Lecture 1 Flashcards
What are the steps of biosignaling
Signal, transduction, response
What is a signal?
NON-COVALENT interaction between ligand and receptor
What is a receptor?
Membrane-bound or soluble protein or protein complex, which exerts an intrinsic effect after binding to its endogenous/natural ligand
What are the features of signal transduction? (6)
- Specificity
- Amplification
- Modularity
- Desensitization/adaptation
- Integration
- Localization
What are the 4 features of specificity?
- Complementarity
- Non-covalent bond
- Tissue specific receptor
- Tissue-specific receptor target
Can there be a permanent interaction between a ligand and a receptor?
No
Give an example of tissue-specific receptor target
E.g. adrenalin. Both liver and adipose tissues have adrenaline receptors. However, receptor in liver results in a different response (hepatocyte interaction with adrenaline stimulates glycogen breakdown) than receptor in adipose tissue (triglyceride breakdown)
Does amplification occur downstream or upstream?
Downstream- from a signal down the cascade
What is a domain of a protein?
Domain or a module is a part of a protein that has a specific role
What is modularity?
Signaling proteins are somewhat modular. This allows cell to mix and match different complexes to have different functions
Are all modules of proteins catalytic?
No, they can just fulfil 3D function
When does desensitization occur and what is it?
When a signal is present continuously, desensitization of the receptor system results
- Blocking of the receptor
or
- Removal of it from the cell surface.
What is integration?
When 2 signals have opposite effects on a metabolic characteristic, the regulatory outcome results from the integrated input from both receptors
What is localisation?
When the enzyme that destroys an intracellular message is clustered with the message producer, the message is degraded before it can diffuse to distant points, so the response if only local and brief
What does interaction of ligand and receptor increase?
Interaction of L & R increases the activity of effectors/ mediators of signal transduction
What are the 4 classes of receptors?
- G-protein coupled receptors
- Receptor enzyme (tyrosine kinase)
- Gated ion channel
- Nuclear receptor
What are the steps of G-coupled receptor?
- Ligand interacts with receptor, which is a transmembrane protein
Transmembrane protein interacts with G proteins–modularity
When Gsα is bound to GDP, it is inactive - As soon ligand binds to receptor, it causes Gsα to become activated by replacing GDP to GTP.
- Gsα dissociates and is an enzyme. It moves to adenylyl cyclase and activates it.
- Adenylyl cyclase catalyzes formation of cAMP
- cAMP activates protein kinase A (PKA)
- PKA can phosphorylate cellular proteins, resulting in cellular response
- cAMP is degraded to AMP, reversing the activation of PKA
What is the most druggable class of proteins?
G-coupled
Which protein is self-inhibitory? Explain
G protein has an unique ability to self deactivate
Gsα is also known as GTPase. It can convert GTP to GDP, deactivating itself.
What happens when Gsα is deactivated?
Gsα goes back to Gsβ
___ is the most common second messenger
cAMP is the most common second messenger
How is ATP converted into cAMP?
ATP is converted by adenylyl cyclase to cAMP by removal of 2 phosphate groups and cycling the remaining phosphate within the sugar
What is a second messenger?
Second messenger is a substance that is released after a ligand-receptor interaction and which brings about a response by the cell.
Are second messneger and mediators the same?
No, as only proteins can be mediators. Second messengers are not proteins
What are the 2 PKA subunits?
- Regulatory subunit regulates catalytic subunit
- Catalytic subunit- has enzymatic function
What are the 2 PKA subunits held together by?
AKAP
How is PKA activated?
- Regulatory subunit needs to be activated. It blocks substrate binding cleft which is at the catalytic subunit
- Needs 4 cAMP to activate 2 regulating subunits
- This opens substrate binding clefts which can now bind ligands
How is PKA disactivated?
Removal of cAMP disactivates PKA
What are the 2 method of G-protein signal termination?
- cAMP is catalyzed by cyclic nucleotide phosphodiesterase, which emoves phosphodiester bond to produce AMP
This results in deactivation of PKA - Modulators of GTPase activity deactivate Ga protein
What are modulators of GTPase activity?
different classes of proteins that can speed up or slow down the process of conversion of GTP to GDP (deactivation of G alpha)
Describe desentization of g-protein
- When G alpha leaves, the receptor is only associated with beta and gamma
Free beta and gamma subunits can attract another protein called βARK, which adds phosphate groups - βARK phosphorylates Ser residues at carboxyl end (cytoplasmic end) o
- β-arrestin (βarr) protein binds to the phosphorylated receptor
- βarr associated receptor is taken in by endocytosis and is now inside the cell. It is no longer available for binding with the ligand
- Inside the cell βarr is removed and the receptor is dephosphorylated
Dephosphorylated receptor can go back to the membrane again and is available for his ligand
Can G protein also be inhibitory?
Yes- Gi instead of Gs
Active Gi inhibits Adenylyl cyclase, reducing cAMP concentration and supressign protein phosphorylation
Describe G-protein localization/nucleation
AKAP proteins e.g. AKAP5 are A kinase anchoring proteins
Adaptor proteins
They physically holds receptor, PKA and adenylyl cyclase
Thus, anything that can happen has to happen within the vicinity of this anchoring protein - localization of the signal
What are the 4 classes of G proteins?
- Gs-alpha- activates adenylyl cyclase
- Gi-alpha - inhibitory to adenylyl cyclase
- Gq-alpha - stimulates phospholipase protein
- G12,13 family - is associated with cytoskeleton
Steps of GPCR signaling through IP3 and Ca
- Hormone (H) binds to a specific receptor.
- Binding results in replacement of GDP by GTP and dissociation of Gq bound to GTP
- Gq moves to PLC and activates it
- PLC cleaves a phospholipid called PIP2 into two products: IP3 and Diacylglycerol (DAG)
- IP3 binds to a specific receptor- calcium channel that is present on endoplasmic reticulum, releasing
sequestered Ca2+ - Diacylglycerol and Ca2+ activate protein kinase C at the surface of the plasma membrane
- Phosphorylation of cellular proteins by protein kinase C produces some of the cellular responses to the hormone.
What is PLC?
Phospholipase C
What are the second messengers in GPCR pathways that uses Gq? What qualifies them as second messengers?
Calcium, IP3 and Diacylglycerol. They are not proteins and their concentration can be measured
Where’s calcium stored?
In endoplasmic reticulum
Neurotransmitters usually act through GPCR by ___ signaling pathway
Neurotransmitters usually act through GPCR by IP3 signaling pathway
What is the common structure shared by GPCRs?
They 7 helical domains that pass through the membrane 7 times- transmembrane proteins that cross the membrane 7 times
What does PKA stand for?
Protein kinase A
G-alpha either ___ or ___ an effector enzyme (AC) changing local ___ ____ concentration thus modulating ____
G-alpha either activates or inhibits an effector enzyme (AC) changing local second messenger concentration thus modulating PKA
Adaptors like ___ ensure localization of the signal
Adaptors like AKAP ensure localization of the signal
Some G-alpha (Gq) activate ___ that signals through ___ and ___ thus activating ___
Some G-alpha (Gq) activate PLC that signals through Ca and diacylglycerol thus activating PLC
What else acts through GPCRs apart from hormone receptors?
Vision, olfaction and gustation
What is the process of PIP2 cleavage? Products?
PIP2 is cleaved in two by phospholipase C.
The ring part is IP3 and the CHO chain is DAG
What does phospholipase C do?
Cleaves PIP2 into DAG and IP3
What does RTK snad for?
Receptor tyrosine kinase
What does RTK always act as?
Always acts as a dimer
What are the 3 important features of RTK?
- Receptors themselves are kinases
- Always act in a dimer
- Activate multiple signaling pathways.
What are the 2 most important multiple signaling pathways activated by RTK?
MAPK and PIP3
Steps of MAPK pathway with insulin as an example
- Insulin binds to RTK. Results in phosphorylation of cytoplasmic domain of the receptor on its carboxyl-terminal Tyr residues
- This phosphorylation results in another phosphorylation of IRS-1 protein on its Tyr residues, activating it
- IRS-1 now acts as a docking protein. It binds to SH2 domain of Grb2.
- Irs-1> Grb2->Sos-> Ras-> Raf-1
- Raf-1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and a Tyr residue, activating it.
Activated ERK can act as kinase - ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them
- Elk1 joins SRF to stimulate the transcription and translation of a set of genes
On which residues does MEK phosphorylate ERK?
On Thr and Tyr
Mnemonic to remember MAPK pathway
rigRRmet Receptor IRS-1 Grb2 Ras Raf-1 MEK ERK Transcription factors or target
MAPK pathways involves cascade of _____
MAPK pathways involves cascade of phosphorylation
Steps of signaling through PIP3- insulin example
- Ligand binds to a receptor, IRS-1 is phosphorylated and acts as a docking protein
- IRS1 allows for PI3K (PI3 kinase) to bind
- PI3K converts PIP2 to PIP3 by adding a phosphate group- phosphorylation of 3rd carbon
- PIP3 allows for docking of PKB (protein kinase B)
- PKB is activated by phosphorylation
- One target of PKB is GSK3. Phosphate group is added to GSK3 on Ser residue, INACTIVATING it.
- GSK3 is a GS (glycogen synthase) kinase. It is active only when it is not phosphorylated. In its active state can add phosphate to glycogen synthase- GS. GS converts glucose to glycogen. GS is active when it is NOT phosphorylated. The moment it is phosphorylated, it becomes inactive. - remover of an activator step
- When GSK3 is phosphorylated, GS remains active. PKB stimulates movement of glucose transporter GLUT4 from internal vesicles to the plasma membrane, increasing the uptake of glucose
Does phosphorylation always activate?
No, phosphorylation is a modulation step - can both activate & deactivate
How is glucose taken up from circulation?
Using GLUT- glucose transporter proteins- e.g. GLUT4
PKB helps in opening of GLUT4
How is PIP3 made?
It is converted by PI3K from PIP2
Which is the most studied RTK?
Insulin receptor
What activates TRK activity?
Phosphorylation of the receptor due to the binding of the ligand
What activates MAPK signaling cascade?
Multiple adaptive protein
What PIP3 serves as?
As an activator of PKB
How does TRK and β-adrenergic receptor interact?
When RTK is activated, it phosphorylates and activates IRS-1.
IRS-1 in turn can activate PKB. PKB can phosphorylate Tyr and Ser on GPCR
RTK can lead to multiple phosphorylations of Tyr and Ser of GPCR tail
And these multiple phosphorylations lead to internalization of GPCR.
GPCR is taken into the cytoplasm thus GPSR become non-available for signaling
RTK can inhibit GPCR signal
What is β-adrenergic receptor?
Class of G protein-coupled receptors that are targets of many neurotransmitters
How does TRK and GPCR interact?
RTK is activated and it can phosphorylate instead of multiple sites, just one Tyr on cytoplasmic domain on GPCR
This one Tyr acts as activator protein, providing a docking site for multiple proteins
This complex proteins ultimately lead to MEK and ERK -> altered gene expression
Multiple MEKs and ERKs are activated-leading to multiple gene expressions
What is the usual name of hormones that trigger RTK? What do they do?
Hormones that trigger RTK are usually called cytokines
Cytokines inhibit other ligand function
What is IRS-1 function?
IRS-1 provides a docking site for many proteins
These complex protein ultimately lead to MAPK
What is protein phosphorylation?
It’s the process in which a phosphoryl group (phosphate), donated by ATP, is transferred to an acceptor protein. The reaction is catalysed by a protein kinase
What are the 3 most common AA phosphorylated?
Serine, Threonine and Tyrosine
Why do we why say that kinases have specificity?
They only target only specific AA
Which AA is target of RTK?
Tyr
What are the 2 characteristic that all kinases have?
- ATP binding site
- Catalytic cleft
What do Raf-1, MEK, and ERK all have in common?
They are all protein kinases
How can ion gated channels be opened or closed?
they may be open or closed, by a specific ligand (a neurotransmitter, for example) or by a change in the transmembrane electrical potential, Vm.
What is the cytosolic calcium concentration normally?
It is usually kept very low
What drives Na+ and Ca2+ movement and in which direction? What effect does it have?
The chemical gradient drives Na+ and Ca2+ inward producing depolarization
What drives K+ movement and in which direction? What effect does it have?
The chemical gradient drives K+ outward producing hyperpolarization
What drives Cl- movement and in which direction? What effect does it have?
The electrical gradient drives Cl- outward, against its concentration gradient (producing depolarization).
What are the ionic movements across plasma membrane and what drives them?
Chemical gradient moves Na+ and Ca2+ inward, and K+ outward
Electrical gradient drives Cl- outward against it’s gradient
What does NaK ATPase? How?
Creates a resting membrane potential (Vm) of -50 to -70 mV by carrying 3 Na+ out of the cell for every 2 K+ carried in making the inside negative relative to the outside. As both of the ions go against their gradient, their movement requires ATP
What dictates the random flow of ions through the membrane via ion channels?
Electrochemical gradient potential across the membrane
Is [Na+] and [K+] higher on the outside or inside?
[Na+] is higher on the outside
[K+] is higher on the inside
Flow of each ion and in which direction can alter the cytosolic concentration of it? Why
As the intracellular concentration of Ca2+ is generally very low (∼10^7M), inward flow of Ca2+ can significantly alter the cytosolic [Ca2+].
Is Ca2+ a second messenger?
Yes, as it is not a protein and has a very low concentration
What is a nicotinic acetylcholine receptor?
This receptor is found in the postsynaptic membrane of neurons and opens in response to the neurotransmitter acetylcholine
Give 2 examples of excitable cells
Neurons
Muscles
What are excitable cells + example
Thus, excitable cells are any cells that secrete hormones e.g hormone insulin that are produced by beta cells in the pancreas
Which side of membrane is positive and which is negative?
Outside is positive
Inside is negative
What is the value of resting membrane potential?
-70 to -50 mV
Name and describe 3 states of membrane potential
- Polarized- When cell is in resting membrane potential - polar membrane
- Depolarized- When is a bit more positive compared to resting membrane potential e.g when sodium or calcium goes from outside to inside, or calcium goes from outside to inside
- Hyperpolarized- when membrane becomes more negative than the resting membrane potential e.g. when chloride goes from inside to outside, the negative charge goes to outside, which might create further negative voltage difference
What are the other ion transporters that can be found in the membrane?
Sodium, chloride and potassium channels
What do sodium, chloride, potassium channels do?
When they are open, they allow ions to move down the electrochemical gradient
Why does chloride move against the electrochemical gradient?
The amount of force created by electrical differential takes over the amount of force created by chemical difference, thus chloride comes from inside to outside, down the electrical gradient (negative chloride moves from negative inside to positive outside)
Describe the process of propagation of neural signal
There are sodium gated ion channels along the axon which are gated by voltage difference
If there is any change in the voltage, they open
When this occurs, sodium goes in from outside to inside which creates less negativity/more positivity on the inside. Thus, wherever these channels are, the membrane becomes depolarized
When this happens, potassium channels open up and potassium leaves due to chemical gradient
This repolarizes the membrane
Opening of potassium channels is slightly delayed compared to sodium channels
This creates the direction for action potential propagation
Once sodium channels open, the membrane becomes depolarized, this is followed by repolarized by opening of potassium channels. However, by then there’s a slight difference in the action potential which opens sodium channels next to that-> Action potential keeps going forward
Describe how is the signal passed from one neuron to another one
This is achieved by voltage gated calcium channels
They are opened by changes in membrane potential and allow calcium to move from outside to inside.
Calcium comes in and due to normally very low concentration, it not only causes depolarization, but also acts as a second messenger
Secretory granule that contain neurotransmitters come to the membrane and are exocytosed into the synaptic cleft in response to calcium concentration changes
Across the synaptic cleft there are receptors for neurotransmitters which are gated ion channels that are gated by a ligand- e.g. acetylcholine
Acetylcholine channels allow cations to go from outside to inside like sodium or calcium
This movement of positive ions from outside to inside depolarizes the membrane and begins the propagation of signal
Is there more calcium on the inside or outside?
Normally there are more calcium on the outside
Where does calcium act as a second messenger
- IP3
- Signal transduction in neurons
Neurotransmitters are ___ substances
Neurotransmitters are hormone substance
How many acetylcholine molecules bind to acetylcholine receptor?
2
What does binding of acetylcholine to its receptor do?
It induces a conformational change in acetylcholine receptor which opens the channel
This allows for influx of ions from outside to inside
Which ions (+ or -) move through acetylcholine receptor
Cations (+ive)
What is the purpose of the intrinsic ability of gated ion channels to close?
Allows for a short window of time during which movement of ions occurs
Name cation gated receptors
Serotonin, acetylcholine, glutamate receptors
Name anion gated receptor
Glycine
Which receptors are not membrane bound? Where are they found
Nuclear receptors
Mostly found in the nucleus
5-10% of them can be in the plasma membrane
Describe ligands of nuclear receptors
They are lipid soluble as they have to be able to move across the cell membrane
Have to be transported by a protein in aqueous environments
Steps of nuclear receptors
- Ligands (usually hormones) carried by serum binding proteins diffuse across the plasma membrane and bind to its specific receptor in the nucleus
- Ligand binding changes conformation of the receptor;; it forms homo- or heterodimers with the other hormone-receptor complexes and binds specific regulatory regions-HREs in the DNA adjacent to specific genes
- Receptor attracts coactivator or corepressor protein(s) and, with them, regulates transcription of the adjacent gene(s), increasign or decreasign the rate of mRNA formation
- Altered levels of the hormone-regulated gene produce the cellular response to the hormone
Describe the 2 types of possible dimers formed by nuclear receptors
Homodimerization- 2 receptors of the same class come together and dimerize Heterodimer- 2 different types of nuclear receptors that are activated by 2 different ligands come together.
Which substances can use nuclear receptors to regulate gene expression?
Steroid and thyroid hormones, retinoids, and vitamin D
Nuclear receptors act as ___ factors
Nuclear receptors act as transcription factors
How fast is the signal transduction by nuclear receptors?
Slow
Are steroid receptors cytoplasmic or nuclear?
Both
What are the 3 essential components that define signal transduction through GPCR?
- a plasma membrane receptor with seven transmembrane helical segments,
- an enzyme in the plasma membrane that generates
an intracellular second messenger, - a guanosine nucleotide–binding protein (G protein)
What does the enzyme, bound by G-alpha generate?
Second messenger
Define agonist
Agonists are structural analogs that bind to a receptor and mimic the effects of its natural ligand
Define antagonist
antagonists are analogs that bind without triggering the normal effect and thereby block the effects of agonist
What does S stand for in G coupled proteins?
S stands for stimulatory
What are the G proteins that G receptor is made up of?
G proteins are composed of Gs alpha, Gs beta and Gs gamma
The interaction between Gsa and adenylyl cyclase is possible only when Gsa i bound to ___
The interaction between Gsa and adenylyl cyclase is possible only when Gsa i bound to GTP
Which residues does PKA phosphorylate?
Ser or Thr
Describe amplification in G proteins
First, the binding of one hormone molecule to one receptor catalytically activates several Gs molecules. Next, by activating a molecule of adenylyl cyclase, each active Gs? molecule stimulates the catalytic synthesis of many molecules of cAMP. The second messenger cAMP now activates PKA, each molecule of which catalyzes the phosphorylation of many molecules of the target protein
Define adaptor protein
non-catalytic proteins that hold together other protein molecules that function together
What is phospholipase C specific for?
PIP2
What is the long name for IP3
Inositol 1,4,5-triphosphate
What are Raf-1, MEK and ERK
Protein kinases
Ion fluxes are __ in contrast to _ transport by the Na+K+ ATPase
Ion fluxes are passive in contrast to active transport by the Na+K+ ATPase
