Cell Bio 11 Flashcards
Signal Transduction
Conversion of 1 Signal into another
A signal from outside of the cell converted into a signal inside the cell
Involves growth factors, cytokines, hormones, ECM, neutrotransmitter, light, sound.
Controls all aspects of normal development and physiology
Initiator of dseases
Key Players in Signal Transduction
Receptor Tyrosine Kinases (RTKs)
G-protein coupled receptors (GPCRs)
Proto-oncogenes
Mitogen activated protein Kinases (MAPKs)
Basic elements of cell signaling
Signal or signaling molecule (ligand; primary messengers)
Receptors:
Cell-surface receptors
Intracellular receptors
Intracellular signaling and effector proteins:
G-proteins, protein kinases + phosphotases
Secondary messengers:
Ca++, cAMP, cGMP, IP3, DAG, NO
Signal or Signaling molecule
Small molecules, epinephrine, acetylcholine, steroids.
Large molecules, growth factors, cytokines.
Cell Signaling: Signaling molecule types
Different types of signaling molecules.
Some are hydrophobic and cross the plasma membrane, binding to receptors in the cell, the complex can move into the nucleus.
Hydrophilic molecules bind to cell surface receptor can effect changes in intermediate proteins.
Nuclear-receptor Superfamily
Lipid soluble hormones bind to intracellular receptores which constitute the NRS of transcription factors.
Intracellular signaling receptors are directly activated by hydrophobic ligands, All receptors have a ligand binding domain and DNA binding domain
Gene activation by a nuclear receptor
Inhibitors binds to receptors from entering the nucleus when glucocorticoid binds it removes the inhibitor allowing GR to enter the nucleus and bind to regulatory sequences in the DNA, the activation domain recruits transcription factors that increase transcription.
Signal Transducton
Hydrophilic proteins have to transmit their signal through an intermediate protein (cell surface receptor), it causes a conformational change in the receptor and then causes the recruitment of other intracellular proteins that transmit signal to effector proteins
Four Forms of intercellular signaling
Endrocine signaling
Paracrine signaling
Autocrine signaling
Signaling by plasma membrane-attached proteins
Endocrine Signaling
Hormone secretion into blood by endocrine gland and target distant cells.
Tissues produce a ligand that enters into the bloodstream and acts distantly on target cells.
Paracrine Signaling
One cell secreting a ligand that acts on an adjacent target cell.
Autocrine Signaling
Target sites on the same cell
Cell producing a ligand that is detected by receptors on the cell surface, autosignaling.
Signaling by plasma membrane attached proteins
They can act locally or at a distance
Signaling by Cell-Surface Receptors (steps)
1- Synthesis and release the signaling molecule by the signaling cell
2- Transport and binding the signal to a specific receptor
3- Initiation of one or more intracellular signal-transduction pathways
4- short term cellular response
5- long-term cellular responses
6- termination of cellular response
Receptor can transmit a signal very quickly by modifying intracellular proteins in close proximity to the receptor affecting metabolism, function or movement.
Rapid change in cell behaviour
Altered protein function
i.e. changes in ion transport, cell movement, secretion or metabolism
Slow change in cell behaviour
A number of steps to activate transcription factors that alter gene expression.
i.e. gene regulated changes in cell growth and division
Animal cell’s dependence on multiple extracellular signal molecules
A cell is bombarded by different signals.
Mitogenic factors: activate cell cycle
Stem cell differentiation in response to signals
Apoptogenic factors or complete absence of trophic factors, absence of survival factors might lead to the cell developing an apoptotic response.
Ligand-receptor interactions
Binding specificity is based on the molecular complementarity between the interacting surface of a receptor (binding interface) + ligand (noncovalent forces)
It triggers a conformational change in the receptor
very often signaling molecules (ligands) induce receptor dimerization.
An extracellular protein has a part that sticks outside the cell and it can interact with the ligand, molecular complementarity. Structure of the ligand and its ability to bind to it, complementary structure, and charge
The dissociation constant
R+L -> RL
at equilibrium we have a simple equilibrium binding equation:
Kd = [R] [L]/[RL]
Kd is the measure of affinity of a receptor to its ligand
Kd is the ligand concentration required to bind 50% of the cell surface receptors
The lower the Kd the higher the affinity of the ligand for the receptor
Functional expression assay to identify a cDNA encoding a cell surface receptor
If you have a transmembrane receptor it is difficult to purify it due to the hydrophobic domain, the hydrophobic domain causes the protein to fold up together.
Cultured cells do not express receptor for ligand X.
Transfect cells with cDNA library and screen for cellular phenotype associated with ligand X.
Identify incorporated cDNA by PCR followed by sequencing
The receptor protein can be deduced from the cDNA sequence
Can perform further studies by mutating specific amino acids to determine essential ligand binding domain
Regulation of protein activity by a kinase/phosphatase switch
Tyrosine kinases/Serine/Threonine kinases or Phosphatases
When a ligand and receptor interact that release a signal related to changes in phosphorylation of intracellular proteins, certain kinases can be activated by these signaling molecules, which adds a phosphate group to specific amino acids within a substrate protein.
It can lead to activation or inactivation.
Protein phosphatases can remove phosphate to turn the protein off.
G-proteins exist in 2 forms
Active-bound to GTP
Inactive-bound to GDP
Activation of G-protein is triggered by
a signal
hormone binding to the receptor
Helped by Guanine nucleotide exchange factor.
leads to GTP binding and activating the G-protein
Gly-60 and Thr-35 residues bind to the phosphate attached to GTP
GEF
Guanine Nucleotide Exchange Factor
Conversion back to inactive state is mediated by
A GTPase (either intrinsic or separate protein)
GTPase-activating proteins (GAP) can accelerate GTP hydrolysis
G Protein-coupled receptor system
A receptor with 7 membrane-spanning domains
coupled trimeric G-proteins
A membrane-bound effector protein
A second messenger in many GPCR pathways
GPCRs are
The most numerous class of receptors in animal cells
vast class of transmembrane proteins
affect a wide array of neurological pathways, they are exploited by frug companies to affect cognition, satisfaction, reward pathways
G protein-coupled receptors/Ligands
Diverse ligands: ligands, hormones, neurotransmitters, chemoattractants, odorants
Major target of pharmaceutical drugs
Trimeric Proteins
The GTPase superfamily of proteins; G refers to the ability to bind guanine nucleotide (GDP and GTP)
Three subunits: Galpha, Gbeta, Ggamma
Galpha and Ggamma are lipid anchored proteins at the cytoplasmic face of the plasma membrane
Galpha-GDP is inactive and Galpha-GTP is active
function in signal transduction by coupling ligand-bound receprots to specific effector molecules
G-alpha
G-alpha subunit controls the activity of the trimeric complex
G-protein coupled receptor steps
1- Binding of hormones induces a conformational change in receptor
2- Activated Receptor binds to Galpha subunit
3- Activated receptor causes a conformational change in Galpha subunit triggering dissociation of GDP
4- Binding of GTP to Galpha triggers dissociation of Galpha both from the receptors from Gbeta/gamma
5- Hormone dissociates from receptor; Galpha binds to effector, activating it
6- Hydrolysis of GTP and GDP causes Galpha to dissociate from the effector and reassociate with Gbeta/gamma
FRET (G-alpha and G-beta/gamma)
Cells are transfected with genes encoding two fusion proteins:
1- G-alpha fused to cyan fluorescent protein
2- G-beta fused with yellow fluorescent protein
Looking at interactions between G-beta and G-alpha using FRET. YFP is emitted so long as the two proteins are interacting.
When a ligand is added, the trimeric complex is recruited and they dissociate form each other, we lose the fret signal,
After adding the ligans we see a drop in yellow fluorescence, this occurs when we have a ligand interacting with the receptor
Total Number of GPCRs
800 in total
21 different G-alpha, 6 G-beta, 12 G-gamma
Effector Proteins
Adenylyl cyclase
G-alpha(s) and G-alpha(i)
G-alpha (s) is stimulating
G-alpha (i) is inhibiting
Activation of muscarinic acetylcholine receptor and K+ channels in the heart
When acetylcholine binds to its receptor leads to the opening of K+ channels and cellular hyperpolarisation.
Slows the rate of heart muscle contraction
G-beta/gamma subunit activated the effector protein (K+ channel)
Regulation of adenylyl cyclase
Catalyzes the formation of cAMP
Alpha subunit will move along and activate the effector
Gai and Gas depending on the isoforms of the g-alpha subunit may have a stimulating effect or an inhibitory effect
Cholera Toxin
1- Cholera- 200-500,000 cases/yr with 20-50% mortality
2- toxin enters enterocytes of the small intestine using a GM1 ganglioside receptor
3- toxin maintains G-alpha (s) in an active state
4- mass activation of adenylyl cyclase
5- massive increase in cAMP
6- Activates protein kinase a
7- increase leads to hyperactive CFTR ion channel
8- Cl- pumped out and Na+ follows and H2O loss to the intestine
9- Massive diarrhea
Pertussis Toxin
Bordella perutsi toxin enters cilitated epithelial cells in the lungs
pertussis toxin maintains G-alpha i in its inactive stae (GDP)
Mass activation of Adenylyl cylclase
mass increase activity in ion pumps
Inhibit the inhibitor you can no longer encourage the basal slow activation of adenylate cyclase, you have a progressive increase of adenylate cyclase, so you get lots of cAMP, which activates CFTR leading to an efflux of chloride and sodium ions leading to osmosis, water enters into the bronchi of lung and the water accumulation causes watery mucous,
mucous secretion and electrolyte/H20 accumulation in lungs
Four common intracellular second messengers (4)
cAMP
cGMP
IP3
DAG
Other secondary messengers
Ca++
NO
Signal Amplification
Small amount of primary messenger can trigger the release of large amounts of secondat messenger
amplification of a signal to trigger a rapid response
cAMP
Activates protein kinase A
Binds to the regulatory subunits (co-operative binding) to release them from the catalytic subunits of PKA that will go on and do the phosphorylation
PKA
Four subunit protein
2 regulatory and 2 catalytic
catalytic protein is only active when the regulatory subunits are released
Regulation of glycogen metabolism by cAMP: Activating phosphorylation
PKA activates and phosphorylates GPK (glycogen phosphorylase kinase)
GPK phosphorylates GP (glycogen phosphorylase)
GP will phosphorylate glycogen to release G1P for energy
Regulation of glycogen metabolism by cAMP: Inhibitory phosphorylation
PKA phosphorylates GS (glycogen synthase)
PKA phosphorylates IP (Inhibitor of phosphoprotein) activating so it will bind PP (phoshoprotein) inhibiting it
Regulation of glycogen metabolism: decreased cAMP
PP (phosphoprotein phosphotase) inactivates GPK and GP
Activates GS
Glycogen metabolism full pathway (SYN)
Epineprhine acts on G protein coupled receptors on our liver and muscle cells leading to the acyivation of adenylyl cyclase that releases cAMP, cAMP binds to and activates PKA.
PKA will phosphorylate proteins involved in Glycogen release and synthesis
Activation of gene transcription by GPCR
Genes regulated by PKA contains a specific nucleotide sequence (TGACGTCA) cAMP response element (CRE)
Activation of the CRE binding protein by phosphorylaion in the neucleus
CREB Signaling Pathway
Involves G-protein activation
Increase in cAMP levels
Activation of PKA
PKA translocation to the nucleus
Phosphorylation of CREB
P-CREB binds as a dimer to cAMP Response Element (CRE)
P-creb also binds CBP/P300 coactivator
CBP/P300 recruits transcriptional machinery
CREB plays a role in
memory formations, neurotransmitters in neurons can bind and lead to the production of cAMP activating CREB and the CREB can activate certain genes responsible for synaptic remodelling, the ability of neurons to be able to signal and connect with one another
This is a very slow process, minutes to hours, once established it’s a stable pathway and it leads to longer lasting effects
Phospholipase C
An effector which is activated by G-alpha subunits
Cleaves sugar lipid PIP2 into DAG and IP3
IP3/DAG signaling pathway and Ca++
Signal Molecules binds its GPCR
Activates G-alpha protein
Stimulation of Pholipase C
PLC cleaves PIP2 into DAG and IP3
IP3 shuttles to ER membrane and open Ca++ channels
Levels of Ca++ ions increase which bind Protein Kinase C
PKC translocates to PM where it interacts with DAG
Active PKC phosphorylates numerous substrates including players that activate MAPK
NO gas
Gas with a short halflife (2-30 seconds)
Present in skeletal, cardiac or smooth muscles
NO Pathway
Stimulation of acetylcholine G-protein coupled receptors on endothelial cells induces an increase in cytosolic Ca++ (PLC, IP3) , and synthesis of NO.
NO diffuses into the surrounding smooth muscle cells.
activates an intracellular NO receptor to synthesize cGMP. The resulting increase in cGMP leads to activation of protein kinase G (PKG), which triggers a pathway resulting in muscle relaxation and vasodilation.
Viagra
Vasodilators can open arteries to allow for increased blood flow, Viagra inhibits an enzyme called phosphodiesterase, this enzyme converts cyclic GMP to GMP, a cofactor for protein kinase g an enzyme critical for relaxation of muscle cells
if cGMP can’t become GMP we get vasodilation, cGMP will build up, as a result the two penal arteries, have smooth muscle cells that are relaxed and this causes increased increase blood flow to the penis, resulting in blood filling up into the corpus cavernosum resulting in the engorgement of the penis.
Mode of Action
Parasympathetic NS releases NO in corpus cavernosum
NO receptors activate Guanylyl Cyclase to Increase cGMP & PKG Activity that causes vasodilation of smooth muscle resulting in increased blood flow
Viagra inhibits the PDE that hydrolyzes cGMP to GMP
Erection is maintained
PDE-5 inhibitor side effects
Side Effects: heart attack, stroke, sudden hearing loss, cyanopsia, priapism
A sudden drop in blood pressure can trigger a heart attack, cuase stimulation of blue rod cells in the retina, priapism is an erection that doesn’t go away.
The blood that flows into the penis, constricts on the veinous system and prevent s blood flow back, no fresh oxygenated blood leading to gangerene, to alleviate that situation the blood has to physically removed from the penis.
