Describe cell signaling:
Internal or external stimuli tell the body to react. This signal needs to be sensed and transmitted to individual cells in target organs and tissues This is done by chemical messengers and is called cell signaling
Describe/Draw a generalized signal-transduction cascade.
1. release of chemical messenger 2. reception of chemical messenger (where is it*) 3. delivery of message inside the cell 4. signal transduction by: a) signal transducer proteins b) second messengers 5. activation of effectors that alter a physiological response 6. termination of the signal (if it can’t get turned off it is diseased)
Explain how signal transduction is amplified:
Secondary messengers amplify the signal, can diffuse, and have a fast response relative to proteins that turn genes on. - A ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell. Enzymatic cascade amplify signal: enzyme 1 turns on multiple enzyme 2, which in turn all of the #2 turn on #3, while #1 continues to turn on more #2’s
Name types of second messengers:
Second messengers are nonprotein molecules 1. Phosphatidylinositol signaling 2.cAMP 3.Ca2+
Identify the five major types of chemical messengers
Neuropeptides: nervous system Hormones: endocrine system Cytokines: immunes system Eicosanoids: injury Growth Factor: cell proliferation a. The nervous system secretes two types of messengers: small-molecule neurotransmitters (such as acetylcholine) and neuropeptides (normally small peptides between 4 and 35 amino acids in composition). b. Endocrine system hormones consist of polypeptide hormones (such as insulin and glucagon), catecholamines (such as epinephrine), steroid hormones (derived from cholesterol, such as estrogen), and thyroid hormone. c. The immune system utilizes the messengers known as cytokines, which are small pro-teins with an average molecular weight of 20 kDa. There are different classes of cytokines (such as interferons, interleukins, tumor necrosis factors, and colony-stimulating factors), but all are secreted by the cells of the immune system and will induce alterations in gene transcription in the target cells. d. The eicosanoids are derived from long-chain fatty acids, and consiste of the prostaglan-dins, thromboxanes, and leukotrienes. e. Growth factors are polypeptides that function through the stimulation of cellular proliferation (hyperplasia) or cell size (hypertrophy).
Describe the three modes of action chemical messengers used to signal
Endocrine: through the blood (hormones) Paracrine: adjacent cell (axon and dendrites, immune system) Autocrine: same cell (often paracrine can be performed by a autocrine cell)
Describe intracellular transcription factor receptors, and where they reside:
Intracellular transcription factor receptor: Receptor can be cytosolic or nuclear and the chemical messenger is lipophilic (can diffuse through membranes). Turns genes on and off. Slow = takes hours to days for effect (thyroid hormones)
How do steroid hormone/thyroid hormone receptors function intracellularly?
1) These lipophilic molecules are transported in the blood bound to serum albumin, or to more specific transport proteins, such as steroid-hormone-binding globulin, or thyroid-binding globulin.(2) Once in the cell the lipophilic messenger binds to its receptor, which will often dimerize (with other intracellular transcription factors) to bind to the promoter-proximal regions of DNA to alter gene expression.
How does cortisol bound to its receptor functions at the level of gene transcription?
Because of the intracellular function, once in the cell and bound to its cytoplasmic receptor, the cortisol will enter the nucleus and will alter gene encoding. Specifically it increases transcription of genes encoding enzymes that raise blood glucose levels
- cortisol binds to GR (glucocorticoid receptor)
- two bound GRs dimerize and undergo confirmational chnage to reveal DNA binding domain
- DNA binds to zinc finger DNA binding domain
- increased of transcription of genes encoding enzymes, raise blood glucose levels
Identify the three major classes of plasma membrane receptors and describe their common feature
Ion channel, kinase or bind kinase, and heptahelical Common features for plasma membrane receptors: 1) Extracellular domain that binds the chemical messenger 2) membrane spanning region 3) conformational change in the receptor 4) intracellular domain that initiates signal transduction/secondary messengers (fast response)
Describe an ion channel:
Ion channel: the neurotransmitter binds with the receptor and alters the conformation of the protein, which opens the ion-channel, allowing extracellular ions to go into the cell. The ion permeability of the plasma membrane is altered, and this will instantaneously convert the extracellular chemical signal into intracellular electric signal, which will alter the excitability of the cell. (fast response) - nervous system, muscles 1. Signal transduction consists of a conformational change when a ligand binds, which allows ion-flow through the channel 2. The acetylcholine receptor is an example of an ion-channel receptor.
Describe the common feature of the kinase/bind kinase receptor and signaling cascade:
The common feature of this class of receptors is that the intracellular kinase domain of the receptor (or the kinase domain of the associated protein) is activated when the messenger binds to the extracellular domain. The signal transduction pathway is propagated downstream through signal transducer proteins that bind to the activated messenger–receptor complex.
Describe heptahelical receptor/g-protein coupled receptor:
In addition to transporting small molecules, membrane proteins can also function as receptors. G-protein coupled receptors, the most important class of cell membrane receptors, are proteins that traverse the plasma membrane seven times (seven-pass receptors). They are coupled to trimeric GTP-binding proteins (G proteins), which are composed of three subunits: α, β, and γ. The G proteins are found on the cytosolic face of the membrane and serve as relay molecules. G-protein coupled receptors have extremely diverse functions and respond to a vast array of stimuli. However, all G-protein coupled receptor signaling is transduced via a similar mechanism. When the receptor is inactive, the α subunit (active subunit) of the G protein is bound to GDP. When the receptor is stimulated, a change in conformation causes the α subunit to exchange GDP for GTP, thereby releasing itself from the βγ complex. Once released, it binds and activates target proteins. α Subunit activity is short-lived, however, because the GTPase quickly hydrolyzes GTP to GDP, resulting in its inactivation. The target proteins activated by the α subunit vary, depending on which of the three main types of G protein is involved.
Describe Ion channels and their biochemical characteristics and functions: Example: the nicotinic acetylcholine receptor
Biochemical characteristic: The channel opens and Na+ ions flood down the electrochemical gradient into the cell Functions: The activation of receptors by nicotine modifies the state of neurons through two main mechanisms. On one hand, the movement of cations causes a depolarization of the plasma membrane (which results in an excitatory postsynaptic potential in neurons), but also by the activation of voltage-gated ion channels. On the other hand, the entry of calcium acts, either directly or indirectly, on different intracellular cascades leading, for example, to the regulation of the activity of some genes or the release of neurotransmitters.
Describe the general structures of the receptor tyrosine kinases and the process that converts them from inactive proteins to active enzymes
Receptor tyrosine kinase (RTK) is composed of an extracellular domain, which is able to bind a specific ligand, a transmembrane domain, and an intracellular catalytic domain, which is able to bind and phosphorylate selected substrates. Binding of a ligand to the extracellular region causes a series of structural rearrangements in the RTK that lead to its enzymatic activation.
Define autophosphorylation and its role in the signal-transduction process:
Occurs by the addition of a phosphate group to serine,threonine or tyrosine residues within protein kinases, normally to regulate the catalytic activity Protein kinases, many of which are regulated by autophosphorylation, are vital in controlling the cellular proliferation, differentiation, metabolism, migration and survival. Mutations in the genes encoding them or their potential activators or repressors can affect any number of functions within an organism. Phosphorylation is easily reversed by phosphatases. Therefore, it is an effective method of turning `on' and `off' kinase activity.
Explain the role of SH2 domains in tyrosine kinase function
The function of SH2 domains is to specifically recognize the phosphorylated state of tyrosine residues, thereby allowing SH2 domain-containing proteins to localize to tyrosine-phosphorylated sites. This process constitutes the fundamental event of signal transduction through a membrane, in which a signal in the extracellular compartment is "sensed" by a receptor and is converted in the intracellular compartment to a different chemical form, i.e. that of a phosphorylated tyrosine.
Why can a mutations in Ras result in cancer?
Mutations can cause Ras to be active all the time, this in turn causes signaling cascade to always be on leading to tumor growth and cancer (ras is apart of the epidermal growth factor kinase cascade)
Describe insulin signaling and its function
It is unique that the insulin receptor is already a dimer and has an alpha and beta part to the receptor. It also requires IRS before a kinase pathway can be activated. The insulin receptor can activate MAP kinase pathway AND the protein kinase B (Akt) pathway
- Insulin signaling increases the number of glucose transporters at the plasma membrane
- The activation of PK B (Akt) promotes cell survival (antiapoptotic), as well as propagating some specific insulin effects
Describe common examples of ion channel pathologies in the following:
- Neuromuscular junction
- General effect:
1.) Neuromuscular junction:
- Cobra venom blocks the receptor, could lead to suffocation (competitive inhibition, decrease muscle tone can lead to suffication)
- Suxamethonium bind receptor: short term muscle relaxant that aid endotracheal intubation
2) CNS - nicotine binds receptors (increases neurotransmitters available to bind and activates reward circuits)
- Myasthenia gravis is a neuromuscular disorder due to an autoimmune condition that produces antibodies against the acetylcholine receptor. The receptor cannot be activated by acetylcholine, and the muscle cell cannot respond to the neurotransmitter, and contraction will not occur. The overlying symptom of this disorder is muscle fatigue. The treatment consists of acetylcholines-terase inhibitors (such that acetylcholine is present for an extended period at the neuromuscular junction) and immunosuppressants to reduce the production of the antibodies targeted against the receptor. (competitive inhibition)
3) General Effect:
- Malathion poisoning (insecticide) bind acth prohibiting breakdown and termination of signal
- Sarin gas, same as above, but more toxic (competitive inhibition)
Describe the structure of the insulin receptor (IR) and the significance of the insulin-dependent dimerization of the receptor.
Always present as a dimer, insulin binds - conformation change through to the transmembrane protein, autophosphorylation of beta subunits. IRS binds to the receptor.
• already a dimer
• needs signal to bind to activate tyrosine kinase activity
• example of PI3 kinase signaling
Draw and describe the two different signaling pathways initiated by activation of the insulin receptor (MAP kinase and PI3 kinase)
After insulin activates alpha receptor and IRS attaches either Grb2 or PI3-kinase attach: Grb2 binds with GAP1 PIP and activates Ras/MAP kinase pathway, which is the same cascade as the EGF PI3-Kinase PIP
(a) The activation of PI-3-kinase leads to the generation of phosphatidylinositol 3,4,5- trisphosphate, which is a membrane-binding site or proteins with pleckstrin homology domains, such as PDK1 and PK B
(b) the activation of PK B (Akt) promotes cell survival (antiapoptotic), as well as propagating some specific insulin effects
Provide a general overview of the following kinase receptors: serine-threonine kinase
Receptor serine-threonine kinase
- Heterodimerizes after signal binding
- Serine kinase activity poised
Provide a general overview of the following kinase receptors: Cytokine Receptors
- Receptor kinases have no intrinsic kinase activity
- JAK binding provides tyrosine kinases activity
- Example of direct signaling to nucleus (STATs)
Provide a general overview of the following kinase receptors: Tyrosine Kinase receptors
Tyrosine Kinase receptors
- Dimerizes are signal binding
- Tyrosine kinase activity poised
- Example of ras/MAP kinase signaling
Provide a general overview of the following kinase receptors: Insulin receptors
- Already a dimer
- Needs signal to bind to activate tyrosine kinase activity
- Example of PI3/Akt kinase signaling
What is a transcription factor:
- A protein that binds to a specific site on DNA and regulates the rate of transcription of a gene
- EX. STATs, p53,
What type of receptor is involved with transforming growth factor and what process is it involved in?
serine-threonine kinases & cell growth and differentiation
Describe and explain how the signal is transduced with TGF-ß (transforming growth factor), starting with ligand binding and going to the nucleus through SMADs
- Functional receptor is type II and once the TGFB binds
- Then type I receptor binds to dimerize -> phosphorylates
- Type I binds to Smad, which it phosphorylates on a serine residue
- The phosphorylated R-SMAD dissociates from the receptor, and dimerizes with SMAD 4, the common SMAD5. The SMAD complex translocates to the nucleus to alter gene transcription 6. One of the genes activated is an inhibitory SMAD, which regulates how long the signal is active (signal termination) - chromosomal deletion of smad4 is in 30% of pancreatic cancer (predictor of metastasis and decreased survival)
Describe: JAK/STAT signaling by cytokine receptor
Signal transduction by cytokine receptors: use of JAK-STAT proteins
- Cytokine receptors do not have an intrinsic kinase domain (means they are not ready to go and need require something else to start cascade = JAK kinases)
- When cytokines bind to the receptors dimerization occurs, cross-phosphorylation between JAK kinases occurs, allowing the SH2-domain proteins STAT (signal transducer and activator of transcription) to bind to the receptor–kinase complex
- The STATs are phosphorylated, dimerize, and travel to the nucleus to alter gene transcription
- STAT signaling is modulated by the SOCS (suppressors of cytokine signaling) and PIAS (protein inhibitors of activated SA) family of proteins, some of which are induced by SA.
Why is JAK/STAT signaling important for immune function?
JAK/STAT is used by many immune cells and cytokines are secreted to direct the process of inflammation/immunity response.
Severe combined immunodeficiency syndrome (SCID) exists in two types, either as adenosine deaminase deficiency, or the X-linked SCID that is missing a cytokine receptor subunit (the defective gene is the IL2RG gene, which stands for interleukin 2 receptor, gamma). This subunit is common to many different cytokine receptors, and when it is defective the immature blood cells cannot appropriately respond to growth and differentiation signals, resulting in the lack of a functional immune system.
What is the purpose of kinase?
transfers phosphate groups from high-energy donor molecules
Describe the topology of heptahelical receptors
(a) these receptors contain seven membrane-spanning domains. (b) Ligand binding to the receptor is transduced through the activation of heterotrimeric G-proteins, of which there are many classes.
Draw and describe how heterotrimeric G-proteins function, and how they play a role in how. Include the point at which cholera toxin acts [Heptahelical receptor]
1. G-alpha bound to GDP
2. Ligand binds to receptor (alpha, beta, or gamma)
3. Receptor becomes guanine nucleotide factor (GEF)
4. G-alpha exchanges GDP for GTP
5. alpha dissociates from beta/gamma
6. signaling occurs
Cholera toxin is an enzyme that catalyzes ADP ribosylation of the αs subunit. This blocks GTPase activity so it is continuously bound to GTP and therefore continuously active. The resulting activation of adenylyl cyclase causes large effluxes of Na+ and water into the gut lumen, resulting in severe diarrhea.
Compare and contrast bordetalla pertussis vs cholera toxin effect on G proteins: [Heptahelical receptor]
The bacteria Bordetalla pertussis colonizes the lungs and secretes a toxin that enters the lung cells. Pertussis toxin is an enzyme that catalyzes the ADP ribosylation of the αi subunit, blocking its dissociation from the βiγI complex so it is unable to inhibit adenylyl cyclase. Thus, adenylyl cyclase is permanently activated resulting in whooping cough.
Contrast to that of cholera toxin: catalyzes ADP ribosylation of the αs subunit. This blocks GTPase activity so it is continuously bound to GTP and therefore continuously active.
Describe the roles of G proteins in coupling a hormone-receptor complex to adenylyl cyclase and in amplifying the stimulus: [Heptahelical receptor]
G-alpha-GTP complex binds to adenylyl cyclase. Adenylyl cyclase converts ATP to cAMP. cAMP is a secondary messenger because its concentration reflects changes in the concentration of the hormone (first messenger).
Describe the mechanism by which cAMP modulates the activity of protein kinase A (PKA) [Heptahelical receptor]
cAMP binds the regulatory subunit of Protein Kinase A allowing the catalytic subunit to dissociate.
Describe signal transduction through heptahelical receptors:
Signal transduction through heptahelical receptors
- these receptors contain seven membrane-spanning domains.
- Ligand binding to the receptor is transduced through the activation of heterotrimeric G-proteins, of which there are many classes.
- The basic scheme ex is a hormone that leads to adenylate cyclase activation.
- The α subunits of the heterotrimeric G-proteins, in addition to binding GTP, also have an intrinsic GTPase activity, which self-regulates how long the G-protein is active.
- There are five major classes of G-proteins. Note that there are G-proteins that can activate their target, as well as G-proteins that inhibit their target.
- cAMP levels are also modulated through the regulation of the cAMP phosphodiesterase, the enzyme that converts cAMP to 5’-AMP. Methylxanthines, such as caffeine and theophylline, inhibit the cAMP phosphodiesterase and act by maintaining elevated levels of cAMP.
- PI signaling through heptahelical receptors occurs through the G`q family of G-proteins. G`q targets phospholipase Cβ, which hydrolyzes PIP2 into DAG and IP3.
- IP3 binds to the sarcoplasmic or endoplasmic reticulum, which triggers calcium release into the cytoplasm. The calcium release activates the enzymes containing the calcium–calmodulin subunit, including a protein kinase.
- The DAG, which remains membrane-bound, activates protein kinase C, which propagates the response by phosphorylating appropriate target proteins.
Draw a generic signal transduction pathway and identify the mechanisms responsible for termination
Do Gs and Gi increase/decrease cAMP levels? (G-protein signaling)
Gs→↑ adenylyl cyclase →↑ cAMP →↑ PKA activity
Gi→↓ adenylyl cyclase →↓ cAMP →↓ PKA activity
Gs (stimulatory G protein) = ↑ cAMP levels
Gi (inhibitory G protein) = ↓ cAMP levels
Gq = activates phospholipase C (PLC)
Both the Gs and Gi proteins signal through the adenylyl cyclase pathway. Adenylyl cyclase is a plasma-membrane-bound enzyme that synthesizes cyclic AMP (cAMP) from ATP. Receptors coupled to Gs result in the activation of adenylyl cyclase and an increase in cAMP. Receptors coupled to Gi result in the inhibition of adenylyl cyclase and a decrease in cAMP.
Describe GQ Protein signaling: (phospholipase C (PLC) pathway
Occurs via the phospholipase C (PLC) pathway. Phospholipase C is a plasma membrane–bound enzyme that, when activated, cleaves the inositol phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is present in the inner leaflet of the plasma membrane in small amounts (see discussion of plasma membranes earlier under The Cell). This cleavage results in the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 causes Ca2+ release from the ER, which activates the Ca2+/calmodulin-dependent protein kinase (or cAM-kinase). cAM-kinase then phosphorylates certain intracellular proteins, resulting in a specific cellular response. DAG activates protein kinase C (PKC) directly, which also phosphorylates certain intracellular proteins, resulting in a specific cellular response
Gq →↑ PLC activity → PIP2 → IP3 + DAG IP3→↑ Ca2+ →↑ CaM-kinase activity DAG →↑ PKC activity