Signal Transduction Flashcards
Juxtacrine mode
direct contact
juxtacrine signal
- membrane bound surface proteins (surface protein/receptor interaction)
- transmembrane protein channels (gap junction: docking connexons)
juxtacrine target
adjacent cells
endocrine mode
secreted molecules
endocrine signal
hormones
endocrine binding affinity
very high
endocrine range
long
endocrine target
distant cells
paracrine mode
secreted molecules
paracrine signal
local mediators/large proteins (growth factors, cytokines)
paracrine binding affinity
low to high
paracrine range
short
paracrine target
neighboring cells
synaptic mode
secreted molecules
synaptic signal
neurotransmitters
synaptic binding affinity
very low
synaptic range
very short
synaptic target
postsynaptic cells
autocrine mode
secreted molecules
autocrine signal
local mediators/large proteins (growth factors, cytokines)
autocrine binding affinity
low to high
autocrine range
short
autocrine target
same cell
3 aspects of signal transduction
selective response, amplification system, coordination of handling numerous signals
crosstalk
component from one pathway interacts/influences a component of another pathway
4 signal characteristics
specificity, relatively small molecules employed for signal, rapid deployment, can be turned off (often rapidly)
on demand vs pre made signal
on demand: intracellular signal is made when extracellular signal arrives
pre-made: intracellular signal stored in vesicles, vesicles released when extracellular signal arrives
Ways to turn off signal
- decreased concentration of first messenger
- receptor desensitization (inactivation via structural modification like phosphorylation; down-regulation via receptor internalization and degredation which is more common)
connexon
- 4 transmembrane domains, 2 extracellular loops (3 cysteines/loop)
- connect two cells via gap junctions
endocrine on/off response time
slow (minutes to hours) because of tight binding and time required to decrease blood concentration
paracrine on/off response time
rapid
synaptic on/off response time
very rapid (milliseconds)
autocrine on/off response time
rapid
neurotransmitters system
synaptic
neurotransmitters physical characteristics
hydrophillic molecules, very small <1kDa, fast diffusion
hormones system
endocrine
hormones physical characteristics
-hydrophillic, small, <5kDa, usually charged
OR
-hydrophobic, very small, <1kDa, membrane permeable
growth factors system
paracrine, autocrine
growth factors physical characteristics
polypeptides (some multimers), small-large, 6-80kDa, local acting
cytokines system
paracrine, autocrine
cytokines physical characteristics
polypeptides, usually multimers, small to large, 8-70kDa
agonist vs antagonist
agonist: ligand that activates normal response (excitatory or inhibitory)
antagonist: ligand that induces no response (blocks normal response)
Coupling
intracellular protein that transmits the signal of an activated receptor to an effector protein
adaptor
intracellular protein that lacks intrinsic enzymatic activity but contains several domains that mediate protein-protein interactions
SH2 binding affinity
phosphorylated tyrosines
PTB binding affinity
phosphorylated tyrosines
SH3 binding affinity
prolines (-X-P-p-X-P-)
PH binding affinity
phosphorylated inositol phospholipids (in plasma membrane, inositol portion hangs into cytosol)
Protein phosphorylation
Phosphorylate–>via protein kinases that use ATP to replace hydroxyl group with phosphate group
Dephosphorylate-> via protein phosphatases that dephosphorylate via hydrolysis
Guanine nucleotide binding (g protein cycle)
ON
GDP exchanged for GTP
Assisted by activated receptor for trimeric G; guanine nucleotide exchange factors (GEFs) for monomeric G
Guanine nucleotide binding (g protein cycle)
OFF
Hydrolysis back to inactivated GDP form
Catalyzed by intrinsic GTPase for trimeric G; intrinsic GTPase with help from GTPase-activating proteins (GAPs) for monomeric G
Relationship of KD and binding affinity
small Kd=high binding affinity=low concentration
large KD=low binding affinity=high concentration
Intracellular receptor class
- hydrophobic hormones enter through plasma membrane
- bind to receptor (which is bound to HSP before)
- receptor/ligand dimer enter nucleus and bind to DNA
- direct
Cell surface receptor class
more common
- hydrophillic hormones/NT/Growth factors/cytokines bind to cell surface receptor and cause conformational change
- receptor affects second messangers
- indirect
ligand gated ion channel
system
synaptic
ligand gated ion channel
ligands
neurotransmitters
ligand gated ion channel
binding
very low affinity
high KD
KD=10^-6 to 10^-3
ligand gated ion channel
examples
cation selective: excitatory (nicotinic ACh, glutamate)
anion selective: inhibitory (glycine, GABA)
ligand gated ion channel
structure
multimeric ring-like complex of 3-5 polypeptides with multiple transmembrane domains
opens internal water filled pore
ligand gated ion channel
termination
Rapid ligand removal by diffusion, enzymatic degredation, reuptake
Also can form inactive ligand-bound state via RECEPTOR INACTIVATION (inactive despite the fact ligand is bound, causes channel closing and NT release)
G protein coupled receptor
system
synaptic, endocrine, paracrine, autocrine
G protein coupled receptor
ligands
NT, hormones, cytokines (particularily chemokines)
G protein coupled receptor
binding
intermediate range
KD=10^-9 to 10^-6M
G protein coupled receptor
Examples
muscarinic ACh, beta adrenergic, rhodopsin
G protein coupled receptor
structure
N terminous outside cell: site of glycosylation
7 transmembrane alpha helixes
c terminus inside cell, site of phosphorylation and g protein binding
large ligands bind to extracellular loops, small ligands bind in pocket (of rhodopsin receptor)
G protein coupled receptor
heterotrimeric g proteins/coupling proteins
Structure
alpha subunit: largest, hydrophillic, covalent attachment to plasma membrane with lipid anchor, GDP/GTP binding site and GTPase activity, interact with effector proteins
beta-gamma complex: smaller dimer, hydophobic, covalently attached to plasma membrane with lipid anchor, some interaction with effector proteins
alpha has many different forms; beta gamma dimer similar for differ g protein subtypes
G protein coupled receptor
Action
- ligand binds and causes conformational change in receptor
- recognition site exposed and g protein binding occurs (location of amplification)
- GDP/GTP exchange and g protein dissociates
- alpha subunit binds to enzyme (like adenylate cyclase) causing release of second messangers (Site of most amplification)
- intrinsic GTPase activation, hydrolysis of GTP to GDP and release from enzyme
- g protein reforms
G protein coupled receptor
termination
- extracellular enzymes inactivate ligands
- receptor mediated endocytosis
- receptor phosphorylation by protein kinases (MAJOR MECHANISM OF DESENSITIZATION): PKA phosphorylate with or without ligand bound, GPCR specific protein kinases (GRKs) phosphorylate only with bound ligand.
Caffeinated alcohol drinks
alcohol keeps GABA ligand channel open longer alosterically, decreases membrane potential, increases neural supression, increase dopamine
caffeine blocks adenosine binding on g protein receptor (antagonist), cancels adenosines effect, allows increased neural activity, increases dopamine
enzyme linked receptor
system
endocrine, paracrine
enzyme linked receptor
ligands
hormones, growth factors
enzyme linked receptor
binding
very high affinity
KD=10^-12 to 10^-9
enzyme linked receptor
examples
receptor tyrosine kinase (RTK): EGF, insulin, and less important: FGF, PDGF
Receptor serine/threonine kinase: TGF-beta, and less important BMP
enzyme linked receptor
structure
-subunits are single polypeptide chain with large extracellular N terminal domain for ligand binding, single transmembrane domain, intracellular c terminal domain with catalytic domains
enzyme linked receptor
RTK structure/action
- dimer
- inactive RTK monomers in membrane
- growth factor (ligand) binds to membrane receptor causing dimerization and activation of kinase domain
- autophosphorylation (cross phosphorylation) of tyrosine residues in tyrosine kinase domain
- SH2 and PTB domains bind to phosphorylated tyrosines, set up large signalling cascades (like MAP kinase cascade)
Map kinase cascade
Mitogen activated protein kinase cascade; after RTK activation or cytokine receptor activation
- adaptor protein binds it’s SH2 domain to phosphorylated tyrosine domain
- SH3 domain of adaptor protein binds RAS-activating protein
- RAS activating protein exchanges GDP for GTP on inactive RAS at membrane
- Active RAS binds to N-terminal of MAPKKK (brings MAPKKK from cytoplasm to membrane)
- MAPKKK activated, attracts MAPKK to membrane and phosphorylates serine/threonine using ATP
- Activated MAPKK phosphorylates threonine/tyrosine of MAPK using ATP
- Activated MAPK phosphorylates serine/threonine using ATP either in cytoplasm to change enzyme activity or nucleus to change gene expression
Enzyme linked receptor
Serine/threonine kinase structure/action
- tetramer
- inactive type 1 and type 2 monomers in membrane
- growth factors (ligand) bind to type 2 and dimerize with type 1, causing activation and cross phosphorylation of Ser/Thr of type 1
- SMAD binds to and gets phosphorylated by receptor, unfolds and becomes active
- SMAD dissociates from receptor, dimerizes with different SMAD subtype, NLS revealed and taken to nucleus where gene expression is altered
Enzyme linked receptor
termination
Receptor mediated endocytosis
- adaptin binds to intracellular sequence on ligand/receptor sequence, clatherin binds to adaptin
- clatherin polymerizes and vacuole is formed
- vacuole released into cytoplasm, clatherin coat shed, fused with endosome, ligand-receptor complexes dissociated
- receptors either recycled to plasma membrane or transferred to lysosome for degredation
Cytokine receptor
system
paracrine, autocrine
cytokine receptor
ligands
cytokines, some growth factors
cytokine receptor
binding
intermediate
KD=10^-9 to 10^-6M
cytokine receptor
examples
Class I, interleukin (IL2 [through IL7 and IL9), dimers]
Class II, interferon (IFN-gamma, [IFN-alpha, IFN-beta, IL-10), multimers]
Tumor necrosis factor (TNF-alpha, [TNF-beta), trimers]
cytokine receptor
structure
- diverse structure
- single polypeptide with large extracellular N-terminal domain for ligand binding, single transmembrane domain, intracellular c-terminal domain with different protein-protein interaction motifs but NO INTRINSIC ENZYMATIC ACTIVITY
- multimeric complexes
What differentiates structure of cytokine receptor and enzyme linked receptor?
intracellular C domain is catalytic in enzyme linked receptor but has no intrinsic enzymatic activity in cytokine receptor
cytokine receptor
action
- inactive monomeric receptors in membrane have associated JAKs on prolines
- cytokine binds and causes receptor dimerization
- JAK activated (via phosphorylation) and cross phosphorylation of tyrosine in subunits
- STAT SH2 domains binds to phosphorylated tyrosines
- JAK phosphorylate STAT, STAT activated
- STATs dissociate from receptor and dimerize
- STAT dimer translocated to nucleus to alter gene expression, or could start MAP kinase cascade
cytokine receptor
termination
- phosphatases remove tyrosine phosphates from receptor and/or active STAT
- SOCS (supressor of cytokine signaling) proteins inhibit STAT phosphorylation by binding/inhibiting JAK or competing with STAT for receptor binding sites
- endocytosis triggered by multimer formation
Ebola virus
- replaces STAT in import complex (importin-alpha5 adapter and Importin-beta receptor)
- virus (VP24 protein) translocated to nucleus instead of STAT dimer
- complex dissociates by Ran-GTP
- antiviral response supressed
Intracellular receptor structure
- polypeptide dimer with DNA-binding domains
- binds as dimer to DNA sequence
intracellular receptor examples
Progesterone, thyroxine, retinal
Ligand-gated ion channel structure/action Cation selective (nicotinic ach)
4 transmembrane domains, 2 intracellular loops
M3 transmembrane domain has large hydrophobic aa and small polar aa (ligand binding causes polar aa to face inside/open channel)
requires binding of 2 ligands to alpha subunits
KD equation and units
KD=koff/kon=([L][R])/[LR]
Units are concentration
Small KD meaning vs large KD meaning
small KD=receptor has high affinity for ligand
large KD=receptor has low affinity for ligand
Ligand/receptor binding
Saturation binding relation
[LR]=([R]o[L]o)/(KD+[L]o)
or
Bound=Bmax(Free/(KD+free))
where Bmax is total number of receptors
ligand/receptor binding
saturation plot
On free (x) vs bound (y) plot
KD is x/free value when bound=1/2*Bmax
So if Free»Kd, bound=Bmax
if free=Kd, bound=0.5Bmax (effective receptor)
Assumptions in saturation binding relation (5)
- equilibrium conditions
- homogeneous, monovalent (1:1) populations of ligand and receptor
- negligible ligand depletion (bound<10% of free)
- negligible inactivation of ligand and receptor
- negligible cell surface interactions
Scatchard relation
[LR]/{L]o=([R]o/KD)-(1/Kd)[LR]
or
Bound/free=Bmax/Kd - 1/Kd*bound
Scatchard plot
slope/yint/xint and implications
bound (x) vs bound/free (y)
slope= -1/Kd
yint=Bmax/Kd
xint=Bmax
steeper slope=smaller Kd=better binding
Scatchard plot advantages/disadvantages
Advantage: easily visual evaluation for comparing different ligand/receptors or checking original assumptions
Disadvantage: bound on both axes magnifies experimental error–> should get Kd and Bmax from non linear reg
Dose response plot
ligand concentration (x) vs fraction of maximum response or binding (y)
response can be anything downstream from receptor
EC50 (half maximal effective concentration) is x value where R/Rmax=1/2* Rmax=0.5
If EC50
Saliva stimulation in diabetes
Showed that diabetic rats had fewer ACh receptors with normal KDs in parotid gland, could contribute to reduced saliva stimulation.
All other results shaky at best due to poor fits of plots.
Ideal properties of second messangers (3)
- rapid generation
- small size and ability to easily diffuse
- quick removal from system
3 classes of second messangers
ions, water soluble molecules, membrane associated molecules
cAMP source
cyclic adensosine monophosphate
second messanger
ATP
cAMP effector enzyme
cyclic adensosine monophosphate
second messanger
adenylate cyclase at plasma membrane. Both N and C terminal catalytic domains in cytoplasm
cAMP main function
cyclic adensosine monophosphate
second messanger
activates PKA (cAMP dependant protein kinase)
cAMP location
cyclic adensosine monophosphate
second messanger
cytoplasm
cGMP location
second messanger
cytoplasm
cGMP source
second messanger
GTP
cGMP effector enzyme
second messanger
guanylate cyclase (soluble, or catalytic domain of membrane associated)
cGMP main function
second messanger
activates PKG
IP3 location
1,4,5-inositol triphosphate
second messanger
cytoplasm
IP3 source
1,4,5-inositol triphosphate
second messanger
PIP2 cleaved by phospholipase C to form cytosolic IP3 and membrane bound DAG
IP3 effector enzyme
1,4,5-inositol triphosphate
PLC
phospholipase C family
IP3 main function
1,4,5-inositol triphosphate
release Ca2+ from ER
can cause PKC activation
DAG location
diacylglycerol
membrane
DAG source
diacylglycerol
- mainly from PC (PC cleaved by phospholipase D to form PA and choline, PA cleaved by PAP to form DAG and phosphate)
- also from PIP2 (PIP2 cleaved by phospholipase C to form cytosolic IP3 and membrane bound DAG) and PE
DAG effector enzyme
diacylglycerol
PLC (for PIP2), PLD/PAP (for PC and PE)
DAG main function
diacylglycerol
activates PKC
AA location
Arachidonic acid
membrane
AA source
Arachidonic acid
mainly from PC
also from PE, PI (and derivatives PIP, PIP2)
phospholipase A2 cleaves and leaves membrane bound AA
AA effector enzyme
Arachidonic acid
PLA2
phospholipase A2 family
AA main function
Arachidonic acid
percursor for eicosanoids (for paracrine/autocrine signalling)
Source for membrane lipid reformation
PIP3 location
phosphatidylinositol-3,4,5-triphosphate
membrane
PIP3 source
phosphatidylinositol-3,4,5-triphosphate
PIP2
PIP3 effector enzyme
phosphatidylinositol-3,4,5-triphosphate
PI-3K
phosphoinositide-3-kinase family
PIP3 main function
phosphatidylinositol-3,4,5-triphosphate
activates PKB, PDK1 (phosphoinsoditide-dependant protein kinase 1)
–>activator of kinases recruited to membrane via PH domains
Calcium concentrations for on/off
low <10^-7 is OFF
higher >10^-6 ON
Calcium pumps (Ca signalling OFF)
Plasma membrane:
NCX: Na/Ca antiporter (low affinity, high rate)
PMCA: Ca ATPase (high affinity, low rate, 1ATP/Ca)
Sarco/ER:
SERCA: Ca ATPase (high affinity, low rate, 2ATP/Ca)
Calcium channels (Ca signalling ON)
Plasma membrane:
- ligand gated (cation selective) in nerve and smooth muscle
- voltage gated (AP responsive) in nerve, muscle, endocrine
Intracellular:
- IP3 receptors everywhere, need Ca and IP3
- ryanodine receptors (RyR) in skeletal/cardiac muscle only need Ca
CICR
calcium induced Ca2+ release
part of calcium ON mechanism
Ca2+ sensors (name/general location)
Troponin C (TNC): skeletal/cardiac muscle, controls actin-myosin interaction
Calmodulin (CaM): in all cells, mediates lots
Calmodulin structure/action
2 loops in 2 domains have negative amino acids to bind Ca2+ and cause conformational change (stretched out with long alpha helix in center)
Interacts with downstream serine/threonine specific protein kinases, phosphatases, PMCA pumps, adenylate cyclases
Ca/CaM dependant protein kinase
CaM-Kinase II
- Ca binds to CaM, with complexes with the kinase to form an activated complex
- complex autophosphorylates using ATP to become fully active
- complex dissociates into 3 parts
- kinase still 50-80% active and is Ca independant
- deactivated by phosphatase
Toxin effect on cAMP (cholera and pertussis)
Cholera: stimulatory alpha subunit of g protein cannot unbind from adenylate cyclase so increase in cAMP (increased PKA activation, Cl channel opening, lots off loss of water and Na into intestine)
Pertussis: inhibitory g protein cannot dissociate (stays as alpha, beta gamma complex) so cannot bind adenylate cyclase (increased PKA activation, high insulin, low glucose causing seizures and high histamine, low pressure causing shock)
cAMP termination
cAMP phosphodiesterase uses H20 to make 5’-AMP
cGMP action
Membrane associated guanylate cyclase: hormone binds, cGMP formed.
Soluble guanylate cyclase: NO diffuses through membrane into cytoplasm causing formation of cGMP
PKG activated, enzymes phosphorylated
cGMP termination
cGMP phosphodiesterase uses H2O to make 5’-GMP
PIP/PIP2/PIP3 formation
PI to PIP via PI-4 Kinase
PIP to PIP2 via PI-5 kinase
PIP2 to PIP3 via PI-3 kinase
(reverse via phosphatases)
Phospholipid derive second messanger compounds (4)
IP3, DAG, AA, PIP3
IP3 and DAG action
- PIP2 cleaved by phospholipase C to form cytosolic IP3 and membrane bound DAG
- IP3 opens IP3 sensitive Ca channel
- Ca and DAG activate PKC to cause phosphorylation of substrates
Insulin pathway
- insulin binds to alpha subunit of enzyme linked receptor
- conformational change and autophosphorylation of beta subunit
- coupling IRS protein’s PTB domain binds phosphorylated tyrosine
- SH2 domain of PI-3K effector protein binds and becomes active
- PIP2 phosphorylated to PIP3
- PDK1 moves to plasma membrane, activated, phosphorylates PKB (both have PH domain)
- PKB leaves membrane and causes vesicle with GLUT4 to fuse to plasma membrane
- GLUT4 moves glucose into cell
Insulin receptor structure
- receptor tyrosine kinase
- constitutive heterotetramer (2 extracellular alpha, 2 transmembrane beta) held together with disulfide bonds
- alpha have cysteine rich domains
- beta have tyrosine kinase domains
Type 2 diabetes therapy goal
-decrease insulin resistance (caused by elevated insulin in blood) and delay insulin therapy by targeting insulin receptor or post receptor pathway
Salivary glands secretion composition and %volume
Submandibular: 70%, mixed mostly serous
Parotid: 25%, serous
Sublingual: 5%, mixed mostly mucous
Saliva secretion pathway (Fluid)
PSN:
- Ach binds to muscarinic ACh receptor (g protein coupled receptor), causing conformational change
- G protein activated with GTP dissociates
- active alpha subunit activates PLC in membrane
- PLC cleaves PIP2 to form DAG and IP3
- IP3 in cytoplasm binds to IP3 ligand gated ion channel receptor causing release of Ca2+
- other pumps and channels activated forming osmotic gradient causing secretion of water and ions
- termination via PK phosphorylation
Saliva secretion pathway (proteins)
SNS: protein secretion pathway, Norepi, causes increased cAMP
- Norepinephrine binds to beta-adrenergic g protein coupled receptor causing conformational change
- g protein activated with GTP then dissociates
- active alpha subunit activates adenylate cyclase in plasma membrane
- cAMP formed from ATP
- cAMP activates PKA
- exocytosis of preformed protein-containing vesicles and synthesis/packaging of new vesicles
Xerostomia therapies
- want long lasting, selective treatment to increase saliva secretion
- target muscarinic ACh receptor using agonists (cholinomimetics)
Odontoblast layer of human teeth
- bacteria/by-products diffuse through dentinal tubules
- bind to odontoblast receptor
- production/release of cytokines, antimicrobial peptides, chemokines
- amplification via autocrine and paracrine (macrophages/immunecells/dendritic cells) signalling
insulin receptor
- enzyme linked recepter
- constitutive heterotetramer
