediated induction of urokinase-plasminogen activator. Hepatocyte growth factor receptor (HGFR) The HGFR is encoded by the proto-oncogene that was identified as a regulator of a variety of morphogenic processes like cell migration, cell scattering and invasion of extracellular matrices. HGF over-expression was demonstrated in a variety of human tumours, such as thyroid and colorectal carcinomas and seems to have prognostic significance for non-small lung and breast cancer. Most of these mutations lie adjacent to the kinase domain, leading to enhanced enzymatic activity, transformation of fibroblasts and invasive growth. RET receptor tyrosine kinase: Multiple endocrine neoplasia type 2 (MEN2) is a dominant autosomal inherited cancer syndrome which exists in three different subtypes and is characterized by the development of medullary thyroid carcinoma. The gene responsible for MEN2 was identified as the Rearranged during transformation (RET) RTK. RET is expressed during embryogenesis in the peripheral nervous system and in the development of the neural crest and the kidneys. MEN2 cancer syndromes are caused by dominant activating germline mutations in the RET protooncogene. - - Men 2A : Autosomal dominant disorder o Men and women equally effected o Medullary Thyroid carcinoma o Pheochromocytoma o Primary Parathyroid hyperplasia Men 2B: Autosomal dominant o Medullary Thyroid carcinoma o Pheochromocytoma o NOT HYPERPARATHYROIDISM o The tumor develops at an earlier age and is more aggressive than in MEN2A Platelet derived growth factor receptor: (PDGFR) The two RTKs PDGFRs and Kit are members of the PDGFR family. These proteins are characterised by an extracellular domain with five Ig-like domains and an intracellular tyrosine kinase domain. Coexpression has been identified in glioblastoma and other human astrocytotic brain tumours, whereas normal brain tissue does not express proteins. This suggest an autocrine loop that stimulates the uncontrolled growth of human brain tumours. Nuclear Receptors: As mentioned above these hormones bind to intracellular receptors that function in the nucleus of the target cell to regulate gene expression. Classical hormones that utilize intracellular receptors include thyroid and steroid hormones Because nuclear receptor ligands are lipophilic, most are readily absorbed from the gastrointestinal tract. This makes nuclear receptors excellent targets for pharmaceutical interventions. In addition to natural ligands, many drugs in clinical use target nuclear receptors. These range from those used to treat specific hormone deficiencies to those used to treat common multigenic diseases such as inflammation, cancer and type 2 Diabetes. The carboxyl (C-) terminus of the nuclear receptor is responsible for hormone binding. The major structural change induced by ligand binding is an internal folding of the most C-terminal helix (H12), which forms a cap on the ligand-binding pocket. Subclasses of Nuclear Receptor Ligands: Class Classical Hormones Vitamins Metabolic intermediates and products Xenobiotics Hormone Thyroid Hormone Estrogens Testosterone Progesterone Aldosterone Cortisol 1,25-(OH)2-vit.D All-trans-retinoic acid 9-cis-retinoic acid Fatty acids Receptor Thyroid hormone receptor Subtype α, β Estrogens Receptor (ER) subtype α, β Androgen receptor (AR) Progesterone receptor (PR) Mineralocorticoid receptor (MR) Glucocorticoid receptor (GR) Vit.D receptor (VDR) retinoid acid receptor , subtypes α, β and γ Retinoid X receptor (RXR) , subtypes α, β and γ Peroxisome proliferators activated receptor (PPAR) , subtypes α, β and γ Oxysterols Bile acids Liver X receptor (LXR) , subtypes α, β Bile acid receptor (BAR) Pregnane X receptor (PXR) Constitutive androstane receptor (CAR) Vitamins: Vitamin A and D are precursors of important signalling molecules that function as ligands for nuclear receptors. Vitamin D are synthesized and stored in the skin and activated by ultraviolet light; vit D can also be derived from dietary sources. Vit D is then converted in the liver to 25(OH) Vitamin D and in the kidney to 1-25(OH)2-Vitamin D3., the most potent natural ligand of vitamin D receptor (VDR). Vitamin A is stored in the liver and is activated by metabolism to all-trans-retinoic acid, which is a high-affinity ligand for retinoic acid receptors (RARs). Retinoic acid is likely to function as a signalling molecule in both paracrine and endocrine pathways. Retinoic acid is also converted to its 9-cis-isomer, which is a ligand for another nuclear receptor called the retinoid X receptor (RXR). Xenobiotics: The role of the activated nuclear receptor is to induce cytochrome P450 enzymes that facilitate detoxification of potentially dangerous compounds in the liver. Ligands can be regulated by the following mechanisms: - Precursor availability: e.g. iodine deficiency in hypothyroidism - Synthesis - Secretion - Activation (prohormone active hormone) - Deactivation - elimination Hormones that act mainly on the genetic machinery of the cell: Steroid Hormones increase protein synthesis This hormones act by causing synthesis of proteins in the target cells (specifically the steroid hormones secreted by the adrenal cortex, ovaries and testes). These proteins then function as enzymes, transport proteins or structural proteins that in turn provide other functions of the cells. The sequence of events: 1. Steroid hormone enter cytoplasm 2. Bind to specific receptor protein 3. Combined receptor protein-hormone then diffuses into or is transported into nucleus 4. Here the combination bind to a specific points on the DNA strands in the chromosomes, 5. Which activates the transcription process of specific genes to form mRNA 6. The mRNA diffuses into the cytoplasm where it promotes the translation process at the ribosomes to form new proteins Example: Aldosterone enters the cytoplasm of renal tubular cells, which contain specific aldosterone receptor protein..after about 45 min, proteins begin to appear in the renal tubular cells and promote sodium reabsorption from the tubules and potassium secretion into the tubules. Thyroid Hormones increase Gene transcription in the cell nucleus: The thyroid hormones thyroxine and triiodothyronine cause increased transcription by specific genes in the nucleus. These hormones first bind directly with receptor proteins in the nucleus itself; the receptors are probably protein molecules located within the chromosomal complex, and they probably control the function of the genetic promoters or operators. Two important features of Thyroid Hormone function in the nucleus: - They activate the genetic mechanism for the formation of many types of intracellular proteins. Many of these are enzymes that promote enhanced intracellular metabolic activity in virtually all cells of the body - Once bound to the intranuclear receptors, the thyroid hormones can continue to express their control functions for days or even weeks. Mechanisms of Disease: The simplest forms of endocrine disease are are caused by either a deficiency or an excess of a hormone. However, hormone resistance syndromes resulting from defects in the signalling pathways can masquerade the true disease. Genetic Defects in Receptor function 1. Mutations can lead to a decrease in the number of receptors by - Impairing receptor biosynthesis - Inhibiting the transport of receptors to their normal location in the plasma membrane - Accelerating the rate of receptor degradation 2. Mutations can impair the intrinsic activities of the receptor. Auto-antibodies directed against Cell-surface receptors - - - Inhibitory anti-receptor autoantibodies were first identified in myasthenia gravis (where nicotinic acetylcholine receptor impair neuromuscular transmission, apparently by accelerating receptor degradation) Insulin resistance is caused by at least two mechanisms: 1. The anti-receptor antibodies inhibit insulin binding to the receptor 2. The antibodies accelerate receptor degradation Graves-disease provided the first example of stimulatory anti-receptor antibodies, where there are auto-antibodies directed against the thyroid-stimulating hormone (TSH) receptor. These anti-receptor antibodies activate the TSH receptor, thereby stimulating growth of the thyroid gland as well as hypersecretion of the hormone. Receptor Serine Kinases: Receptor serine kinases have several features in common with receptor tyrosine kinases: - N-terminal extracellular domains, which bind ligand - A single transmembrane domain - C-terminal intracellular domains, which possess protein kinase activity However the two classes of receptors differ with respect to enzymatic specificity: Whereas receptor tyrosine kinases phosphorylate tyrosine residues, receptor serine kinases phosphorylate serine and threonine residues in their protein substrates. There are two types of receptor serine kinases: Type I and II. Receptor Activation: Role of Receptor Dimerization Receptor serine kinases mediate the biological actions of a single, large family of ligands: the transforming growth factor (TGF)-β family of ligands, which are characterized by the presence of six conserved cysteine residues. The human genome contains 42 genes encoding cytokines in the TGF-β family, which are divided into two classes: 1. Activin/TGF-β family 2. Mullerian inhibitory substance (MIS)/Bone morphogenic protein(BMP) family Receptor Serine Kinases Phosphorylate other Intracellular proteins Once activated, receptor serine kinases are capable of phosphorylating other protein substrates. Receptor-regulated Smad proteins (R-Smad) function as the immediate downstream effectors of receptor serine kinases. When RI (Type I serine receptor) becomes phosphorylated in its GS domain in response to TGF-β, this increases the binding affinity for R-Smad proteins such as Smad2. This in turn leads to the phosphorylation of the two C-terminus of Smad2. Receptor-mediated phosphorylation of the R-Smad, takes place when Smad2 is bound to the the Smad anchor for receptor activation (SARA)., which is located in early endosomes. Smad Proteins regulate gene expression: Some R-Smads contain lysine-rich nuclear localization signals which bind to importin, thereby mediating translocation into the nucleus. It is possible that direct binding to components of the nuclear pore complex also contribute to the mechanism whereby Smads are translocated into nucleus. Most R-Smads (with the exception of Smad2) bind to DNA in a sequence-specific fashion. CYTOKINE RECEPTORS: Receptors that signal through associated tyrosine kinases: Instead of the tyrosine kinase being intrinsic to the receptor, enzymatic activity resides in a protein that associates with the cytokine receptor. As with receptor tyrosine kinases, ligand binding to the cytokine receptor activated the associated kinase. There are more than 25 ligands that bind to members of the cytokine receptor family. Three of the ligands are hormones: 1. Growth Hormone (GH) 2. Prolactin 3. Leptin – suppresses appetite and stimulates energy expenditure Cytokine receptors are composed of multiple subunits: Most cytokine receptors are composed of two or more subunits with as many as six subunits constituting a single receptor. Some of these receptors are thought to bind ligand dimers. One or more of these receptor subunits is shared by receptors for other cytokines. This phenomenon of mixing and matching receptor subunits is an efficient way for the cell to fine-tune its cellular responses and increase the number of ligands a group of receptors subunits can bind. The two-headed god Janus, originally venerated in the valley of the Lower Tiber, belongs to the oldest members of the Roman pantheon. He was considered to be the "god of the gods" and guarded all kinds of doors and gates. This god was also invoked at the beginning of sacrifices and is therefore thought to have had something to do with beginnings in general. From the reign of the emperor Domitian (81-96) on, Janus was also represented with four heads, called Janus Quadrifrons Cytokine receptors activate members of the Janus family of tyrosine kinases: Members of the cytokine family of receptors do not themselves exhibit enzymatic activity, they bind members of Janus family of tyrosine kinases(JAK) via a proline-rich region. There are four known JAKs (JAK1, JAK2, JAK3, TYK2). As do the cytokine receptors themselves, the JAKs mix and match to activate multiple JAK family members. Activation of JAKs requires receptor oligomerization, presumably to bring two or more JAKs into sufficiently close proximity to transphosphorylate each other on the activating tyrosine in the kinase domain. Both receptor dimerization and ligand –induced changes in receptor conformation appear to be required for receptor activation. Transphosphorylation is believed to cause a conformational change that exposes the ATP- or substrate-binding site or both. Signalling pathways initiated by Cytokine receptor –JAK complexes Phosphorylated tyrosines within cytokine receptor subunits and their associated JAKs form binding sites for carious signalling proteins containing phosphotyrosine binding domain, such SH2 and PTB domain. Each cytokine receptor-JAK complex would be expected to have some tyrosine-containing motifs and some ligand-specific tyrosine containing motifs. Thus ligand binding to cytokine receptors would be expected to initiate some signalling pathways that are shared by many cytokines and some that are more specialized to a particular cytokine receptor. There is one family of signalling proteins that appears to be particularly important for the function of cytokines – signal transducers and activators of transcription (STATs). STAT proteins are latent cytoplasmic transcription factors. STATs bind through their SH2 domains, to one or more phosphorylated tyrosines in activated receptor-JAK complexes. Precise regulation of the cytokine receptors is required for normal function: Ligand binding to cytokine receptors normally activates JAKs rapidly and transiently. Activated JAKs and STATs are associated with cellular transformation. For example, a single acquired activating point mutation in JAK2 is present in the majority of patients with a myeloproliferative disorder. As with the receptor tyrosine kinases, several steps have been hypothesized to serve as points of signal termination for cytokine signalling. These include receptor degradation and dephosphorylation of tyrosines within JAK or receptor. The suppressors of cytokine-signalling (SOCS) are thought to be particularly important players in the termination or suppression of cytokine signalling pathways. SOCS proteins are thought to bind to phosphotyrosines in the kinase domain of JAK and inhibit kinase activity. G Protein – Coupled Receptors: G protein-coupled receptors (GPCR) are an evolutionarily conserved gene superfamily with members in all eukaryotes from yeast to mammals. All members of the GPCR superfamily share a common structural feature, seven membrane-spanning helices, but various subfamilies diverge in primary amino acid sequence and in the domains that serve in ligand binding, G protein coupling and interaction with other effector proteins. All GPCR act as guanine nucleotide exchange factors. In their activated conformation, they catalyze exchange of GDP tightly bound to the α subunit of heterotrimeric G proteins for GTP. This inturn leads to activation of the αsubunit and its dissociation from the Gprotein βγ dimer. Both G protein subunits are capable of regulating effector activity. Identified G protein- regulated effectors include enzymes of second messenger metabolism such as adenyl cyclase and phospholipase C-β and a variety of ion channels. Agonist binding to GPCRs thus alters intracellular second messenger and ion concentrations with resultant rapid effect on hormone secretion, muscle contraction and a variety of other physiologic functions. G Protein – coupled receptor structure and function: Structure: - Seven membrane spanning α helices connected by three intracellular loops and three extracellular loops with an extracellular amino terminus and an intracellular carboxyl terminus. - Other common features: o A disulfide bridge connecting the first and second extracellular loops o One or more N-linked glycosylation sites, usually in the amino terminus but occasionally in extracellular loops o Plamitoyation of one or more cysteines in the carboxyl terminus o Potential phosphorylation sites in the carboxyl terminus and occasionally the third intracellular loop. Ligand Binding: There is considerable diversity in both the sequence and structure of presumptive GPCR ligand-binding domains. G Protein coupling: Because the number of potential G proteins to which GPCR s couple is much more limited than the number of ligands that bind GPCRs, more conservation of the domains involved in Gprotein coupling would be expected. Mechanism of Activation: Movement of the transmembrane helices in turn leads to changes in conformation of cytoplasmic loops that promote G protein activation. A general hypothesis of GPCR activation postulates that GPCRs are in equilibrium between an activated state and an inactive state. These states presumably differ in the disposition of the transmembrane helices and in turn the cytoplasmic domains that determine G protein coupling. Agonists, according to this ternary complex model , are viewed as stabilizing the activated state. Antagonists may be neutral , that is they simply compete with agonists for receptor binding but their binding does not influence this equilibrium. Dimerization: Members of the tyrosine kinase receptor family have long been known to require dimerization as part of their activation mechanism. G protein coupled receptors in disease pathogenesis and treatment: Because of their diverse and critical roles in normal physiology their accessibility on the cell surface and the ability to synthesize selective agonists and antagonists, GPCRs have long been a major targer for drug development. Beyond drug development, defects in GPCRs are an important cause of a wide variety of human diseases. GPCR mutations can cause loss of function by impairing any of several steps in the normal GPCR-GTPase cycle. These include failure to synthesize GPCR protein altogether, failure of synthesized GPCR to reach the plasma membrane, failure of GPCR to bind or be activated by agonist and failure of GPCR to couple to or activate G protein. Most of these diseases are manifested as resistance to the action of the normal agonist and thus mimic deficiency of the agonist. Clinically, diseases caused by activating GPCR mutations therefore mimic states of agonist excess, but direct measurement shows that agonist concentrations are actually low, reflecting normal negative feedback mechanisms. Many disease-causing loss of function mutations in GPCRs lead to defective protein folding and/or protein routing. Receptor V2: Vasopressin ACTH GHRH GnRH GPR54 FSH LH TSH CA 2+ sensing Diseases caused by G protein coupled receptor loss of function mutations Disease Nephrogenic diabetes insipidus Familial ACTH Resistance Familial GH deficiency Hypogonadotropic Hypogonadism Hypogonadotropic Hypogonadism Hypergonodadotropic ovarian dysgenesis Male pseudohermaphroditism Familial Hypothyroidism Familial Hypocalcuric hypercalcemia, Neonatal severe primary hyperparathyroidism Melanocortin Obesity 4 PTH/PTHrP Blomstrand Chondrodysplasia Receptor LH TSH Diseases caused by G protein coupled receptor gain-offunction mutations Disease Familial male precocious puberty Sporadic hyper functional thyroid modules TSH Ca2+ sensing PTH/PTHrP Familial nonautoimmune hyperthyroidism Familial hypocalcemic hypercalcuria Jansen’s metaphyseal chondrodysplasia Inheritance X-linked Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal dominant Autosomal recessive Autosomal recessive Autosomal recessive Inheritance Autosomal dominant Noninherited (somatic) Autosomal dominant Autosomal dominant Autosomal dominant V2 vasopressin Nephrogenic inappropriate antidiuresis Autosomal dominant Second Messenger Mechanisms for mediating intracellular Hormonal functions: The Adenylyl Cyclase – cAMP Second messenger System: The hormone bind with a special transmembrane receptor that then becomes the activated enzyme adenyl cyclase, this enzyme then protrudes to the inside of the cell. This cyclase in turn catalyzes the formation of cAMP, and cAMP has a multitude of effects inside the cell to control cell activity. Thus the only direct effect that the hormone has on the cell is to activate a singly type of membrane receptor. The second messenger does the rest. Binding of the hormones with the receptor allows coupling of the receptor to a G-protein. If the Gprotein stimulates the adenyl cyclase it is called a Gs – protein (stimulatory G-protein). Stimulation of the adenyl cyclase, a membrane-bound enzyme, by the Gs-protein then catalyses the conversion of a small amount of cytoplasmic adenosine triphosphate (ATP) into cAMP inside the cell. This then activates cAMP-dependent protein kinase, which phosphorylates specific proteins in the cell, triggering biochemical reactions that lead to cell response to hormone. Once cAMP is formed, it usually activates a cascade of enzymes. If binding of the hormone to its receptors is coupled to an inhibitory G-protein (denoted Gi-protein), adenyl cyclase will be inhibited, reducing the formation of cAMP and ultimately leading to an inhibitory action in the cell. Depending on the coupling of the hormone receptor to an inhibitory or a stimulatory G-protein, a hormone can either increase or decrease the concentration of cAMP and phosphorylation of key proteins inside the cell. Different functions are elicited in different target cells- such functions as initiating synthesis of specific intracellular chemicals, causing muscle contraction or relaxation, initiating secretions by the cells and altering cell permeability. Hormones that use the cAMP secondary system: - Adrenocorticotropic Hormone (ACTH) - Angiotensin II (epithelial) - Calcitonin - Catecholamines (B receptors) - Corticotropin –releasing hormone (CRH) - Follicle –stimulating Hormone (FSH) - Glucagon - Human Chorionic Gonadotropin (HCG) - Luteinizing hormone (LH) - Secretin - Thyroid Stimulating Hormone (TSH) - Vasopressin The cell membrane phospholipid second messenger system: Some hormones activate transmembrane receptors that inactivate the enzyme phospholipase C attached to the inside projections of the receptors. This enzyme catalyses the breakdown of some phospholipids in the cell membrane, especially phosphatidyllinositol biphosphate (PIP2), into two different second messenger products, inositol triphosphate (IP3) and diacylglycerol (DAG). The IP3 mobilizes calcium ions from mitochondria and the endoplasmic reticulum, and the calcium ions then have their own second messenger effects, such as smooth muscle contraction and changes in cell secretions. DAG activates the enzyme protein kinase C (PKC), which then phosphorylates a large number of proteins, leading to the cell’s response. In addition to these effects the lipid portion of DAG is arachidonic acid, which is the precursor for the prostaglandins and other hormones. Hormones that use the phospholipase C second Messenger System: - Angiotensin II (vascular smooth muscle) - Catecholamines - Gonadotropin-releasing hormone - Growth Hormone releasing hormone - Oxytocin - Thyroid releasing hormone (TRH) - Vasopressin The Calcium-Calmodulin Second Messenger system: Another second messenger system operates in response to the entry of calcium into the cells. The calcium entry may be initiated by - changes in membrane potential that open membrane calcium channels or - hormone interacting with membrane receptors that open calcium channels On entering a cell, calcium ions bind with the protein calmodulin. This protein has four calcium sites, and when three of the four of these sites have bound with calcium, the calmodulin changes its shape and initiates multiple effects inside the cell, including activation or inhibition of protein kinases. Activation of calmodulin-dependent protein kinases, causes, via phosphorylation , activation or inhibition of proteins involved in the cell’s response to the hormone. Referrences: 1. Guyton 2. Williams Textbook of Endocrinology 3. Gether. Uncovering molecular mechanisms Involved in Activation of G protein-coupled Receptors. Endocrine Reviews 21(1): 90-113 4. Zwick, Bange, Ullrich. Receptor tyrosine kinase signalling as a target for cancer intervention strategies. 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