Cell Signaling Flashcards
signal transduction definition
the process whereby one type of signal is converted into another
comparison:
Ie. a mobile phone receives a radio signal which is converted to a sound signal
Ie. individual cells need to sense and respond to their environment
name 3 signal transduction pathways
- epinephrine
- insulin
- epidermal growth factor (EGF)
epinephrine signal transduction pathway
epinephrine + beta-adrenergic receptor –> energy-story mobilization
insulin signal transduction pathway
insuline + insuline receptor –> increases glucose uptake
epidermal growth factor (EGF) signal transduction pathway
EGF + EGF receptor –> expression of growth-promoting genes
what are the 4 general principles of signal transduction?
Between cells, the signaling cell produces a particular type of extracellular signal molecule that is detected by the target cell
Most animal cells BOTH send and receive signals; can act as both signaling cells and target cells
Target cells possess RECEPTORS: proteins that recognize and respond to the signal molecule
Signal transduction begins when the receptor on a target cell receives an incoming extracellular signal and then produces intracellular signaling molecules that alter cell behavior
receptors (aka receptor proteins) definition
proteins that recognize extracellular signal molecules and relay the signal inside the cell
they are specific to the signaling molecules and are categorized as cell-surface receptors and intracellular receptors
what are the steps of signal transduction (4)
signal –> reception –> amplification/transduction –> response
- An environmental signal is received by interaction with a cellular component (most often a cell-surface receptor)
- The information that the signal has arrived is converted into other chemical forms or transduced
- The signal is often amplified before evoking a response
- Feedback pathways regular the entire signaling process
name 4 examples of types of signal molecules
hormones
local mediators
neurotransmitters
contact-dependent signal molecules
what are 6 examples of hormones?
epinephrine (adrenaline)
cortisol
estradiol
insulin
testosterone
thyroid hormone (thyroxine)
what are 5 examples of local mediators?
EGF
platelet-derived growth factor
nerve growth factor (NGF)
histamine
nitric oxide (NO)
what are 2 examples of nuerotransmitters?
acetylcholine
y-Aminobutyric acid (GABA)
what is 1 example of contact-dependent signal molecule?
delta
hormones definition
local mediators definition
neurotransmitters definition
contact-dependent signal molecules definition
type of signal molecule, sit of origin, chemical nature, and action/function of epinephrine
type: hormone
site of origin: adrenal gland
chemical nature: derivative of the amino acid tyrosine
some actions: increases blood pressure, heart rate, and metabolism
type of signal molecule, sit of origin, chemical nature, and action/function of insulin
type: hormone
site of origin: adrenal gland
chemical nature: steroid (derivative of cholesterol)
actions: affects metabolism of proteins, carbohydrates, and lipids in most tissues
type of signal molecule, sit of origin, chemical nature, and action/function of epidermal growth factor (EGF)
type: hormones
site of origin: various cells
chemical nature: protein
actions: stimulates epidermal and many other cell types to proliferate
type of signal molecule, sit of origin, chemical nature, and action/function of acetylcholine
type: neurotransmitters
site of origin: nerve terminals
chemical nature: derivative of choline
actions: excitatory neurotransmitter at many nerve-muscle synapses and in central nervous system
what are the 2 major classes of extracellular signal molecules?
cell surface receptors
intracellular receptors
cell surface receptors definition
proteins located on the PLASMA MEMBRANE of a cell that bind to signaling molecules (ligands) such as hormones, neurotransmitters, or growth factors.
These receptors facilitate the transmission of signals from the extracellular environment into the cell, often triggering intracellular signaling pathways that lead to a specific cellular response.
intracellular receptors
proteins found WITHIN the CYTOPLASM or NUCLEUS of a cell that bind to small, hydrophobic signaling molecules (such as steroid hormones or certain gases).
These receptors typically act as transcription factors, influencing gene expression and cellular processes by directly interacting with DNA or other intracellular targets.
what type of molecules are cell surface receptors for?
Molecules that are too large or too hydrophilic
Cannot cross the plasma membrane of the target cells → needs to rely on receptors on the surface of the target cell to relay message across the plasma membrane
what type of molecules are intracellular receptors for?
Molecules that are small enough or hydrophobic enough
Pass through the plasma membrane and into the cytosol of the target cell, where they can bind to intracellular receptor proteins
what is an example of a signal molecule that can induce different responses in different target cells?
acetylcholine (neurotransmitter that binds to similar receptors of different target cells and induce difference responses)
give 3 examples of acetylcholine binding to different types of cells and their effects
Acetylcholine binding to heart pacemaker cell → decreased rate of firing
Acetylcholine binding to salivary gland cell → secretion of serous saliva
Acetylcholine binding to skeletal muscle cell → contraction of muscle
Cells may require multiple signals… (3)
to survive
to grow
to differentiate
Extracellular signals work in combinations to regulate ___
Extracellular signals work in combinations to regulate cellular behavior
If deprived of the necessary survival signals, what happens to most cells?
If deprived of the necessary survival signals, most cells undergo apoptosis
Extracellular signals can act ___ or ___
Extracellular signals can act rapidly or slowly
what types of cellular responses/processes (resulting from extracellular signals) occurs more quickly?
processes that do not involve changes in gene expression
ie.
Changes in cell movement
secretion
metabolism
(seconds to minutes)
what types of cellular responses/processes (resulting from extracellular signals) occurs more slowly?
processes that involve changes in gene expression and new protein synthesis
ie.
cell differentiation
increased cell growth and division
(minutes to hours)
Extracellular signal molecules definition
the ligands that INITIATE cellular signaling by binding to receptors
how can extracellular signal molecules (aka ligands) be categorized?
chemical nature: determines how the ligands interact with their receptors and how they are transported in the body
distance of action: how far the ligand travels to reach their target cells
describe the chemical nature of extracellular signal molecules
chemical nature: determines how the ligands interact with their receptors and how they are transported in the body
main categories:
- small molecules (ie. amino acids, nucleotides)
- steroid hormones (ie. cortisol, estrogen, testosterone)
- lipids
describe the distance of action for extracellular signal molecules
distance of action: how far the ligand travels to reach their target cells
types of distances:
- endocrine signaling
- paracrine signaling
endocrine signaling
the ligand (usually a HORMONE) is secreted into the bloodstream and travels over LONG distances to reach the target cells in DISTANT parts of the body
ie. insulin, thyroid hormones
paracrine signaling
paracrine signals act on NEARBY target cells within the same tissue or local area; the ligands are usually secreted into the extracellular fluid and diffuse over SHORT distances to bind to receptors on neighboring cells
ie. EGF, NO
effector proteins definition
Effector proteins: the targets of signaling pathways that execute the cellular response
- can be categorized by their functions in the cell and they are often specific to the signaling pathway that is being activated
different types: metabolic enzymes, cytoskeletal proteins
pathway-specific examples: G protein coupled receptors pathway (GPCR), receptor tyrosine kinase pathway (RTK), PI3K pathway
intracellular signaling molecules (aka second messenger molecules) definition
the small INTRACELLULAR signaling molecules generated by target enzymes that relay signals from the cell surface receptors to intracellular targets to help propagate and amplify the signal within the cell; they are produced/released in response to the activation of cell surface receptors
relay, amplify, integrate, distribute, and modulate via feedback an incoming signal
Many of the steps can be modulated via ___ by other molecules or events in the cell
Many of the steps can be modulated via feedback by other molecules or events in the cell
how does feedback regulation affect an extracellular signal?
Feedback regulation within an intracellular signaling pathways can adjust the response to an extracellular signal
Helps adjust the strength, duration, and outcome of a cellular response to extracellular signals and can respond appropriately to changes in the environment or in the signaling conditions
what are the 2 main types of feedback regulation
positive feedback loops
negative feedback loops
positive feedback loops definition
amplifies the signaling response and can lead to a stronger or prolonged response
- the end product of the pathway or a downstream component STIMULATES an earlier step in the pathway
ie. activation of the proteins that trigger cell division
negative feedback loops definition
dampens or limits the cellular response to avoid overactivation or prolonged signaling (serves as a way to restore homeostasis and more common than positive feedback)
- when signaling pathway is activated and the cellular response reaches a certain threshold, downstream signaling proteins or transcription factors often trigger the production of molecules that inhibit upstream components of the pathway
ie. receptor tyrosine kinase RTK pathway, Mitogen activated protein kinase (MAPK) pathway
what are the two classes of intracellular signaling proteins?
phosphorylation vs dephosphorylation
GTP vs GDP
many intracellular signaling proteins act as what?
Many intracellular signaling proteins act as molecular SWITCHES
molecular switches
proteins that “switch” between active and inactive states in response to specific signals
- play a critical role in intracellular signaling
- regulate downstream cellular processes by altering the activity, confirmation, or interaction of proteins within signaling pathways
describe the phosphorylation/dephosphorylation of intracellular signaling proteins
phosphorylation/dephosphorylation: The addition or removal of phosphate groups that alter the protein’s conformation, activity, or interaction with other molecules; REVERSIBLE and regulated process
phosphorylation often ACTIVATES the protein
dephosphorylation often INACTIVATES the protein
protein kinase definition
enzyme that catalyzes the ADDITION of a phosphate group onto the intracellular signaling protein
protein phosphatase
enzyme that catalyzes the REMOVAL of the phosphate group from the intracellular signaling protein
describe the GTP/GDP of intracellular signaling proteins
GTP/GDP switches depend on the binding and hydrolysis of guanine nucleotides GTP or GDP
GTP-binding protein: protein adopts an ACTIVE conformation and can interact with downstream effectors to propagate a signal
GDP-binding protein: protein becomes INACTIVE and dissociates from effectors
how do you classify protein kinases?
based on the amino acid sequence of their CATALYTIC domains
what are 3 types of protein kinases?
serine/threonine kinases
tyrosine kinases
dual-specificity kinases
serine/threonine kinases (include examples)
enzymes that phosphorylate the OH group of serine or threonine residues in proteins
PKA (protein kinase A): activated by cAMP → regulates metabolism and gene transcription
PKC: activated by diacylglycerol (DAG) and calcium ions → regulates cell growth, differentiation, and apoptosis
AKT (PKB): activated in the PI3K/AKT signaling pathway → key regulator of cell survival, growth, and metabolism
tyrosine kinases (and 2 subgroups)
enzymes that phosphorylate tyrosine residues in proteins
subgroups:
1. nonreceptor tyrosine kinases (aka cytoplasmic tyrosine kinases)
2. receptor tyrosine kinases (aka transmembrane receptor-linked tyrosine kinases)
nonreceptor tyrosine kinases (aka cytoplasmic tyrosine kinases)
kinases that do NOT have a membrane spanning domain and instead are located in the CYTOPLASM
Receptor tyrosine kinase (aka transmembrane receptor-linked tyrosine kinases)
transmembrane proteins that have a kinase domain in the intracellular portion of the receptor → when ligands (ie. growth factors) bind to the extracellular domain, the receptor undergoes autophosphorylation of tyrosine residues in the cytoplasmic domain
Ie. insulin receptor, EGF receptor
Dual-specificity kinases
enzymes that act as BOTH tyrosine kinase AND ser/threonine kinase –> aka phosphorylates serine, threonine, or tyrosine residues in proteins
Ie. MEK (MAPKK - MAP kinase kinase): a key component in the MAPK signaling pathway which regulates cell division, differentiation, and stress response
what are the 2 main types of GTP binding proteins?
Small, monomeric GTPases
Large, trimeric GTP-binding protein (aka G proteins)
small monomeric GTPases
single subunit proteins that bind and hydrolyze GTP to GDP (aka act as molecular switches toggling between GTP-bound on state and GDP-bound off state)
- the transition between the 2 states is regulated by 2 families of proteins:
1. guanine nucleotide exchange factors (GEFs) which promote the exchange of GDP for GTP
2. GTPase-activating proteins (GAPs) which promote the hydrolysis of GTP to GDP
ie. Ras protein: involved in controlling cell proliferation, differentiation, and survival
ie. Rho, Rac, Cdc42: regulate the cytoskeleton and affect cell migration, shape, and division
large trimeric GTP binding proteins (G proteins)
G proteins consist of 3 subunits: alpha, beta, gamma
relay messages from G protein coupled receptors (aka these proteins mediate the signaling pathways initiated by G protein coupled receptors GPCRs) –> when a ligand binds to a GPCR, the G protein is activated and leads to a cascade of intracellular events
what are the 2 protein families that regulate monomeric GTPases GTP-bound “on” and “GDP-bound off” states?
Guanine nucleotide exchange factors (GEFs)
GTPase-activating proteins (GAPs)
Guanine nucleotide exchange factors (GEFs) function
promotes the exchange of GDP for GTP, switching the protein ON –> activates monomeric GTPases to trigger downstream signaling processes
GTPase-activating proteins (GAP) function
stimulate the hydrolysis of GTP to GDP, thus switching the protein OFF –> deactivates the monomeric GTPases and terminates the signaling and prevents prolonged activation
give 7 examples of foreign substances that act as cell-surface receptors
- barbiturates and benzodiazepines (valium and ambien)
- nicotine
- morphine and heroin
- curare
- strychnine
- capsaicin
- menthol
what are the 3 main classes of cell-surface receptors?
Ion-channel-coupled receptors
G-protein coupled receptors
Enzyme-coupled receptors
Ion-channel-coupled receptors (aka transmitter-gated ion channels)
type of cell surface receptors that rapidly transmits signals across synapses in the nervous system
These receptors are involved in converting chemical signals (neurotransmitters) into electrical signals by regulating the flow of ions across the plasma membrane → play crucial role in synaptic transmission, muscle contraction, neuron firing, sensory perception
ie. action potential with neurotransmitters: a neurotransmitter binds to ion-channel-coupled receptors on the surface of a target cell –> the receptor alters its conformation to open a channel in the target membrane → specific ions (ie. Na+, K+, Ca+) are permeable to the channel –> changes membrane potential –> generates an action potential that is passed onto the next cell
G-protein coupled receptors (GPCRs); aka seven-transmembrane-helix 7TM receptors
GPCRs form the LARGEST class of cell surface receptors: more than 700 GPCRs in humans; 1000 involved in smell alone in mice
Diverse extracellular signal molecules bind GPCRs
Called G-protein-coupled receptors because they signal through G proteins (guanine nucleotide-binding proteins)
GPCRs are transmembrane proteins
what type of proteins are GPCRs?
transmembrane proteins
describe the structure of GPCRs
All GPCRs have a SIMILAR STRUCTURE:
Each is made of a single polypeptide chain that treads back and forth across the lipid bilayer 7 times (hence the name)
Because 7TM receptors are always associated with G proteins, they are often called G-protein-coupled receptors
what are 13 biological functions mediated by 7TM (GPCR) receptors?
hormone action
hormone secretion
neurotransmission
chemotaxis
exocytosis
control of blood pressure
embryogenesis
cell growth and differentiation
development
smell
taste
vision
viral infection
GPCR superfamily definition and 2 examples
GPCR superfamily: a group of cell surface receptors that are involved in transmitting extracellular signals to the inside of the cell through interactions with G proteins and various intracellular signaling pathways
examples
1. rhodopsin: a light-activated photoreceptor protein in vertebrates’ eyes –> responsible for the initial step in the visual signaling pathway that allows organisms to detect light
- binds to light/photons and undergoes a conformational change that activates a G protein called transducin –> triggers a cascade of intracellular events, ultimately leading to an electrical signal being generated and sent to the brain for visual processing
- Olfactory (smell) receptors in the vertebrate nose:
Responsible for detected odorants (smell molecules) in the environment and are located in the olfactory epithelium of the vertebrate nose
- An odorant binds to an olfactory receptor → receptor undergoes conformational change that activates an associated G protein → leads to a signaling cascade, ultimately resulting in an electrical signal generated that the brain interprets as a smell
beta2-adrenergic receptor
Activated by the binding of epinephrine or norepinephrine and involved in the fight-or-flight response
what are 4 similarities in the overall structures of rhodopsin and the beta2-adrenergic receptor?
Both are members of the G protein coupled receptor superfamily (GPCR) and share a similar overall structural framework despite having distinct roles and different ligand binding sites
- Both possess 7 transmembrane alpha-helices that span the plasma membrane and form a hydrophobic core than anchors the receptor in the lipid bilayer of the membrane → the arrangement of these 7 helices creates a binding pocket for ligands to interact with the receptor
- Both have extracellular loops that interact with the ligand (recognition and binding) and intracellular loops that intact with G proteins (to propagate the signal into the cell)
- For both, ligand binding induces a conformational change in the receptor’s structure
- Both share highly conserved amino acid sequences within the transmembrane regions and intracellular loops
describe the general structure for G proteins (subunits)
Composed of 3 protein subunits: alpha, beta, gamma
describe the Galpha subunit
Alpha subunit:
- largest subunit oo the G protein
- has the ability to bind to GTP or GDP
- Ga bound to GDP = INACTIVE state
- Ga bound to GTP = ACTIVE state
- Responsible for interacting with and activating downstream effectors
describe the Gbeta and gammas subunit
- Together these 2 subunits form the stable complex that helps anchor the G protein to the cell membrane and also play a role in signaling by modulating the activity of certain effectors in the cell
- When Ga is in its INACTIVE state, it interacts with the Gbeta/gamma dimer to keep the G protein inactive
→ prevents Ga from interacting with downstream effects - The binding of GTP to Ga triggers a conformational change in the Ga subunit
→ Ga subunit dissociates from the Gbeta/gamma dimer, which allows both the Ga-GTP and Gbeta/gamma complexes to interact with and activate various downstream signaling pathways
When extracellular signal molecule binds to its receptor (in this case GPCR), which 2 things happen?
- The receptor protein undergoes conformational change AND…
- Activates G protein: the alpha subunit affinity for GDP is reduced → the alpha subunit binds to GTP instead –> alpha subunit detaches from the beta/gamma complex, which is also activated
what are the 3 steps for inactivation of G protein?
- Activation of a target protein by the activated alpha subunit
- HYDROLYSIS of GTP by the alpha subunit inactivates this subunit and causes it to dissociate from the target protein
- Inactive alpha subunit reassembles with the beta-gamma complex to reform an inactive G protein
The G protein alpha subunit controls the amount of time that the G protein stays active for
The hydrolysis of GTP inactivates the alpha subunit
The alpha subunit has an intrinsic GTPase activity → hydrolyzes its bound GTP to GDP
The whole G protein returns to an inactive G protein
the alpha subunit has an intrinsic ___ that allows it to ___
The alpha subunit has an intrinsic GTPase activity → hydrolyzes its bound GTP to GDP
describe step 1. Activation of a Target Protein by the Activated Gα Subunit
when a G protein coupled receptor GPCR binds to the alpha subunit, the Ga exchanges GDP for GTP to become active –> this causes a conformational change in Ga that causes it to dissociate from the Gβγ dimer
The activates Ga-GTP subunit can now interact with specific effector proteins (ie. adenylyl cyclase which produces cAMP from ATP, phospholipase C which generates 2nd messengers like IP3 and DAG, and ion channels or other enzymes)
these effectors then initiate intracellular signaling cascades –> leads to cellular responses (ie. changes in metabolism, gene expression, ion flow)
describe step 2. Hydrolysis of GTP by the Gα Subunit
the Ga subunit has an intrinsic GTPase activity (aka it can HYDROLYZE bound GTP into GDP and inorganic phosphate Pi)
–> this hydrolysis reaction is slow so it is accelerated by GTPase-activating proteins (GAPs) which bind to Ga and enhance its GTPase activity
THIS HYDROLYSIS REACTION MAKES THE ALPHA SUBUNIT INACTIVE AGAIN
after GTP is hydrolyzed to GDP, the conformation of Ga changes back to its inactive form which reduces its affinity for target effector proteins –> Ga DISSOCIATES FROM THE TARGET PROTEIN
describe step 3. Reassembly of the Inactive G Protein
the inactive Ga-GDP subunit now has a high affinity for the Gbeta/gamma complex and reassociates with it
–> the reformation of heterotrimeric G protein (aka Ga-GDP + Gbeta/gamma) restores the G protein to its INACTIVE STATE –> now ready to respond to another signal from an activated GPCR
many G proteins activate what enzymes that produce what?
Many G proteins activate membrane-bound enzymes that produce small messenger molecules
the 2 most frequent target enzymes that different types of G proteins activate are what?
- adenylyl cyclase (aka adenylate cyclase, adenyl cyclase)
- phospholipase C
what is adenylyl cyclase and what does it generate?
A membrane bound enzyme/protein with 2 intracellular domains that converts ATP into cAMP (aka generates cAMP)
A key 2nd messenger in math signaling pathways
The 2 intracellular domains: C1 (functions with C2 domain to form the active site for ATP conversion to cAMP) & C2 (works with C1 to catalyze the cyclization of ATP to cAMP)
Activation: adenylate cyclase is activated by the Gas subunit of a G protein which binds to the intracellular domains and induces a conformational change to facilitates catalytic activity
Inhibition: can be inhibited by Gαi proteins or by specific regulatory proteins (PKA-mediated phosphorylation)
what is phospholipase C and what does it generate?
A membrane bound enzyme that generates small molecules inositol triphosphate IP3 and diacylglycerol DAG (which are second messengers)
first messengers definition
the EXTRACELLULAR signals that activated the target enzymes in the first place
name 5 common second messengers
cAMP, cGMP, calcium ion, IP3, DAG
what synthesizes cyclic AMP (cAMP)
adenylyl cyclase
what are the steps of the epinephrine signaling pathway?
- Epinephrine binds to the beta-adrenergic receptor
- The beta-adrenergic receptor is activated
- The activated beta-adrenergic receptor activates adenylate cyclase
- Adenylate cyclase produces cAMP
- Undergoes the epinephrine signaling pathway (ie. stimulates glycogen breakdown in skeletal muscle cells
- binding of epinephrine: Epinephrine binds to the beta-adrenergic receptor located on the plasma membrane of target cells –> binding induces a conformational change in the receptor and activates it
- activation of G protein: The activated beta-adrenergic receptor acts as a guanine nucleotide exchange factor (GEF) and interacts with a heterotrimeric G protein –> causing the alpha subunit (Gαs) of the G protein to exchange GDP for GTP –> The activated Gαs-GTP dissociates from the β and γ subunits (Gβγ)
- activation of adenylyl cyclase: The Gαs-GTP subunit binds to and activates adenylyl cyclase
- production of cAMP: Adenylyl cyclase catalyzes the conversion of ATP to cAMP –> cAMP levels rapidly increase in the cytosol
- Activation of Protein Kinase A (PKA): cAMP binds to the regulatory subunits of protein kinase A (PKA), causing them to dissociate from the catalytic subunits –> catalytic subunits of PKA are now active
- phosphorylation of Target Proteins: activated PKA phosphorylates phosphorylase kinase which activates glycogen phosphorylase which catalyzes the breakdown of glycogen –> glucose-1-phosphate –> glucose
- cellular response: the breakdown of glycogen provides rapid energy for muscle contraction during fight or flight response
- signal termination: Gαs hydrolyzes GTP to GDP using its intrinsic GTPase activity enhances by GAPs; cAMP is degraded to AMP by phosphodiesterase (PDE); beta-adrenergic receptor is desensitized through phosphorylation by GPCR kinases (GRKs) and binding of arrestin
beta-adrenergic receptor
a G-protein-coupled receptor (GPCR) that mediates the cellular response to epinephrine (adrenaline)
When epinephrine binds to this receptor, it triggers a signaling cascade that leads to the breakdown of glycogen in skeletal muscle cells, providing energy for rapid physical activity (plays a role in fight-or-flight response)
heterotrimer
a macromolecule made up of three subunits, at least one of which is different from the other two
what is Gas?
Gas is the alpha subunit of the G protein responsible for activating adenylyl cyclase (Gas is the GTP form of GDP)
relationship between cAMP and protein kinase A (PKA)?
cAMP stimulates the phosphorylation of many target proteins by activating protein kinase A (PKA) `
protein kinase A (PKA)
a cAMP dependent protein kinase (aka it is activated by second messenger cAMP) whose primary function is to PHOSPHORYLATE TARGET PROTEINS, and thus altering their activity → leads to different cellular effects (ie. metabolic regulation, gene expression, ion channel regulation, cell growth and differentiation)
Activation: cAMP levels in the cell increase due to the activation of adenylyl cyclase by Gas → cAMP binds to the regulatory subunits of PKA and each regulatory subunit binds to cAMP and causes a conformational change → the regulatory subunits dissociate from the catalytic subunits → the free catalytic subunits are now active and can phosphorylate target proteins
name 4 examples of cell responses mediated by cAMP (include the extracellular signal molecules, target tissue, and major response involved)
- extracellular signal molecule: epinephrine –> target tissue: heart –> major response: increase in heart rate and force of contraction
- extracellular signal molecule: epinephrine –> target tissue: skeletal muscle –> glycogen breakdown
- extracellular signal molecule: epinephrine, glucagon –> target tissue: fat –> major response: fat breakdown
- extracellular signal molecule: adrenocorticotropic hormone (ACTH) –> target tissue: adrenal gland –> cortisol secretion
- although all these examples of signal molecules above are hormones, some responses to local mediators and neurotransmitters are also mediated by cAMP
how does the cAMP signaling pathway turn on genes?
- A rise in intracellular cAMP activates PKA
- The activated PKA enters the nucleus and phosphorylates specific transcription regulators
- Once transcription regulators are phosphorylated, these proteins stimulate the transcription of a whole set of target genes
transcription regulators definition
proteins that control the expression of specific genes by binding to DNA sequences in the promoter or enhancer regions of a gene
→ play a key role in turning genes “on” or “off” and influence cellular functions
How is the signal initiated by epinephrine switched off?
Galpha subunit has an intrinsic GTPase activity that HYDROLYZES bound GTP to GDP and Pi
This hydrolysis of GTP → GDP + Pi is SLOW but is made quicker with the help of GTPase-activating proteins (GAPs)
–> as a result, activation of adenylate cyclase is terminated
GTPase
the enzyme that hydrolyzes GTP to GDP and inorganic phosphate
GTP –> GDP + Pi
name 4 examples of cell responses mediated by phospholipase C (include signal molecule, target tissue, and major response)
- signal molecule: vasopressin (a peptide hormone) –> target tissue: liver –> major response: glycogen breakdown
- signal molecule: acetylcholine –> target tissue: pancreas –> major response: secretion of amylase (a digestive enzyme)
- signal molecule: acetylcholine –> target tissue: skeletal muscle –> major response: muscle contraction
- signal molecule: thrombin (a proteolytic enzyme) –> target tissue: blood platelets –> major response: aggregation
what are the steps for the IP3 and Calcium signaling pathway?
- Activation of GPCR → Activation of Gq protein.
- Gq protein activation → Activation of phospholipase C (PLC).
- PLC cleaves PIP2 → Produces IP3 and DAG.
- IP3 diffuses to the ER, leading to Ca²⁺ release into the cytosol.
- DAG remains in the plasma membrane and, together with Ca²⁺, activates PKC.
- PKC activation → Phosphorylation of target proteins, leading to cellular responses
protein kinase C
PKCs are serine/threonine kinases ⇒ aka they PHOSPHORYLATES serine or threonine residues in target proteins, altering the target proteins’ activity, location or interaction → affects cellular behavior
describe IP3 and its function
inositol 1,4,5-triphosphate: a WATER-SOLUBLE molecule consisting of an inositol ring with three phosphate groups attached at positions 1, 4, and 5
–> Since it is water-soluble ⇒ hydrophilic, it diffuses away from the membrane and goes to the endoplasmic reticulum (ER)
Function: releases Ca2+ ions from the ER
IP3 binds to IP3 receptors of ligand-gated calcium channels on the ER membrane → these channels open and allow Ca2+ to flow from the ER into the cytosol → increase in Ca2+ concentration in the cytosol which is a crucial signal that can trigger various cellular processes
Ca2+ also serves as another 2nd messenger than can activate various calcium-dependent proteins and enzymes (ie. calmodulin, calcium-dependent kinases)
describe DAG and its function
diacylglycerol: a HYDROPHOBIC molecule composed of a glycerol backbone bound to two fatty acid chains
–> Since DAG is hydrophobic, it stays embedded in the plasma membrane
Function: activation of protein kinase C (PKC)
DAG works together with the released Ca2+ from the ER to activate protein kinase C: when DAG binds to PKC and CA2+ levels rise in the cytosol, PKC is activated at the plasma membrane → activated PKC phosphorylates serine and threonine residues in various target proteins, changing their activity, localization, and interactions
→ PKC regulates many cellular processes (ie. cell growth, differentiation, apoptosis, immune response)
how are IP3 and DAG generated?
Both generated through the cleavage of PIP2 (phosphatidylinositol 4,5-bisphosphate) by the enzyme phospholipase C
which G protein is involved in the activation of phospholipase C?
Gq
–> Both the alpha subunit and the beta-gamma complex of the G protein Gq are involved in activating phospholipase C
what are 2 properties of calcium ion that allow it to be widely uses as an intracellular (second) messenger?
The concentration of free Ca2+ in the cytosol of an unstimulated cell is kept low: 10-7M
→ thus transient increases in Ca2+ content produced by signaling events can be readily detected by the cell
Ca2+ binds tightly to proteins and induces STRUCTURAL conformational changes in proteins
How to detect changes in Ca2+ concentrations inside cells (aka intracellular Ca2+ concentrations)?
use special dyes that bind to Ca 2+
what is an example of a special dye used that binds to Ca2+ to measure its concentration inside the cell?
Fura-2 AM
Fura-2 AM (Fura-2-acetoxymethyl ester) definition and relation to Ca2+
Fura-2-acetoxymethyl ester (fura-2 AM): a MEMBRANE-PERMEABLE, non-invasive derivative of the radiometric calcium indicator fura-2 → a calcium sensitive fluorescent radiometric dye
- Fura-2 AM crosses the cell membrane freely because of the addition of the AM (acetoxymethyl ester) group which makes it lipophilic (fat-soluble)
- Once inside the cell, the intracellular esterases cleave the AM group, leaving the activated form Fura-2 –> Fura-2 is the form one that binds to Ca²⁺ and undergoes fluorescence changes
–> Fura-2 molecule exhibits different fluorescence characteristics depending on whether it is bound to Ca²⁺ or not → this difference allows the measurement of intracellular calcium concentrations through fluorescence intensity ratios
When Ca²⁺ binds to fura-2, the molecule undergoes a change in its excitation and emission spectra.
- Ca2+ bound Fura-2 AM & Ca2+ free Fura-2 AM
Ca2+ bound Fura-2 AM: excitation maximum, typical excitation wavelength, and emission maximum
excitation maximum: 335 nm
typical excitation wavelengths used: 340 nm
emission maximum: ~510 nm
Ca2+ free Fura-2 AM: excitation maximum, typical excitation wavelength, and emission maximum
excitation maximum: 363 nm
typical excitation wavelengths used: 380 nm
emission maximum: ~510 nm
explain the ratiometric measurements for Fura-2 AM dye
Ratiometric measurement can provide a quantitative assessment of intracellular calcium levels based on the ratio of fluorescence intensities at two different excitation wavelengths: bound = 340nm; free = 380nm
–> The fluorescence emitted at 510 nm (the emission maximum) is measured at both of these wavelengths → compare the ratio of fluorescence intensity at these 2 wavelengths to determine the intracellular calcium concentration
⇒ the ratio is directly related to the amount of Ca²⁺ present inside the cell
- A higher ratio indicates a higher Ca²⁺ concentration inside the cell, and a lower ratio indicates lower calcium levels
**The ratios 510 nm/340 nm and 510 nm/380 nm are directly related to the amount of intracellular Ca2+
The effects of Ca2+ in the cytosol are ___ by interacting with Ca2+ - responsive proteins (give an example of a Ca2+ responsive protein)
The effects of Ca2+ in the cytosol are indirect by interacting with Ca2+ - responsive proteins
calmodulin
calmodulin (number of Ca2+ binding sites): name 4 things about it
- A 17KDa protein with 4 Ca2+ binding sites: 2 Ca2+ binding sites (aka EF hand motifs) in each of the globular ends of the protein
- Serves as a Ca2+ sensor in nearly all eukaryotic cells
^^ because calmodulin DIRECTLY responds to changes in intracellular Ca2+ concentrations - When the cytoplasmic Ca2+ level is raised above 500 nM, the binding of Ca2+ activates CaM
– Ca2+ ions bind to the EF hand domains in calmodulin → causes conformational change that activates the protein and allows it to interact with its target proteins (ie. protein kinases, phosphatases) - CaM a member of the EF hand protein family
EF hand motif definition
a helix-loop-helix structural domain that is the binding site of Ca2+ in many calcium-sending proteins
–> Each EF hand can bind 1 Ca2+ ion ⇒ Calmodulin can bind a total of 4 Ca2+ ions (2 at each of the globular ends of the proteins)
what is the concentration threshold of Ca2+ in the cytoplasm for the binding of Ca2+ to activate calmodulin?
500nM
–> When the cytoplasmic Ca2+ level is raised above 500 nM, the binding of Ca2+ activates CaM
EF hand protein family: definition, structure, and activation
definition: a group of proteins that share a common structural motif called the EF hand
structure: EF hand motif is composed of… 2 alpha helices (E helix and F helix) connected by a loop region (calcium-binding loop) which contains conserved amino acid residues that coordinate the binding of a Ca2+ ion
- E helix: positioned BEFORE the loop the binds and calcium ion & plays a structural role to help stabilize the conformation of the motif
- F helix: positioned AFTER the loop and works with E helix to create the helix-loop-helix structure
- Calcium-binding loop: typically contains 12 residues with specific positions coordinating the binding of a single Ca2+ ion
activation: upon binding Ca2+, EF hand proteins undergo a conformational change that exposes hydrophobic regions or alter their surface properties to interact with other proteins or target molecules
give 2 examples of biochemical targets that the Ca2+ - calmodulin complex activates
the plasma membrane Ca2+ ATPase
the calmodulin-dependent protein kinase (CaM kinase)
calmodulin-dependent protein kinase (aka CaM kinase) definition and function
CaM kinases: a class of serine/threonine protein kinases activated by the Ca2+/calmodulin complex (and an important class of targets for calmodulin)
Function of activated CaM kinases: phosphorylate specific serine and threonine residues in target proteins
kinase definition
an enzyme that catalyzes the transfer of a phosphate group from ATP to a specified molecule
plasma membrane Ca2+ ATPase definition and function
Plasma membrane Ca2+ ATPase: a pump that actively transports (aka uses ATP hydrolysis) Ca2+ ions out of the cytosol (either out of the cytosol or into intracellular stores - ER)
Function: helps regulate intracellular Ca2+ levels by returning the cytosolic Ca2+ concentration to resting levels after a signaling event
what are the 3 steps for the activation of CaM kinases?
- Ca2+ increases → Ca2+ binds to CaM
- Ca2+ levels in the cytosol rise during to an extracellular signal
- The Ca2+ binding to calmodulin changes calmodulin’s shape to enable target protein interaction - The activated CaM complex with Ca2+ binds to CaM kinases → the CaM kinases are activated
- Binding induces conformational changes in CaM kinase, activating its kinase domain - The activated CaM kinases phosphorylate selected proteins
enzyme-coupled receptors definition and one example
definition: transmembrane proteins that display ligand-binding domains on the OUTER surface of the plasma membrane
- aka a major class of cell-surface receptors that, when activated by ligand binding, either act as enzymes themselves or associate with enzymes to mediate intracellular signaling
ie. receptor tyrosine kinases (RTKs)
enzyme-coupled receptors structure
Usually have only 1 TRANSMEMBRANE segment which spans the lipid bilayer as a SINGLE alpha helix (aka crosses the cell membrane only once)
Extracellular domain: binds the signal molecule (aka ligand)
Transmembrane domain: anchors the receptor to the plasma membrane
Intracellular domain: contains or associates with enzymatic activity (Acts as a tyrosine kinase which catalyzes the transfer of phosphate from ATP to the amino acid tyrosine)
Instead of associating with a G protein, the cytoplasmic domain of the enzyme-coupled receptors either act as… (2)
An enzyme itself (have their own enzyme activity) OR…
Forms a complex with another protein that acts as an enzyme
Receptor tyrosine kinases (RTKs)
the LARGEST class of enzyme-coupled receptors that consists of receptors with a cytoplasmic domain that functions as a tyrosine kinase
what is one of the most common intracellular signaling pathways that receptor tyrosine kinases (RTKs) trigger?
MAP kinase signaling pathway
The superfamily of RTKs includes… (6)
EGFR: epidermal growth factor receptor
Insulin receptor: Insulin receptor
PDGFR: platelet-derived growth factor receptor
VEGFR: vascular endothelial growth factor receptor
FGFR: fibroblast growth factor receptor
NGR: nerve growth factor receptor
what are the steps for the activation of Ras by RTKs?
Adaptor protein binding: An adaptor protein (ie. Grb2) docks on a specific phosphotyrosine on the activated RTK via its SH2 domain
Recruitment of Ras-GEF: The adaptor protein recruits a guanine nucleotide exchange factor (GEF) specific for Ras aka Ras-GEF
GDP-GTP exchange: Ras-GEF catalyzes the exchange of GDP for GTP on the Ras protein → activating Ras
Activated Ras: Ras which is now bound to GTP activates downstream signaling pathways (ie. the MAP kinase (MAPK) cascade, which regulates gene expression and cellular processes)
Each phosphorylated tyrosine serves as a specific docking site for a ___
Each phosphorylated tyrosine serves as a specific docking site for a different intracellular signaling protein
SH2 domain
Src homology 2 (SH2) domain: protein interaction motifs on intracellular signaling proteins that recognizes and binds to specific phosphorylated tyrosines on the cytosolic tail of an activated RTK or on another intracellular signaling protein
adaptor protein definition and one example
Adaptor protein: a types of intracellular signaling molecule that facilitates the interaction between different proteins in a signaling pathway (typically do not have enzymatic activity but instead act as molecule bridges to help organize signaling complexes and ensuring the correct proteins are brought into proximity for signaling)
ie. Grb2
Ras-GEF
Ras guanine nucleotide exchange factor (Ras-GEF) recruited by the adaptor protein
Ras-GEF: stimulates Ras to exchange its bound GDP for GTP
mitogen definition
Mitogen: extracellular signal molecules that stimulate cell proliferation and bind to RTK
Activated Ras activates which signaling pathway?
Activated Ras activates a MAP kinase signaling pathway
MAPK (mitogen activation protein kinase)
MAPK (mitogen activated protein kinase): a serine/threonine-specific protein kinase that regulates proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis
Found only in EUKARYOTES
RAF: (the final kinase in the pathway) activated by Ras-GTP; phosphorylates and activates MEK
MEK: activated by RAF; phosphorylates and activates MAPK
MAPK: (the final kinase in the pathway) activated by MEK; translocates to the nucleus and activates various transcription factors
name 3 MAPK families
ERK (MAPK): extracellular signal regulated kinase
Involved in cell proliferation, survival, and differentiation
SAPKs (JNKs): stress-activated protein kinase (c-Jun NH2-terminal kinase, Jun kinase)
Involved in regulating apoptosis, stress responses, and inflammatory processes
P38 MAPKs
Regulate inflammation, cell differentiation, and apoptosis
what are the steps of the MAPK pathway?
- Activation of RTKs (Receptor Tyrosine Kinases)
- recruitment of adaptor protein3
- activation of Ras
- activation of Raf
- activation of MEK
- activation of MAPK
- termination of the signaling
what do these abbreviations mean?
MKKK
MEKK
MKK
MEK
ERK
MAPK
MKKK: MAP kinase kinase kinase
MEKK: MAP/ERK kinase kinase
MKK: MAP kinase kinase
MEK: MAPK/ERK kinase
ERK: extracellular signal regulated kinase
MAPK: mitogen activated kinase
EGFR signaling pathway
EGFR is an example of a RTK
follows the RTK pathway where EGFR –> ERK (MAPK)
- The prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs)
- EGFR regulates cell growth and differentiation and are implicated in many human cancers
what are the 5 steps of PI3K/AKT signaling?
- Insulin-like growth factor-1 (IGF-1) activates an RTK (ie. IGF-1-receptor), which recruits and activates PI3-kinase
- PI3-kinase then phosphorylates an inositol phospholipid (in the cytosolic side of the plasma membrane)
- The phosphorylated inositol phospholipid attracts intracellular signaling proteins (ie. AKT; in this Fig, here called protein kinase 1) that have a special domain that recognizes it
- Akt (protein kinase B) is activated at the membrane by phosphorylation by protein kinases 1 & 2
- The activated AKT is released from the plasma membrane and phosphorylates downstream proteins on specific serines and threonines → promote cell growth, cell survival
How does AKT stimulate cells to grow in size and what are the steps (2)?
AKT stimulates cells to grow in size by activating the serine/threonine kinase TOR
The activated AKT (PKB) phosphorylates and inactivates (INHIBITS) proteins that promote apoptosis
TOR is activated
TOR (Ser/Thr kinase): stimulate cell growth by enhancing protein synthesis and inhibiting protein degradation
TOR (Target of Rapamycin)
a Ser/Thr kinase that stimulates cell growth by enhancing protein synthesis and inhibiting protein degradation
Rapamycin
an anticancer drug that inhibits Tor (Target of Rapamycin)
Gleevec
an example of a protein kinase inhibitor acting as an anticancer drug
treats chronic myeloid leukemia CML by specifically INHIBITING the BCR-ABL kinase that is found on the Philadelphia chr (Ph) which carries the BCR-ABL fusion gene
–> the BCR-ABL fusion gene encodes for the oncogenic BCR-ABL kinase (which drives the uncontrolled proliferation of white blood cells in CML)
