1
Q
What is paracrine signalling?
A
A local form of signalling where the signalling molecule is released from the signalling cell into the local area like the matrix between a tissue. The target cell for these local mediators are cells with the specific receptors that are close by.
2
Q
What is synaptic signalling?
A
A specialized form of electrochemical communication where neurons transmit signals to other neurons, muscles, or glands across a tiny gap called a synapse. An example of these signallers are neurotransmitters.
3
Q
What is endocrine signalling?
A
Endocrine signalling utilises hormones synthesised in various tissues + organs within our bodies. Hormones are released in the bloodstream and travel through the blood to their target cells and they target cells that could be a range of cells throughout the body.Involves glands like the hypothalamus, pituitary gland, pineal gland, parathyroid gland, thyroid gland, thymus, adrenal gland, pancreas, kidney, testis and ovary.
4
Q
What is intracellular signalling?
A
As a whole, intracellular signalling occurs once the external/extracellular signalling molecule reaches the cell and triggers a signalling pathway or the activation of a signalling pathway within the cell. Once the signalling molecule reaches the receptor, it triggers the activation of a pathway within the cell that could actually include quite a number of different signalling molecules. Several molecules may be involved before effector proteins are actually activated.
5
Q
What are effector proteins?
A
Effector proteins carry out the role that the signalling protein is intending to perform. These effector proteins contribute to the target/desired cell responses, e.g. metabolic enzyme (altered metabolism), cytoskeletal protein (altered cell shape/movement) & transcription regulator (altered gene expression). Examples of effector proteins include metabolic enzymes, cytoskeletal protein and transcription regulators
6
Q
What are the 4 types of signalling?
A
Contact-dependent (signal requires a membrane-bound signal molecule), paracrine, synaptic, and endocrine.
7
Q
What are key features of cell signalling?
A
The specific targets of cell signalling allows for coordinated, and tightly controlled responses. It describes situations in which signal molecules fit the binding site on its complementary receptor whilst other signals do not fit. The activation of a receptor by a single signalling molecule quickly generates or activates an enzyme cascade, meaning that the no. of affected molecules increases geometrically. So, 1 signalling molecule can activate multiple enzymes
8
Q
What is amplification in regards to enzymes?
A
Each time the next target enzyme is activated, the number of activated molecules increase. In an example, 1 molecule of glucagon doesn’t result in the generation of only 1 cAMP as that would fail in generating this cascade. This results in 10,000 molecules of glucose being generated
9
Q
What is feedback in regards to enzymes?
A
Occurs when the downstream effect of a stimulus signals back to the signalling cell and either turns off or enhances the signal further. Provides feedback to the original cell responding to the stimulus. Once the stimulus enters a system, a particular signalling molecule may be released. Positive = increases change, negative = decreases/counteracts change
10
Q
What is signal transduction?
A
The response time to this cell signalling can either be rapid (almost instant) or taking several hours. A fast response will occur if the signalling molecule works to activate/alter the function of a protein that might already be present in the cell. Slower reactions involve synthesising new proteins (transcription, translation or post-translational modifications) which is why the response takes longer. But, some reactions have both slow and fast elements
11
Q
what are the fundamental where and whats of adrenaline?
A
the site of origin is the adrenal gland and it acts to increase blood pressure/ heart rate/metabolism. also, it is a derivative of the amino acid tyrosine
12
Q
what are the fundamental where and whats of insulin?
A
found in the B cells of the pancreas, is a protein by nature. it works to stimulate glucose uptake, protein synthesis and lipid synthesis in various cell types
13
Q
what are the 3 main classes of hormones?
A
Hormones can be divided into 3 main classes: polypeptide/protein hormones (insulin), amine (epinephrine), and steroid hormones (estrogen/testosterone)
14
Q
what does the hypothalamus do?
A
Hypothalamus synthesises and release many releasing factors that act on the anterior pituitary.
15
Q
what are polypeptides or protein hormones?
A
Once synthesised they’re stored in secretory vesicles for up to 1 day. Many are stored as prohormone. Vesicles fuse with the membrane which are released by exocytosis of secretory granules into the extracellular matrix. Secretion is regulated by other hormones, metabolites and CNS. They circulate freely in the blood. Have a lifetime of minutes
16
Q
Is insulin a protein?
A
Yes, it’s a small protein that consists of 2 polypeptide chains (a and B). when it’s translated it is translated as a long single chain called proinsulin. When it’s synthesised it undergoes some post-translational modifications in the ER and is ultimately packaged into vesicles in the form of proinsulin. Once glucose levels rise and the beta cells within the pancreas need to secrete insulin, proinsulin is cleaved into mature insulin and released into the bloodstream.
17
Q
What are peptide-amine hormones derived from tyrosine?
A
Amine = nitrogen base. Epinephrine & norepinephrine are synthesised in the Adrenal medulla. These are stored for several days, are secreted in response to signals from the CNS and are free in the blood with a lifetime of seconds.
Thyroxine and triiodothyronine are synthesised in the thyroid gland by the process of iodination of tyrosine residues followed by further modifications within the protein thyroglobulin. They are released by proteolytic cleavage.
18
Q
what are lipophillic hormones?
A
They can diffuse straight through the cell membrane and pass directly through without needing to bind to a cell surface receptor. The receptors for these hormones are intracellular and examples are steroids (cortisol,testosterone, estradiol) and vitamin D
19
Q
what is intracellular signal transduction?
A
Activation of a cell surface receptor initiates a relay, amplified, distributed, and modulated signal into the cell
20
Q
how is phosphorylation used as a molecular switch?
A
Particular signalling molecules can be turned on through phosphorylation. Phosphatases remove phosphate groups. Some enzymes are turned off when phosphorylated and turned on when dephosphorylated
21
Q
how are GTPases used as molecular switches?
A
Some molecules need to bind to GTP to become active. A GTPase is a molecule that becomes active upon the exchange of GDP for GTP. It functions to activate subsequent signalling molecules. In its inactive form, GTPase remains bound to GDP and a Guanine Nucleotide Exchange Factor (GEF) is required to promote the removal of GDP and exchange for GTP which in-turn facilitates the activation of GTPase. GAP (GTPase-activating protein) increases the rate at which a GTPase hydrolyses GTP to GDP. This converts the GTPase from its active (GTP-bound) form to its inactive (GDP-bound) form. After hydrolysis, GDP remains tightly bound to the GTPase, keeping it inactive until it is exchanged for GTP again.
22
Q
what is the Ras family?
A
There is a Ras family of GTPases with examples like H-Ras, K-Ras, and N-Ras that promote cancer by causing an an overactive stimulation of cell grown & survival pathways but it overall functions to relay signals from RTKs
23
Q
what are receptors? how many types are there, and what are they?
A
There are either cell-surface receptors or intracellular receptors which differ greatly in size (the first sends signals from its membrane whereas the second sends them from its nucleus). Cell surface receptors typically have 3 regions, an extracellular region, transmembrane, and intracellular region
Intracellular receptors are found either within the nucleus or within the cytoplasm. The ligands/molecules that will bind to these are generally lipophilic and can pass directly through the membrane into the cell where the receptor is.
24
Q
what are cell surface receptors?
A
in this sub-class, there are ion-channel-coupled receptors, G-protein-coupled receptors, and enzyme-coupled receptors. In ion channel-coupled receptors, the signalling molecule induces the opening or closing of an ion channel that allows ions to pass through. This is common for neurotransmitters + signalling related to neuronal cells.
In enzyme-coupled receptors, receptors are enzymes themselves or very closely associated with an enzyme. When the signalling molecule binds it leads to the activation of those enzymes, which in-turn leads to the propagation of that signal
25
What are the 2 main types of enzyme-coupled receptors?
Receptor tyrosine kinase: these phosphorylate specific Tyr residues on the intracellular segment
Tyrosine-kinase-associated receptors: couple to separate proteins that have Tyr kinase activity
Other examples include receptor serine/threonine kinases that phosphorylate Ser/ Thr residues, Histidine-kinase-associated receptors, receptor tyrosine phosphatases that remove phosphate groups from tyr and receptor guanylyl cyclases which catalyse the production of cGMP
26
what are common features of receptor tyrosine kinase?
Common features of these subclasses are an extracellular & intracellular domain, tyrosine-kinase domain but some have a split tyrosine-kinase domain meaning they have 2 regions that have this catalytic capacity
27
what are subclasses of receptor tyrosine kinases?
Include subclasses like insulin receptors, insulin-like growth factor receptors and epidermal growth factor receptors (EFG) which is often mutated in cancers. Generally these subclasses promote responses such as cell growth, differentiation, and proliferation
28
what is the process of the mechanism of receptor tyrosine kinases?
In their inactive form, receptor tyrosine kinases may exist as monomers (single proteins) or as dimers (e.g 2 monomers bound together but they can also remain in the membrane in their dimer form but without their signalling molecule). Once the signalling molecule binds to the receptor, transautophosphorylation occurs where one monomer is phosphorylating the other. Here, the binding of the signalling molecule either induces this by bringing the 2 monomers together or it induces a conformation change facilitating the process. This signalling event initiates activation via phosphorylation. Once the initial phosphorylation event occurs, subsequent phosphorylation occurs whereby additional residues within the receptor become phosphorylated. These particular phosphorylation sites then become binding sites for downstream signalling molecules facilitating the binding between signalling molecules & certain residues. After binding, these molecules become activated and can propagate the signal within the cell
29
what are receptor tyrosine kinase domains?
Receptor tyrosine kinases (RTKs) signal through proteins containing conserved binding domains.
30
what is the SH2 domain in receptor tyrosine kinases?
SH2 domain (Src homology 2 domain) is around 100 amino acids long. These recognise and bind phosphorylated tyrosine (Tyr–PO₄) residues on activated receptors or proteins.
31
what is the SH3 domain in receptor tyrosine kinases?
SH3 domain (Src homology 3 domain) is around 50–60 amino acids long and it binds to proline-rich regions on target proteins. SH2 and SH3 domains were first identified in the Src protein kinase.
32
what is the PTB domain in receptor trysoine kinases?
PTB domain (phosphotyrosine-binding domain) recognise specific phosphorylated tyrosine residues, often in a sequence-specific context. Hence, SH2 and PTB bind phosphorylated tyrosine residues
33
what is the PH domain in receptor tryrosine kinases?
PH domain (pleckstrin homology domain) binds phosphorylated inositide lipids in the cell membrane.
34
what is the function of adaptor proteins in receptor tyrosine kinases?
These work to link/bridge 1 protein to another, or couples proteins with receptors that may not have their own SH2 domains. Example: Grb2
35
what is the function of scaffold proteins in receptor tyrosine kinases?
This bring together multiple interacting signalling molecules. These can allow sequential activation, rapid activation of multiple downstream targets, and prevents signalling from mixing with other pathways
36
what initiates insulin receptor signalling and what binds first?
Insulin (signal molecule) binds to the insulin receptor, activating it in its dimeric form. The insulin receptor substrate (IRS-1) will only bind to the insulin receptor and is the first intracellular protein to bind. This involves a docking protein interaction, where IRS-1 docks between the activated receptor and a membrane-associated signalling environment
37
How is IRS-1 activated and what does it enable?
IRS-1 is phosphorylated by the activated insulin receptor. This phosphorylation provides multiple binding (docking) sites for downstream signalling molecules. These phosphorylated sites allow recruitment of adaptor proteins such as Grb2, which contains 2 recognition domains
38
what role do adaptor proteins like Grb2 play?
Grb2 binds to phosphorylated IRS-1 via its recognition domains. It functions as an adaptor protein, linking IRS-1 to downstream signalling pathways. This reflects a sequential activation process: insulin receptor → IRS-1 → Grb2
39
how are scaffold proteins and Sos recruited?
Proline-rich regions recruit scaffold proteins and Sos by binding to them. This brings these proteins into close proximity with the receptor and other signalling molecules. This spatial organisation enables efficient interaction and activation of downstream signalling components
40
what is the sequence of activation in the MAPK pathway?
Ras is activated downstream of these adaptor interactions. Activated Ras leads to the activation of RAF. RAF becomes activated and phosphorylates MEK
MEK then phosphorylates ERK. The MAPK pathway results in cell division and proliferation
41
what does ERK do once activated?
ERK translocates into the nucleus. It targets and activates specific transcription factors. This leads to gene transcription and protein synthesis
42
what is the platelet derived growth factor receptor (PDGF)?
There are multiple tyrosine residues that can be phosphorylated, meaning that there are multiple sites within the receptor that signalling molecules can bind to, dock with, and subsequently activate. Signalling molecules are specific to certain tyrosine residues because of the overall structures of the receptors. The platelet derived growth factor receptor has a split tyrosine kinase domain, so there are 2 regions within that receptor that can phosphorylate its target
43
what is the purpose of the Ras family of GTPases?
These provide a crucial link in the intracellular signaling cascades activated by receptor tyrosine kinases
44
what is autophosphorylation?
Autophosphorylation is a post-translational modification where a protein kinase phosphorylates itself, adding a phosphate group, typically from ATP, to its own serine, threonine, or tyrosine residues.
45
what is the process of signal transduction in MAPK pathway?
Commences with a signalling molecule binding with the receptor tyrosine kinase activating the receptor through dimerisation + autophosphorylation which leads to the subsequent phosphorylation of several residues on the intracellular region of the receptor. The phosphorylated tyrosine residues provide a binding site for the SH2 domain in the adaptor protein Grb2
Grb2 has 2/3 domains that bind proline rich regions in Sos (Ras Binding Nucleotide Exchange Factor). Bound to GTP, Ras is able to propagate subsequent signalling molecules (e.g. MAPK pathway). Once kinases are active, the enzymatic capacity is to phosphorylate subsequent target molecules
Erk will then phosphorylate its target proteins which can relate to transcription, gene expression and changes in protein activity ERK is a MAP kinase (mitogen-activated protein kinase), and it is phosphorylated and activated by MAP kinase kinase (MEK).
46
what does Sos do?
Sos is capable of exchanging a GDP bound to an inactive GTP for a GTP molecule, leading to the activation of the Ras protein
47
what is the MAPK pathway and what is it made up of?
MAPK pathway consists of 3 kinase molecules (Raf, Mek and Erk - activation occurs in this order) . This pathway is called a kinase cascade where 1 molecule activates the next, so there is a sequential event of activation that occurs in a specific order
48
what does the the PI3K/Akt Cell Survival Pathway?
This pathway leads to the activation of mechanisms within the cell that promote cell survival, such as the inhibition of apoptosis.
49
what does the PI3K/Akt pathway require?
This requires Phosphoinositide-3-kinase (PI3K) which is recruited and activated by RTKs, its role is phosphorylate inositol phospholipids in the plasma membrane. Once these specific membrane bound phospholipids are activated, they provide docking sites for subsequent signalling proteins like Akt. Akt is also known as protein kinase B (PKB)
50
what is Akt?
Akt promotes growth and survival by inhibiting the protein Bad, which encourages apoptosis. Akt is phosphorylated by PDK1 And mTOR
51
what is the process of the PI3K/Akt pathway?
The binding of the signal molecule (growth factor like insulin) & subsequent activation leads to the activation or the hyperphosphorylation of phosphoinositide molecules. These molecules then provide docking sites for subsequent signalling molecules that then become active, and can propagate that signal. Similar to the MAPK pathway, the PI3K/Akt pathway is activated after the signalling molecule reaches the cell, activating the receptor, tyrosine kinase, leading to the phosphorylation of those intracellular domains. The key molecule in this pathway PI3K is able to bind to a phosphorylated residue, which enables its own activation
PI3K activity allows it to phosphorylate a phosphoinositide molecule in the membrane like bis- and tris-phosphate. Once the phosphorylation event occurs, it provides docking sites for key signalling molecules like PDK1 and Akt. Once bound PDK1 phosphorylates and activates Akt . Once Akt becomes phosphorylated it dissociates from the membrane but remains active, and is able to move through the cytosol to other signalling molecules in order to activate them. Overall, the dimerisation of a receptor tyrosine kinase can lead to the activation of both Ras/MAPK and PI3K/Akt pathways simultaneously because there are multiple docking sites for those key molecules that activate the pathways. Upon the binding of the ligand/signalling molecule, multiple cellular effects could be produced
52
what is the process of the PI3K/Akt pathway with insulin?
Insulin receptor has its own substrate that is activated by the receptor before the subsequent signalling pathway is activated. Here, instead of PI3K docking with the receptor, it actually docks with IRS-1. Once this is activated by the receptor & has its own phosphorylated residues, PI3K binds to its SH2 domain, leading to the conversion of PIP2 to PIP3. This then provides the docking site for PKB which is responsible for phosphorylating Akt. Once activated, Akt can target multiple downstream effectors. For glycogen synthesis, PKB leads to the phosphorylation of GSK3 (glycogen synthase kinase inactivated via phosphorylated). Without glycogen synthase kinase being activated, glycogen synthase is not phosphorylated which actually activates it & allows for the synthesis of glycogen. Therefore, in the presence of insulin, the cell is stimulated to release glycogen. An additional function for Akt is to stimulate the movement of vesicles containing GLUT4 transports to the membrane where they fuse, embedding the GLUT4 transported into the membrane, and allowing the movement of glucose into the cell.
53
where does the PI3K/Akt pathway occur when it uses insulin?
This function occurs in adipocytes (fat cells) and muscle cells, not the liver as they do not contain insulin-dependent GLUT transporter.
54
how does the PI3K/Akt pathway work to inhibit apopotosis?
Akt can lead to the promotion of cell survival. Bad binds with Bcl2 to inactivate it to promote cell death. In the absence of Akt, Bad is the active protein. When phosphorylated Akt is present, it leads to the dissociation of these 2 molecules which inactivates Bad and activates Bcl2 which results in cell survival
55
is Bad pro-apoptoic?
yes, Bad is pro-apoptotic
56
is Bcl2 anti-apoptotic?
Yes, Bcl2 is anti-apoptotic
57
how can RTKs promote disease?
RTK signalling typically induces growth and proliferation. When overactive this can result in uncontrolled growth. Some RTKs can be active, despite the absence of their ligand. This can lead to the onset of cancer. RTKs and their downstream molecule (especially cell surface receptors) are key targets for many anti-cancer therapeutics. The receptor can be targeted to block or inhibit its downstream signalling, or to activate it. However,receptors themselves can be problematic
58
what is HER2?
HER2 (Human Epidermal Growth Factor Receptor 2) is a receptor tyrosine kinase in the EGFR family encoded by the ERBB2 proto-oncogene
59
how is HER2 activated?
HER2 has no known ligand and is activated through heterodimerisation with other EGFR family receptors. Activation of HER2 leads to signalling through the MAPK pathway (cell proliferation) and PI3K/Akt pathway (cell survival)
60
what does the overexpression of HER2 result in?
Overexpression of HER2 results in increased cell growth and is associated with cancers such as breast cancer
61
how is cancer caused by HER2 combatted?
Trastuzumab binds to the extracellular domain of HER2 and inhibits receptor activation and downstream signalling. This blocks MAPK and PI3K/Akt pathways, reducing tumour cell proliferation and survival. Trastuzumab is used in the treatment of HER2-positive breast cancer. In breast cancer, there are too many HER2 receptors which send more signals causing cells to grow too quickly. To combat this, Herceptin can be used to bind to HER2 receptors & block their signalling pathways, by blocking these pathways apoptosis may be induced allowing for the targeting + treatment of breast cancer
62
what is Trastuzumab?
Trastuzumab (Herceptin) is a monoclonal antibody that specifically targets HER2
63
what are tyrosine-kinase-associated receptors?
Not enzymes themselves but instead associate with proteins that are tyrosine kinases. These include cytokine receptors. The associated kinases include members of the Src kinase family or of the Janus kinase family
64
what is the Src kinase family?
has at least 8 members - Hck, Lyn, Fyn, Lck, Yes, Fgr, Bk which can associate with receptors
65
what is the Janus kinase family?
Has at least 3 members: JAK1, JAK2 and Tyk2, which are involved in signaling from receptors including the Growth Hormone receptor. Signal transducer and activator of transcription (STAT) protein family describes a family of transcription factors activated by the JAK proteins
66
how do cytokine receptors work?
Prior to ligand binding, the cytokine receptor is already associated with one of the Jak members, so in an inactive form the Jak family member remains bound upon binding of the ligand
In the case of cytokines, the receptor dimerises, and Jak protein associated with the 1 receptor phosphorylates the Jak protein associated with the other. This results in cross-phosphorylated, as once the Jak proteins are phosphorylated they then phosphorylate the receptor protein itself. Here, the receptor is not autophosphorylated like with receptor tyrosine kinases. The receptor is not an enzyme, but the phosphorylation of the tyrosine residues provide binding sites for proteins that can attach to the H2 domains. The proteins that bind here are STAT proteins (transcription factors). Upon STAT binding to the receptor, JAK proteins phosphorylate the STAT proteins, causing them to dissociate from the receptor and dimerise with each other. The STAT dimers then translocate into the nucleus, where they act as transcription factors and initiate gene transcription
Depending on the receptor and specific combination of STAT proteins that are activated, different genes will be activated
67
what is the process of activation of GTP binding proteins?
Involves the guanine nucleotide exchange factors and GTPase activating proteins. GTPase activating protein (GAP) leads to the inactivation of the GTPase, hydrolysis of GTP to GDP and GDP remains tightly bound. Ultimately, both the GPCRs and the RTKs can lead to the activation of processes such as transcription and activating series of target proteins promote cell proliferation, cell growth and cell survival.
68
what is GEF?
Guanine nucleotide exchange factor (GEF) stimulates the release of GDP, readily available for GTP to quickly bind, which in-turn leads to the activation of the GTPase and GTP exchanged for GDP
69
what are G-Protein-Coupled receptors?
Very unique and different structure than the receptor tyrosine kinase. However, common elements between the 2 include: the presence of an extracellular & intracellular region, and a transmembrane region. Has 7 transmembrane segments. Has over 800 different receptors with most being uncharacterised for their ligands/function, and belongs to a large gene family. Signalling molecule can activate multiple GPCRs, meaning adrenaline can activate 9. All use G-proteins to relay a signal
70
what is the structure of G-proteins?
Once active, a GPCR interacts with G-proteins. G-proteins are associated with the plasma membrane, so they’re docked/fitted with specific phospholipids. Trimeric G-protein complex
A, B and y subunits. The alpha subunit has a GDP-GTP binding site, so it’s activated through the exchange of GDP with GTP. Many different G-proteins, specific for different sets of GPCRs, meaning that all GPCRs won’t necessarily activate the same G protein. It contains a GDP/GTP binding site
71
what are the 7 steps of G-Protein Activation
1. Ligand binds to a G protein–coupled receptor (GPCR)
2. Receptor changes shape and activates a nearby GDP-bound G protein
3. GDP is released and replaced with GTP
4. GTP binding activates the G protein (conformational change)
5. Activated G protein dissociates from the receptor
6. G protein interacts with effector proteins to produce a cellular response
7. Signal is terminated when the G protein hydrolyses GTP → GDP (inactivates itself)
72
how does G protein signalling work?
When an appropriate protein ligand binds to the domain, the receptor undergoes a conformational change that is transmitted to its cytosolic regions, activating a trimeric GTP-binding protein. In some cases, the inactive G-protein is associated with the inactive receptor whereas in other cases, it only binds after the receptor is activated. An activated receptor induces a conformational change in the alpha subunit causing the GDP to associate, meaning GTP can now readily bind in the place of the GDP. GTP binding causes a further conformational change in the G protein, activating both the alpha subunit and the beta-gamma complex. In some cases, GTP + alpha complex can dissociate from the beta-gamma subunit. Both complexes can regulate the activity of target proteins in the plasma membrane and then relay the signal to downstream components/ targets of the signalling cascade. Eventually, the alpha subunit hydrolyses the bound GTP, transforming it into GDP which inactivates the subunit. This step can be catalysed (sped up) by the binding of RGS. Then the molecule returns to its initial inactive state/form
73
what is the GTP-binding protein?
This protein is composed of alpha, beta and gamma subunits, alpha and beta subunits which have covalently attached lipid tails that anchors the G protein in the plasma membrane
In the absence of a signal, the alpha subunit has a GDP bound and the GTP is inactive
74
what are 2nd messengers?
Effector enzymes are activated in response to the activation of the receptor.
Effector enzyme examples are Adenylate cyclase and Phospholipase C. Second messenger examples include cAMP, diacylglycerol and Inositol 1,4,5-triphosphate (latter 2 are released upon cleavage of an inositol phospholipid).
75
what happens once 2nd messengers are activated?
Once activated, these lead to either the synthesis or the generation of a 2nd messenger which is often a small molecule or ion that is released within the cell upon activation of those affected enzymes.
76
what is the inositol phospholipid pathway?
Commences with the activation of the G-protein as a signal molecules enters the system activating & inducing a conformational change in the GPCR + inducing an exchange of GDP for GTP in the alpha subunit of the G-protein. This would lead to the dissociation of the alpha subunit from the beta-gamma subunits and their subsequent activation . The beta-gamma subunit component works to activate phospholipase C which is an effector enzyme and acts on the target (PI 4,5-bisphosphate). These targets are membrane bound phospholipids that can become phosphorylated. The effect of this is to cleave the phospholipid molecules into 2 molecules known as inositol 1,4,5-triphosphate (IP3) and diacylglycerol which are 2nd messenger molecules that have different targets. Diacylglycerol remains in the membrane whereas IP3 is released from the membrane, moving through the cytosol to its target (gated calcium release channels in the lumen of the endoplasmic reticulum). Here, IP3 binds to the gated channels, allowing the release of calcium ions that are present within the endoplasmic reticulum. These calcium ions bind to and target the molecule, protein kinase C. Upon the split of diacylglycerol and IP3, diacylglycerol recruits protein kinase C and this coupled with the binding of calcium, leads to the activation of protein kinase C. Protein kinase C activates a variety of downstream targets
77
how does the cAMP commence pathway?
the cAMP pathway begins when an alpha subunit dissociates from the beta gamma subunit. When this is activated it is associated with adenylyl cyclase. Adenylyl cyclase is the effector protein for this pathway, which leads to the generation of the second messenger cAMP
78
following the dissociation of the alpha subunit & generation of the 2nd messenger cAMP, what happens next in the cAMP pathway?
cAMP is generated from ATP, and targets downstream molecules. cAMP leads to the activation of a protein called protein kinase A, made up of regulatory subunits and catalytic subunits. Once cAMP enters the system, it binds to the regulatory subunits and allows their dissociation from the catalytic subunits, thus giving rise to activated PKA
79
what are catalytic subunits, what do they do in their inactive form?
Catalytic subunits implies that they’re the ones with the enzymatic activity. In its inactive form, catalytic subunits remain bound to the regulatory subunits. When bound to the regulatory subunits, the catalytic subunits are inactive so they cannot perform the enzymatic activity
80
what do mutations in G-Protein Coupled Receptors cause?
These can cause their downstream signalling pathways to trigger diseases
81
what do loss of function mutations lead to in G-protein coupled receptors?
Leads to the inhibition of the signalling pathway and may present with symptoms comparable to a hormone deficiency.
82
what do gain of function mutations lead to in G-protein coupled receptors?
Lead to an overactive, constitutively active pathway and may present with symptoms similar to an excess of hormone or signalling molecule and consequently resulting in the overstimulation of target tissues
83
what is hypothyrodisim?
Hypothyroidism is a condition characterised by low levels of thyroid hormone, regulated by the hypothalamus and pituitary gland via thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH).
84
what is Thyrotropin Releasing Hormone (TRH)?
Thyrotropin Releasing Hormone (TRH) is a releasing hormone secreted from the hypothalamus into the hypophyseal portal system, a capillary network that connects the hypothalamus with the anterior pituitary. TRH stimulates cells that produce and secrete thyroid stimulating hormone (also known as thyrotropin or TSH)
85
How is TRH and TSH regulated?
The hypothalamus secretes releasing hormones into the bloodstream that then act on hormone releasing cells of the anterior pituitary gland.
86
what does TSH do in hypothyrodism?
TSH then in-turn stimulates cells in the thyroid gland that ultimately release Thyroid Hormones (TH).
87
what can mutations in TRHR cause?
Mutations in the Thyrotropic-releasing hormone receptor (TRHR) can lead to hypothyroidism. This mutations leads to a loss of function. A loss of function mutation in this receptor results in the inability for these cells to be stimulated by TRH released from the hypothalamus. Results in reduced or no secretion of TSH and subsequently reduced or no secretion of thyroid hormones (TH). Typically, the activation of phospholipid C and subsequently PKC leads to the stimulation and secretion of TSH However, in the case of a loss of function mutation, the entire signal breaks down. For example, if the TRH is inactive or incapable of activation, then there is a block in the pathway which leads to a reduction of TSH secretion, this results in the significant reduction of TH
88
where is TRHR located?
The Thyrotropin Releasing Hormone Receptor is located in the cell membrane of thyrotropic cells in the anterior pituitary.
89
symptoms of low/no TH, and TSH in particular, secretion
Symptoms are dry/coarse hair, lateral eyebrows thinning, periorbital edema and a puffy or dull complexion on dry skin.
Low thyroid levels that can lead to fatigue, cold sensitivity, constipation, dry skin and unexplained weight gain
90
what is Cushing Syndrome?
in general, this involves an increase in the hormone cortisol. Cushing syndrome is a rare hormonal disorder caused by prolonged exposure to high levels of cortisol (hypercortisolism), often referred to as the stress hormone.
91
what is cortisol?
is a hormone secreted by the adrenal cortex in stressful situations as a result of the body's natural flight or fight response. this increases blood sugar levels and aids in metabolism.
92
what is cortisol secretion controlled by?
Cortisol secretion is controlled by a mechanism between the hypothalamus, anterior pituitary, and adrenal cortex, called the Hypothalamic-Pituitary Adrenal Axis
93
what is Corticotropin Releasing Hormone (CRH)?
Corticotropin Releasing Hormone (CRH): produced and released by the hypothalamus, acts on the anterior pituitary gland
CRH acts on corticotroph cells stimulating the production and release of adrenocorticotropic hormone (ACTH) which acts on the adrenal gland.
94
what does ACTH do?
ACTH stimulates cells in the adrenal cortex to produce and secrete the steroid hormone, cortisol. The ACTH Receptor is a G-Protein Coupled receptor and leads to the activation of the cyclic AMP second messenger pathway.
95
What activates the cAMP pathway and what is its role in the adrenal cortex?
it is activated by ACTH and stimulates signalling that leads to cortisol production
96
how do gain of function mutations affect the cAMP pathway?
these occur in signalling molecules (e.g. receptors, G proteins, enzymes), cause constitutive (always-on) activation of the pathway and leads to the overproduction of cortisol
97
how can loss of function mutations cause the over activation of the cAMP pathway?
PKA regulatory subunits normally inhibit catalytic subunits. Loss-of-function mutations prevent this inhibition. Catalytic subunits become overactive → pathway overactivation. Results in oversecretion of cortisol
98
what causes Cushing Syndrome?
Sparked by a mutation in the intercellular signalling pathways activated by GPCRs. Caused by the chronic exposure to excess glucocorticoids (cortisol) which is a steroid hormone synthesised in the adrenal cortex
99
what is the treatment of Cushing Sundrome?
Treatment: depends on the cause but can involve adrenalectomy (1 or 2 remove of adrenal glands), medication, hormone replacement
100
what are symptoms of Cushing Syndrome?
Symptoms include weight gain, hypertension, decreased growth in children, diabetes, osteoporosis, and muscle weakness
101
what mutations trigger Cushing Syndrome?
Somatic and germline mutations that lead to adrenal tumours with hypercortisolism. Gain of function: G-Protein Coupled receptor, the α subunit of the trimeric G-protein or the catalytic subunits of Protein Kinase A (PKA). Loss of Function: Regulatory subunits of PKA
102
what are categories of Cushing Syndrome?
1. benign adrenal adenoma, 2. PBMAH (primarily bilateral macronodular adrenal hyperplasia), 3. PPNAD (primarily pigmented nodular adrenocortical disease)
103
what are the characteristics of Benign Adrenal Adenoma (tumours)?
Detected by 24hr cortisol level, ACTH levels normally low, CT scan of adrenal gland, and often unilateral adenoma, sometimes bilateral (could affect just one or both kidneys)
104
what are characteristics of primary bilateral macronodular adrenal hyperplasia (PBMAH)
these are mostly associated with mutations in tumour suppressor gene, ARMC5, or G-protein genes . These mutations would be gain of function ones
105
what is PPNAD (primarily pigmented nodular adrenocortical disease) associated with?
Mostly associated with mutation in the PKA regulatory subunit
106
what are examples of intracellular nuclear receptors?
steroids & Vitamin D
107
where are nuclear receptors found?
Are intracellular, meaning they are found either in the cytoplasm or the nucleus within the cell. Even if they are found in the cytoplasm, they will move into the nucleus where they will act as a transcription regulator, oftentimes increasing gene transcription
108
what are nuclear receptors?
Nuclear receptors (NRs) are a large family of ligand-dependent transcription factors that play functional roles in reproduction, integrated metabolism, and homeostasis. Given their intracellular receptors, their ligands are able to pass through the cell membrane. many function as ligand-dependant transcriptional regulations.
109
how do cell surface receptors differ from nuclear ones?
Unlike cell surface receptors, nuclear receptors participate in the signalling within the cell once activated.
110
what are features of ligands of nuclear receptors?
Their ligands are lipophilic, meaning they dissolve straight through the cell membrane, dissolving through the phospholipid bilayer, moving through the cell towards their target receptor. They mediate signals from lipophilic ligands (hormones) to the nucleus of cells to alter gene expression and eventually physiology.
111
what are characteristics of nuclear receptors?
Since they’re lipophilic, they’re also hydrophobic which means some don’t dissolve well in the blood. They then require carrier (transport) proteins in the bloodstream (e.g. albumin or specific binding globulins) to remain soluble and be transported to target tissues.
Upon the binding of their ligands, gene transcription is activated. Hence, in the absence of that ligand, the genes that these receptors regulate tend to be repressed. There are 48 nuclear receptors in humans
Repression is a characteristic of the apo-nuclear receptor (no ligand)
112
what are steroid hormones all synthesised from?
These are all synthesised from cholesterol, and through a series of enzymatic steps this is modified to form different hormones
113
where are steroid hormones synthesised?
These are synthesised primarily in the adrenal gland, ovaries, and testes. In the adrenal gland, steroid hormones are synthesised in the adrenal cortex. The adrenal cortex predominantly syntheses Glucocorticoids, Mineralocorticoids, and small amounts of testosterone. The enzymes required to synthesise the androgens are present in the adrenal cortex as well. Ovaries = synthesises estrogen, & some testosterone in females. Testes = synthesises testosterone, and some estrogen in males
114
what are mineralocorticoids?
Mineralocorticoids = affect mineral (e.g. potassium) and salt (e.g. sodium) balances within the body affecting
115
what do Glucocorticoids do?
Glucocorticoids = role in general metabolism & metabolic rate
116
what do sex steroids do and how are they produced?
Sex steroids play a role in the development of sex steroids.
The production of sex steroids, Glucocorticoids, and Mineralocorticoids is stimulated from hormonal signalling from the anterior pituitary. This begins with the hypothalamus that secretes releasing hormones targeting the anterior pituitary which subsequently target tissues like the adrenal cortex, testes and ovaries to trigger the production of these hormones.
117
what is congenital adrenal hyperplasia?
Congenital adrenal hyperplasia stems from the deficiency or absence of these particular enzymes (hydroxylases). In an individual with congenital adrenal hyperplasia, where a hormone like cortisol was really low, there will be no feedback going back to the hypothalamus or anterior pituitary to turn off the secretion of ACTH. In the case of an enzyme deficit (e.g. lacking 17 hydrolase) which means that cortisol couldn't be produced, there will be a large amount of mineralocorticoid produced.
Without cortisol, there will be an increased level of ACTH, stimulating the adrenal cortex, compounding the immense production of a mineralocorticoid like aldosterone
118
what occurs when cortisol is low if they have congenital adrenal hyperplasia?
If cortisol is low, ACTH will remain high as there’s no cortisol to feed back to these organs. If this is blocked, ACTH will continue to be very high, which will further stimulate the adrenal cortex but because of the blockage it will also increase levels of aldosterone
119
how are steroid hormones synthesised?
Cholesterol can generate pregnenolone which can either become progestorate to result in the generation of a Mineralocorticoid, or 17-OH-Pregnenolone can either result in the production of Glucocorticoid or Androgens. Without 17a-hydroxylase/17,20 lyase, the pathways for producing Glucocorticoid & Androgens are blocked, this means that all the cholesterol coming into the adrenal cortex and being converted to steroid hormones is only used in the production of Mineralocorticoid, which in-turn leads to an overproduction of hormones like aldosterone. Aldosterone is a steroid hormone produced by the adrenal glands that acts as a key regulator of blood pressure, blood volume, and electrolyte balance. Congenital adrenal hyperplasia can develop from the deficiency of any of these enzymes involved in the synthetic pathway . A deficiency in 21 hydroxylase would lead to a significant increase in androgens being produced in the adrenal cortex. For congenital adrenal hyperplasia, regardless of the enzyme deficiency, each leads to the overproduction of one particular group of hormones. An individual may have a complete or partial deficiency in any of these steroid hormones.
120
what is 21 hydroxylase?
21 hydroxylase is only present in the adrenal cortex so in the absence of this enzyme in the testes and ovaries, only androgens can be synthesised.
121
what is the structure of nuclear receptors?
Overall structure: (1) transcription activation domain, (2) DNA binding domain, (3) hinge, (4) ligand binding domain, and (5) C-terminus domain.
122
what are the specific domains of nuclear receptors?
At the end-terminus, there is the A and B domain which is the transcription activation domain. This is highly variable but typically contains the region that leads to gene transcription activation. C domain = region that encodes the part of the protein responsible for binding directly with DNA. Following the C domain, there is a Hinge. After the hinge, there is a ligand binding domain which is a region of protein responsible for binding specifically to the ligand. At the very end is the C-Terminus Domain
123
what is the hinge of the nuclear receptor?
The hinge region is flexible, allowing the conformal change triggered by a ligand binding to the ligand binding domain to occur, exposing different domains. The hinge region ensures that this is a reversible process. The conformation change referenced describes the ligand-induced conformational change of the nuclear receptor.
124
what is the mechanism of action for nuclear receptors?
The signalling molecule that binds to these receptors are capable of passing through the cell membrane. Following this, the signalling molecule diffuses through the cell membrane into the cell. Once the ligand meets its receptor, it induces a conformation change in the receptor, allowing for the formation of a dimer, and the exposition of the specific DNA binding site on the receptor, so that the receptor can bind to hormone response elements (HREs) in the DNA adjacent to specific genes. To bind to their specific regions, a particular receptor may need to attract a coactivator protein to help them bind DNA or promote gene transcription. Receptors attract coactivator or corepressor protein/s and with them regulates transcription of the adjacent gene/s, increasing or decreasing the rate of mRNA formation. This results in the nuclear receptor being bound to the ligand in its dimeric form (can be a homo- or heterodimer). If a coactivator is bound, this will lead to an increase in transcription & production of mRNA, along with the production of a new protein, and consequently an altered cell function in response to the presence of that protein/ Altered levels of the hormone-regulated gene product produce the cellular response to the hormone
125
what are signalling molecules in nuclear receptors?
Signalling molecules are often carried through the bloodstream via specific serum binding proteins which function to support the signalling molecules as they travel through the bloodstream, ensuring they are not degraded, and then releasing them upon reaching the cell.
126
what is the structure of nuclear receptors with regards to the DNA binding domain?
All nuclear receptors contain a DNA binding domain in both active and inactive forms of the receptor. In the inactive form of the DNA binding domain, the ligand binding domain is unbound (no ligand attached to that domain). When the ligand is introduced to the cell, it binds to the ligand binding domain, inducing a conformal change that results in the recruitment & binding of coactivator proteins. The conformational change exposes the DNA binding domain, allowing it to bind to the hormone response element
127
What are the 2 classes of nuclear receptors?
Type II - Form homodimers: which includes steroid hormone receptors, are located in the cytoplasm prior to the ligand binding, and when the ligand enters the cell & meets the receptor in the cytoplasm, it will then be transferred to the nucleus. Typically exists with an inhibitory complex/ protein prior to binding of the ligand.
Type II - Form heterodimers with Retinoid x Receptor (RXR): Predominately found in their inactive form in the nucleus . Here, the ligand enters the cell through the plasma membrane + nuclear membrane where it encounters the receptor within the nucleus. It doesn’t bind the ligand
128
what are some examples of Type II - Form heterodimers with RXR?
Examples: Thyroid Hormone Receptor, Retinoic Acid Receptor, Vitamin D Receptor, Peroxisome proliferator activated receptor, and Retinoid X receptor
129
what are some examples of 'Type II - Form homodimers' receptors?
Examples: Androgen receptor, Progesterone receptor + Estrogen Receptor
130
what are coactivators?
Coactivator Proteins include Histone Acetyl Transferases (HATs), they promote the dissociation of DNA from histones and increase gene transcription. Coactivators may also involve chromatin remodelling. These bind after the conformational change has occurred. multiple coactivators may be recruited
131
what are corepressors?
Corepressor proteins may help decrease gene transcription. Receptors attract coactivator or corepressor protein/s and with them regulates transcription of the adjacent gene/s, increasing or decreasing the rate of mRNA formation. Coactivators and Corepressors work by mediating the association of DNA with histones. Corepressor Proteins include Histone de-acetyl transferases (HDACs), promotes the association of DNA with histones, and decrease gene transcription
132
what are HATs (which are involved with coactivators)?
HATs help promote the binding of nuclear receptors to DNA by acetylating histones, which weakens the interaction between DNA and histones. This causes the chromatin to become more open, exposing regions of DNA and allowing nuclear receptors to bind.
133
what are estrogen receptors (ERs) and how do they function?
Estrogen receptors (ERs) are nuclear receptors that mediate the biological effects of estrogens (especially estradiol). Function as ligand-activated transcription factors that regulate gene expression. Estradiol binding triggers a conformational change in the receptor
ERs dimerise (form homodimers) after ligand binding. Dimerised receptors bind to estrogen response elements (EREs) on DNA. This leads to regulation of transcription of target genes
ER activity is facilitated by chromatin remodelling (e.g. histone acetyltransferases - HATs). Chromatin remodelling increases DNA accessibility, allowing receptor binding and transcriptional activation
134
hat are ERα and ERβ and how do they differ?
Two main subtypes: ERα and ERβ, are encoded by different genes and have distinct but sometimes overlapping roles. These exhibit different tissue expression patterns. Relative levels of ERα and ERβ influence cellular response to estrogen. The balance determines whether estrogen effects are proliferative, protective, or regulatory.
135
what exactly is ERa?
ERα: Major mediator of estradiol effects in female reproductive tissues. Important for mammary gland development and function. Also involved in bone maintenance, cardiovascular function, and brain activity
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what exactly is ERB?
ERβ: Expressed in ovary, bone, breast, brain, and male prostate. Can modulate or counterbalance ERα-mediated effects depending on tissue context.
137
Why is estrogen receptor signalling important?
Estrogen receptor signalling is essential for normal development, reproduction, and overall systemic physiology. It plays key roles in processes such as reproductive function, bone development and maintenance, cardiovascular health, and brain activity. Although estrogen levels are higher in females, estrogen receptor signalling is important in both males and females, demonstrating that the presence and function of the receptor is critical regardless of circulating hormone levels.
138
How does dysregulation of estrogen receptor signalling contribute to disease?
Dysregulation of estrogen receptor signalling is associated with a range of diseases, particularly hormone-dependent cancers such as breast cancer. Increased estrogen receptor activity leads to enhanced gene transcription, which promotes cell growth and proliferation and contributes to tumour development. ERα is often linked to these proliferative effects, while ERβ may have modulatory or opposing roles depending on the context. Overall, disruption or imbalance in estrogen receptor signalling can result in significant pathological consequences.
139
What does ERα deficiency reveal about estrogen function?
A reported case of a male lacking ERα provides important insight into estrogen receptor function, as the individual had high circulating estrogen levels but was completely resistant to estrogen, demonstrating that receptor presence is essential for hormone action. This individual, who was 22 years old and 6 feet 10 inches tall, exhibited continued linear growth due to unclosed epiphyseal plates. This finding highlights the critical role of estrogen in bone maturation and epiphyseal closure and demonstrates that estrogen is essential for bone development in both males and females, not just females.
140
How do agonists and antagonists affect estrogen receptor signalling?
Agonists are drugs or agents that bind to and activate estrogen receptors, and excessive activation can lead to severe developmental complications due to overstimulation of gene transcription pathways. Antagonists are drugs or agents that bind to the receptor and block estrogen from binding, thereby preventing receptor activation. An example is Tamoxifen, which inhibits estrogen receptor-mediated gene transcription and reduces cell proliferation in estrogen-dependent cancer cells, particularly in ER-positive breast cancer.
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How do endocrine disrupting chemicals affect estrogen receptor signalling?
Endocrine disrupting chemicals interfere with normal hormone signalling pathways by mimicking, blocking, or altering estrogen receptor activity, which leads to abnormal gene transcription and physiological effects. Many of these chemicals were historically used in the manufacture of plastics but are now banned or heavily regulated due to their harmful effects. Examples include DDT, Polychlorinated biphenyls, and Phthalates, all of which can disrupt estrogen receptor signalling and contribute to disease.
BMS2021
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