Cell Biology - Signal Tranduction

Front 1

What are the different types of chemical signals?

Back 1

Ligand - substance that binds to specific receptor initiating a particular event

Primary Messenger - ligand is example

Second Messnger - transmit signlas from extracellular signaling ligands to cell interior (ex. cAMP, Ca2+, Inositol triphosphate, diacylglycerol)

Messenger molecules include AA, Ach, peptides, steroid hormones, retinoids, nucleotides

hydrophilic ligands bind to exterior, hydrophobic on nucleus or cytosol

first message - ligand binds to signal

second message - what happens inside

Front 2

What are some characteristics of Signal Transduction?

Back 2

definition:
-detection of signal on cell surface and mechanism by which signal is transmitted into cell's interior, resulting in changes in cell behavior and gene expression

Properties:
-because they bind and release like enzymes they have kinetics, Km
-for lipid soluble, receptor in cytosol or nucleus

Front 3

What are factors that affect the interactions between ligands and their receptors?

Back 3

Receptor Affinity:
-high affinity = low conc. of ligand (called 'potency' in drug companies)

Cognate receptor - receptor for specific ligand

Dissociation constant (Kd) conc. of free ligand needed to produce 1/2 receptors occupied, small Kd = higher affinity between ligand and receptor

receptor down regulation:
-cell responds to high ligand conc.
-removal of receptor for surface (internalization or tolerance)
-raises Kd
-tachyphylaxis - alters second messenger

Front 4

What are some clinical features of Receptor Down-Regulation?

Back 4

Tolerance:
-prolonged use leads to loss of effectiveness due to receptor down-regulation
ex. prolonged use of neosynephrine, which stimulates alpha-adrenergic receptors, causing blood vessels in nose to constrict to relieve sinus congestion

Tachyphylaxis:
-occurs on first dose
ex. nitroglycerin as vasodilator to treat heart, vasoconstriction action of topical steroids on skin, not as effective second time.

Front 5

What is the sequence of the Signaling Pathway?

Back 5

Reception:
Binding of epinephrine to G protein-linked receptor

Transduction:
1. Inactive G protein --> Active G Protein
2. Inactive adenylyl cyclase --> active adenylyl cyclase
3. ATP --> cAMP
4. Inactive protein kinase A --> active protein kinase A
5. Inactive Phosphorylase kinase --> active phosphorylase kinase
6. Inactive glycogen phosphorylase --> active glycogen phosphorylase
7. Glycogen --> Glucose 1 Phosphate

Front 6

What are some types of receptors?

Back 6

Ligand-gated channels - ionotropic

Intracellular recptors

Second Messenger receptors - metabotropic

Front 7

What is the structure of GPCRs?

Back 7

G Protein Copuled Receptors

Seven-membrane spanning receptors

N-terminus: extracellular
C-terminus: cytosolic (intracellular)
G-protein interacts at cytosolic loop between 5th and 6th transmembrane helices

2 classes of G Proteins:
Large heterotrimeric G-proteins:
-alpha, beta, gamma
-mediates signal transduction (Ga binds GTP, Gbg activates other 2nd messengers or channels)
-Gs (stimulators) and Gi (inhibitors)
-Ga exchanges GDP for GTP and dissociates from Gbg when ligand interacts with receptor
Small Monomeric G Proteins:
Ex. Ras

Front 8

What are the different Heterotrimeric G Proteins?

Back 8

Gs:
Function - stimulates adenylate cyclase --> formation of cAMP
Result - activation of protein kinases, ie. PKA
Examples - binding of epi to BETA adrenergic receptors increasing cAMP levels in cytosol

Gi:
Function - inhibits adenylate cyclase preventing formation of cAMP
Result - Protein kinases remain inactive
Examples - binding of epi to ALPHA adrenergic receptors decreasing cAMP levels in cytosol

Gq (Gp):
Function - activates Phospholipase C (PLC), leading to formation of Inositol triphosphate (IP3) and diacylglycerol (DAG)
Result - Influx of Ca into cytosol and activation of protein kinase C
Examples - binding of antigen to membrane bound IgE causing release of histamine by mast cells

Front 9

What is the G Protein Activation/Inactivation Cycle?

Back 9

1. Ligand binds to receptor

2. G-alpha releases GDP and binds GTP, activating G protein

3. Subunits separate

4. G Protein subunits activate or inhibit target proteins (this is the difference between Gs and Gi)

5. G alpha subunit hydrolyzes its bound GTP to GDP and becomes inactive

6. Subunits recombine to form inactive G protein

Front 10

How is cAMP formed/degraded? What is its importance?

Back 10

Formed:
-Adenylate cyclase forms cAMP from cytosolic ATP

Degraded:
-Phosphodiesterase

Target:
-PKA, adds P to serine/threonine; regulatory subunits dissociate when cAMP binds to PKA

cAMP pathways important in:
1 - glycogen metabolism
2 - heart contraction
3 - blood clotting
4 - secretion of salt and water in gut

methylxanthines (caffeine and theophylline) and viagra are phosphodiesterase inhibitors

NO released by neurons of penis cause dilation responsible for erection. Viagra helps maintain elevated cGMP in erectile tissue

Front 11

What are some diseases results from disruption of G-protein signaling?

Back 11

Cholera toxin poisoning:
-Increase cAMP
-alters salt secretion in intestine
-chemically modifies Gs so that it can no longer hydrolyze GTP
-vibrin cholera bacteria secrete toxin
-water follows salt leading to severe dehydration

Whooping cough (pertussis toxin)
-increase cAMP
-B. Pertussis - Gi is inactivated by pertussis toxin; Gi can no longer inhibit adenylate cyclase

Front 12

What is the Inositol-phospholipid-calcium pathway?

Back 12

Gq uses IP3 and DAG as second messengers

Pathway:
1. Ligand binds to Gq-linked receptor
2. Gq is activated
3. Gq activates PLC-Beta
4. PIP2 is cleaves to IP3 and DAG
5. IP3 binds to IP3 receptor in ER
6. Calcium is released into cytosol and binds calmodulin
7. DAG activates protein kinase C (PKC)
8. PKC stimulates other pathways

Front 13

How is Calcium important in signaling?

Back 13

Ca pumps keep Ca conc. low in cytoplasm

Ca ionophores:
-A23187: antibiotic against gram positive bacteria and fungi, mobile Ca carrier

Fura-2 a flouroescent dye binds to calcium and can use this dye for experiment

calcium-calmodulin complex:
-in cytosol Ca binds and activates calmodulin which target and regulate kinases and phosphatases

Front 14

What is the pathway for the action of NO on blood vessel?

Back 14

1. G-linked receptor (Ach) --> InsP3
2. InsP3 --> Ca
3. Ca activates Calmodulin
4. Calmodulin goes to NO synthase
5. NO synthase creates NO
6. NO activates Guanylyl cyclase
7. Guanylyl cyclase goes to cGMP
8. cGMP activates Protein kinase G

causes muscle relaxation (dilation of blood vessels)

Front 15

What are Protein Kinase-Associated Receptors?

Back 15

Receptor Tyrosine Kinases RTK aggregate and undergo autophosphorylation

structure of tyrosine kinases:
-single pass
-tyrosines on cytosolic tail of receptor

activation:
-regulatory proteins with SH2 domains recognize and bind to phosphorylated tyrosines on receptor
-this binding stimulates regulatory proteins

Front 16

What is the RAS pathway?

Back 16

Activation:
-GRB2 (contains SH2 domain) interacts with phosphorylated receptor
-Sos is associated with GRB2 and becomes activated
-Sos (Son of Sevens) stimulates Ras to release GDP and bind GTP, Ras now active
-Ras activates mitogen-activated protein kinases (MAPK)
-MAPK phosphorylates transcription factors (AP-1)

MAP kinases respond to extracellular stimuli (mitogens) and regulate gene expression, mitosis, cell survival/apoptosis

Ras is inactivated by GTPase activating protein (GAP)

Front 17

What are Oncogenes?

Back 17

proteins that ac as growth factors (EGF) , growth factor receptors (ErbB) and intracellular signaling molecules (Ras and Raf)

ERK MAP kinase lead to induction of genes that encode potentially oncogenic transcriptional regulatory proteins

Front 18

What are mermbers of the Ras superfamily?

Back 18

Ras - cell proliferation

Rho - cytoskeletal dynamics/morphology

Rab - membrane trafficking

Rap - vesicular transport

Arf - vesicular transport

Ran - nuclear transport

Miro - mitochondrial transport

Front 19

What are the different growth factors?

Back 19

Epidermal growth factor (EGF):
target = epithelial and mesenchymal cells
receptor complex = tyrosine kinase

Transforming growth factor - alpha (TGFa):
Target = same as EGF
Receptor = same as EGF

Platelet-derived growth factor (PDGF):
Target = mesenchyme, smooth muscle, trophoblast
Receptor = tyrosine kinase

Transforming growth factor-Beta (TGFb):
Target = fibroblastic cells, involved in cell-cycle regulation
Receptor = serine-threonine kinase


Fibroblast growth factor (FGF):
Target = mesenchyme, fibroblasts
receptors = Tyrosine kinase

IL-2:
Target = cytotoxic T lymphocytes
receptors = 3 subunits

Colony stimulation Factor 1 (CSF-1):
Target = macrophage precursor
Receptor = tyrosine kinase

Wnts:
Target = many embryonic cells
Receptor = Seven-pass protein

Front 20

What is dominant negative mutation?

Back 20

one polypeptide chain in protein disrupts function even though other ones are normal

disruption of growth factor through tyrosine kinase is example

Front 21

What is Achondroplasia?

Back 21

dwarfism that results from autosomal dominant negative mutation of FGFR-3 gene

Front 22

What are some disruptions of growth factor?

Back 22

EGF:
-breast cancer, glioblastoma, fibrosarcoma

TGF beta 1 - 1/3 ovarian cancers

TGF beta 2 - colorectal cancer

Smad4 - pancreatic cancers

Front 23

What do Adrenergic receptors do?

Back 23

alpha receptors:
-bind both hormones
-interact with Gq proteins (phospholipase C activation)
-shuts down blood flow

beta-adrenergic receptors:
-binds epinephrine
-interacts with Gs proteins (adenylate cyclase activation)

Front 24

What are examples of cell function regulated by cAMP?

Back 24

Epinephrine:
function1 - glycogen degradation
target tissue1 - muscle, liver
function2 - FA production
target tissue2 - adipose
function3 - heart rate, BP
target tissue3 - CV

ADH:
Function - water reabsorption
Tissue - Kidney

PTH:
Function - bone resorption
Target tissue - bone

Front 25

How is Glycogen degradation controlled?

Back 25

1. Epi binds to Beta-adrenergic receptor

2. Gs alpha binds GTP, dissociates from Beta and Gamma

3. Activated Gs alpha activates adenylate cyclase

4. Adenylate cyclase produces cAMP from ATP

5. PKA binds cAMP and beocomes activated

6. PKA phosphorylates and actiaves Phosphorylase kinase

7. Phosphorylase kinase phosphorylates b into active a form

8. glycogen is cleaved into glucose-1-phosphate

Front 26

What is Cyclic-AMP inducible gene expression?

Back 26

protein kinase A translocates to nucleus and phosphorylates transcription factor CREB leading to expression of cAMP-inducible genes

Front 27

How are alpha adrenergic receptors and Inositol-phospholipid-calcium pathway related?

Back 27

Alpha1 adrenergic receptors cause InsP3 pathway to release calcium via Gq

elevated calcium causes smooth muscle contraction and constriction of blood vessels

opposite from beta adrenergic

Front 28

Where are alpha and beta adernergic receptors located?

Back 28

alpha - surround peripheral veins and constrict

beta - around heart and bronchioles blood vessels and relax

Front 29

What are good examples of Paracrine regulation?

Back 29

Histamine:
-IgE mast cells stimulate increase of Ca which induces secretion of histamine
-causes allergy locally

Prostaglandins:
-typically active G protein receptor
-aspirin reduces blood clots by blocking COX inhibiting prostaglandins from arachidonic acid

Front 30

What are the two types of cell death?

Back 30

Necrosis:
-severe destruction along with inflammatory response (heterophagy)
-hypoxia, toxins


Apoptosis:
-programmed cell death
-usually at single cell level
-autophagy - NO inflammatory response
-physiologic or pathologic
-helps prevent tumor growth, maintains immune system

Front 31

What are the stages of Apoptosis?

Back 31

-DNA segregates to nuclear periphery

-cell produces organelle fragments

-DNAses digest chromatin (shrinkage of cytoplasm)

-cell fragmentation

-cell is dismantled in apoptotic bodies

Front 32

What are the different mechanisms for proteins during apoptosis?

Back 32

Protein cross-linking:
-mediated by transglutaminase

Protein cleavage (fragmentation):
-mediated by Caspases
-laddering effect

Loss of membrane asymmetry:
-phosphotidylserine

Front 33

What are caspases?

Back 33

zymogens

inactive until apoptotic signal activates one which cause a cascade leading to activation of others

3 classes:
Initiator (caspases 8,9)
Effector (executioner) - 3
Inflammatory response

Front 34

What are the major Caspase-dependant apoptotic routes?

Back 34

Death receptor pathway:
-Fas ligand-Fas receptor
-TNF

Mitochondrial pathway (death-receptor independant):
-innate
-cyt c (stops ATP production)

Front 35

What is Fas Signaling?

Back 35

recruit FADD

FADD recruits procaspase-8 (activates by self-cleavage)

Caspase 8 activates Caspase 3

Front 36

How is Apoptosis triggered by DNA damage?

Back 36

p53 activates production of BAX

Bax interacts with mitochondria

cytc recruits procaspase 9

caspase 9 stimulates caspase 3

Front 37

What are some factors released by mitochondria?

Back 37

Calcium:
-activation of caspases associated with ER

Apoptosis Induced Factor (AIF):
-stimulates large DNA fragmentation and condensation of chromatin

Endonuclease G:
-directly mediates nuclear DNA fragmentation

Front 38

What is Cytolytic Granule-mediated Apoptosis?

Back 38

can circumvent requirement of FAS or TNF receptors

granzymes or NK cells activate effector caspases inside virus-infected cells

also target:
-Lamins
-Histones
-Mcl-1 (leads to activation of mitochondrial apoptotic pathway

Front 39

What are Cancers caused by Apoptosis?

Back 39

Follicular B-cell Lymphoma:
-chromosomal translocation leads to up-regulation of Bcl-2

Melanoma: associated with Apaf-1

Autoimmune lymphoproliferative syndrome (ALPS):
-mutation in Fas ligand or receptor