Chapter 15

Cell signaling

Cells respond to each other and their environment through signaling
1. Most signal molecules are polar
  1. A cell will send out a signaling molecule into the ECM, and it will have complementarity for a receptor in the membrane of another cell
  2. Once the signal binds to the receptor, the receptor will activate a cascade of events
  3. It usually will result in 2 of 3 major effector pathways
    1. alter metabolism
    2. alter gene expression (transcription factors)
    3. affect the cytoskeleton
  4. interactions between signals and their receptors have extremely high specificity
Forms of intercellular signaling
1. contract dependent
  1. 2 cells have to be in physical contact with each other
    1. Both the signal molecule and receptor are transmembrane proteins
      1. Example: antigen presentation in the immune system
        One cell is attached to an antigen that is being presented, and T cells have a receptor attached that binds to that
2. paracrine
  1. close local signaling
    1. a cell will release a signal molecule, but it will not travel far, so it can only bind to receptors on nearby cells
      1. example: blood clot pathway
        when you get a cut, blood cells in that vicinity are triggered to form a clot
3. synaptic signaling
  1. reserved for cells with neuron-like properties
    1. cells with axons or dendrites
    2. signaling is at the molecular level, it is a type of close contact signaling
      1. the signal molecule is released from the end of an axon through a small cleft called the synapse, where it will bind to the receptor on the next protein
        molecules are not traveling far between cells, but because the length of some neurons’ cell bodies they may travel a long way within the cell
        long distance travel in terms of body, but short distance for the signal molecule to travel
4. endocrine signaling
  1. when a hormone is used as the signaling molecule
    1. the hormone is distributed into the bloodstream
      1. this is very long-distance signaling
    2. cells will not respond to a hormone without the appropriate receptor
receptors
1. Cells receive signal molecules through receptors
  1. very complex structures shaped perfectly to recognize a signal with high specificity
2. 3 classes of transmembrane receptors
  1. ion channel coupled receptors
    1. has ions that the channel is specific for, but will not open for the ions unless the channel is bound to a ligand (the signal molecule)
  2. G-protein coupled receptors
  3. enzyme couple receptors
3. Cell surface and intracellular receptors
  1. intracellular receptors
    1. have a hydrophobic signal molecule that can go through the membrane on its own
      1. this also means that the signal is not stable in ECM, so it is usually carried by a carrier protein
    2. signal will bind to an internal receptor that is either in the cytosol or the nucleus
      1. pathways that use this include the steroid hormone pathways such as those that respond to testosterone or estrogen
Molecular switches
1. can turn on/off the actions of certain proteins
2. 2 of the most common ways
  1. phosphorylation
    1. proteins are phosphorylated by kinases and phosphates are removed by phosphatases
      1. example: phosphate is taken off of ATP, turning on a protein by phosphorylation and off be dephosphorylation
      2. Different proteins can be activated by either phosphorylation or dephosphorylation
  2. GTP binding
    1. G-proteins are on when GTP bound and off when GDP bound
    2. proteins that help hydrolyze GTP→GDP are called GAPs and those that help exchange GDP→GTP are GEFs
Complexity (specific and precise)
1. need to be able to have large numbers of specific and efficient responses
  1. cells respond to combinations of signals
    1. ex: survival
      1. say cells have to have signals A,B, and C to survive
      2. if the cell receives a different combination of signals such as A, B, C, D, and E it will survive, grow, and divide
      3. if instead it receives A, B, C, F, and G the cell will survive and differentiate
      4. If they do not get a combination of the survival signals they will die, usually by apoptosis
        what if the combinations are incomplete
        the cell will die or at very best be very sick
One signal different responses: acetylcholine
1. is a neurotransmitter that has different receptors in different cell types
  1. can bind to a receptor in a heart pacemaker cell that will slow down the heart rate
  2. can bind to a receptor in a salivary cell and it will cause the secretion of survival
  3. can bind to a receptor in skeletal muscle cell and cause contraction
    1. binding sites for ligands are often only comprised of a few amino acids, so all three receptors have the same binding site, but the rest of the protein is different and that is why there are different responses
Always activation? double negatives
1. signals binding to receptors do not always activate a pathway
  1. sometimes signal is sent to activate a cell signaling pathway
  2. othertimes the signal can be sent to shut off a pathway
2. when trying to figure out the end result of a pathway, you have ot look at how many activation and inhibition steps are present
  1. inhibiting an inhibitor = activation
Many signals affect a cascade of proteins
1. signaling complexes
  1. scaffold protein
    1. signal binds to receptor
    2. receptor binds scaffold proteins that have binding sites for first few downstream molecules (which are typically proteins), bringing them together so they are then activated in a series
  2. receptor acts as scaffold protein
    1. will not recruit downstream proteins unless signal is bound
      1. signal binds receptor
      2. receptor tail ggets phosphorylated
      3. recruits downstream proteins
  3. PIPs
    1. pips are used as scaffold
      1. signal binds to receptor
      2. receptor phosphorylates pips
        pips are anchored to the membrane
      3. pips recruit downstream proteins
2. roles of phosphorylation
  1. ability to activate/inactivate proteins downstream
  2. create new binding sites
    1. some proteins only bind to binding partner if the binding parter is phosphorylated
3. singaling complexes increase efficiency
  1. they do not always bring all downstream proteins but they bring the first few
  2. Phosphorylation can affect binding affinity
Speed of response - turnover rate of effectors
1. fast if just changing the function of a protein
2. slow if altered protein synthesis
3. turnover: balance between synthesis and degradation
Signal processing
1. 3 processing curves
  1. all-or-none
    1. off or on entirely, slight delay but once activated will activate completely
  2. hyperbolic
    1. sensitive to amount and presence of signal
  3. sigmoidal
    1. reach a threshold amount of signal to start, but is then dependent on concentration of signal
Positive feedbakc control
1. something in a pathway interacts with an earlier step in pathway and ramps up that pathway
2. sustained signal after initial signal
  1. ex: oxytocin pathway during chilldbirth
negative feedback
1. something in the end of the pathway blocks something else earlier on
2. short delay
  1. enzyme phosphorylates inhibitor to shut off
  2. happens at a fairly similar rate as enzyme is activated by signal
3. long delay
  1. inhibition step is slow
  2. will cause a back and forth on and off response
All-or-none response determination
1. could be an actual sigmoidal response where none respond, then all slightly respond, then all a bit more until all are fully responding
2. could be that no cells are responding, then a few are, then more are, then all are
desensitization of cell signals (adaptation)
1. respond to changes in signal concentration because cells can get used to a signal being around and learn to ignore it
  1. group 1
    1. receptor sequestration
      1. cell endocytose signal and receptor and destroys the signal because it does not want it anymore then recycles the receptor
  2. group 2
    1. receptor down-regulation
      1. cell will endocytose signal and receptor then destroy both
  3. group 3
    1. all activate an inhibitor to shut down pathway
      1. receptor inactivation
        inhibitor thats activated with signal inhibits the receptor
      2. inactivation of signaling protein
        inhibitor inhibits something that is not the receptor
      3. production of inhibitory protein
        specifies what type of inhibitory protein you have
G-protein-coupled receptors
1. They all have a G-protein-coupled receptor with 7 transmembrane domains that can act as a GEF
  1. 2 types of G-proteins
    1. monomeric
    2. trimeric
2. When a signal activates a trimeric G-protein, it will split into 2
Trimeric g-protein
1. alpha subunit
  1. has a lipid tail
  2. When activated, it kicks off the beta and gamma subunit
Activation of the G-protein
1. Signal binds to the GPCR, and that activates the alpha subunit
2. GPCR acts as a GEF to activate and exchange its GDP for GTP
3. Once alpha is activated, it will kick off beta and gamma
Second messenger - cAMP
1. A second messenger is a non-protein molecule that’s roughly the same size as a building block for a macromolecule
2. Second messengers are small molecules that spread and amplify signals
  1. examples: ions, single nucleotides, a pair of two amino acids, single amino acids
  2. not include any macromolecules
  3. can release 100s or thousands of secondary messengers very quickly
cAMP production
1. comes from ATP nucleotide
2. adenylyl cyclase will take off 2 phosphates from ATP and make the third cyclic
3. To get rid of cAMP, the cell can activate cyclic AMP phosphodiesterase, which will undo the cyclic part and make it normal
Signals can alter cAMP levels
1. Cholera toxin
  1. enzyme that transfers ADP ribose from NAD+ to the stimulatory G-protein alpha
  2. stimulatory G-protein can no longer hydrolyze GTP, so it is always active
  3. Too much cAMP causes Cl- and water efflux to the gut, causing the severe diarrhea associated with the disease
2. pertussis toxin
  1. catalyzes ADP ribosylation of a G alpha inhibitory protein
  2. prevents the inhibitory G-protein from binding to a receptor, so there is no response
PKA pathway
1. protein kinase A
  1. has 2 regulatory subunits and 2 inactive catalytic subunits
  2. it is usually inhibited when found ina cell
  3. 2 cAMP bind to each regulatory subunit and change their conformation, and they leave the kinase subunits alone
  4. active kinase subunits phosphorylate things
Concentration of cAMP alters gene transcription
1. A signal activates the GPCR, turning it into a functional GEF
2. GPCR activates the G-protein (alpha subunit of the stimulatory pathway)
3. The alpha subunit of the G-protein will activate the next enzyme in the pathway, adenylyl cyclase
4. Adenylate cyclase will convert ATP to cAMP
5. cAMP binds to the regulatory subunits of PKA and removes them, activating the 2 PKA enzymes
6. PKA goes into the nucleus and phosphorylates the CREB protein
7. active CREB binds to an enhancer and brings CBP
8. The name of the enhancer is cAMP response element (CRE)
9. This activates transcription of target genes
PLCbeta pathway
1. utilizes pips
2. PLCbeta acts on PI(4,5)P₂ and cuts it into 2 pieces, diacylglycerol and inositol 1,4,5 - triphosphate (IP₃)
3. pathway
  1. Signal binds activating GCPR, which activates the alpha subunit of the trimeric G-protein
  2. The alpha subunit activates the beta gamma subunit
  3. beta gamma activates PLCbeta
  4. PLC beta finds PI(4,5)P₂ and cuts it in half
  5. diacylglycerol remains in the membrane, and IP3 leaves for the cytosol
  6. IP3 is a ligand for a gated channel on the ER membrane
  7. IP3 will bind and open the channel, and the channel will spill out Ca2+ ions into the cytoplasm
  8. Ca2+ ions bind and activate PKC, which is recruited and tethered to the membrane by diacylglycerol
phototransduction
1. vision depends on GCPRs
2. uses cyclic AMP
3. rod receptors
4. fastest G-protein response in vertebrates
5. The pathway takes place in the outer segment
6. b
7. rod receptors respond to light
  1. off state
    1. in dark / not active
    2. The receptor is rhodopsin
    3. rod cells are releasing neurotransmitters that block the optic nerve
    4. The cell is depolarized
    5. Sodium channels are open
  2. on state
    1. A photon of light is the signal activating rhodopsin
    2. Rhodopsin shuts all sodium channels, and the membrane becomes hyperpolarized
    3. The membrane stops releasing neurotransmitters, so there is no more inhibition of the optic nerve
8. Pathway
  1. Rhodopsin has a cofactor called retinal, and it’s in the 11-cis form
  2. a photon of light changes retinal to trans form
  3. This conformation change activates rhodopsin
  4. Rhodopsin is a GCPR, so it is a GEF
  5. Rhodopsin activates the alpha subunit of the trimeric G-protein transducin
  6. The alpha subunit activates a phosphodiesterase (PDE6B)
  7. PDE6B turns cGMP to normal GMP
  8. The ligand for the sodium channel is cGMP, so GMP can not substitute, and the channel will close without cGMP
  9. The channel is CMGA1/CMGB1
  10. membrane becomes hyperpolarized
9. signal amplification
  1. 1 rhodopsin absorbs 1 photon of light
  2. 1 rhodopsin activates 500 transducin
  3. 500 transducin activate 500 cyclic GMP phosphodiesterase
  4. Phosphodiesterases hydrolyze 10⁵ cGMPs
  5. GMPs close 250 ion channels
  6. Between 10⁶ and 10⁷ Na+ ions per second are prevented from entering the cell
enzyme-coupled receptors
1. Normally are present in the membrane as monomers
2. activate by forming a dimer
  1. has a monomer or a dimer signal molecule
3. The receptor is an enzyme or is attached to one
Receptor tyrosine kinase (RTK)
1. The receptor is an enzyme
2. has diverse structures
  1. GF= growth factor
General steps for RTKs
1. inactive monomers
2. signal comes and they dimerize
3. transautophosphorylation (they phosphorylate each other), causing hyperactivation of kinase domains
4. phosphorylate its own tail
5. The phosphorylated tail makes new binding sites
6. The next couple of enzymes bind to the phosphorylated receptor
Ras pathway
1. signal binds to the receptors and they forms dimer
2. activate and transautophosphorylate
3. The adaptor protein Grb2 will bind to the phosphorylated receptor
4. Grb2 recruits Sos (a Ras-GEF)
5. Sos finds Ras and exchanges its GDP for GTP
6. Ras is activated
  1. Just because Ras is a G-protein does not mean this is G-protein receptor-coupled signaling
Ras activates a kinase cascade
1. a cell survival pathway
  1. Substrates are phosphoinositides (PIPs)
  2. PI 3-kinase always adds a phosphate to carbon 3
  3. Pathway
    1. signal binds
    2. transautophosphorylation
    3. phosphorylates tail
    4. receptor tail recruits PI 3-kinase
    5. PI 3-kinase phosphorylates PI(4,5)P₂ into PI(3,4,5)P₃
    6. PI(3,4,5)P₃ recruits pH domains PDK1 and Akt
    7. phosphorylation and activation of Akt by both PDK1 and mTOR
      1. before the pathway is on, inactive apoptosis inhibitory protein is bound to Bad (when Bad is bound the cell willl die because it inhibits the inhibitor)
    8. Active Akt finds and phosphorylates Bad and Bad can not bind apoptosis inhibitor anymore
    9. apoptosis inhibitor will go stop a pathway so the cell stays alive
JAK-STAT pathway
1. signaling through cytoplasmic kinases
2. JAK proteins closely associate with receptor
3. Cytokines activate the JAK-STAT pathway (immune cells have the most active JAK-STAT signaling)
4. Pathway
  1. cytokine, the signal, binds the receptor and the receptor dimerizes
  2. JAKs phosphorylate one another, then the receptor tails
  3. JAKs recruit and phosphorylate STAT1 and STAT2, and they dimerize
    1. can dimerize as a homodimerize because it can be a combination of any STATs, depending on what is available
  4. STAT dimer goes into the nucleus
  5. It is a transcription factor, so it binds to an enhancer called the cytokine response element (CRE)
  6. Any downstream genes are activated
    1. The pathway is heavily modified by negative feedback because the immune system can cause damage if it is on for too long
TGFbeta
1. signaling through serine/threonine kinases and Smads
2. forms heterodimers from type 1 and 2 (kinase) receptors
3. pathway
  1. TGFbeta signal binds receptor
  2. Type II TGFbeta kinase phosphorylates type I TGFbeta domain
  3. Activated TGFbeta I recruits receptor smads 2 or 3 (rSmads) and phosphorylates them
  4. Smad 2 and 3 form a trimer with Smad 4
  5. trimer goes into the nucleus and binds TGFbeta response elements, causing transcription and translation
    1. Technically, TGFbeta receptors are homodimers that form a heterotetramer
Turning off the TGFbeta pathway
1. 2 ways endocytosis of receptor/ ligand complex
  1. activation route (clatherin coat)
    1. uses protein SARA
    2. SARA is gentle and will bring in the receptor if it does not want the signal anymore, and pull off the signal, and then wait to recycle the receptor
  2. Inactivation route
    1. uses cavaolae coats
    2. Brings the receptor using internal vesicles and destroys it
2. Inhibitory Smads 6 and 7
  1. competition with rSmads
3. Smurf
  1. destroys rSmads and other proteins
4. recruitment of phosphates
  1. this also regulates many other pathways
Alternative signaling
1. 2 developmental pathways
  1. notch pathway
    1. activated by proteolytic cleavage
      1. cleavage happens at site 1 in golgi
      2. receptor goes into the plasma membrane
      3. receptor binds to signal delta, which is embedded in the membrane of a neural cell
      4. 2nd cleave happens on notch
      5. 3rd cleave happens on notch and the receptor tail is a transcription factor
      6. tail goes into the nucleus
      7. tail finds protein Rbpsuh, if Rbpsuh is alone it is a transcription repressor, but if it is bound to the tail it is a transcription activator
    2. 3rd enzyme that cuts in pathway is gamma-secretase and it has high connections to Alzheimers
2. Wnt pathway
  1. off
    1. the signal wnt is not present and the receptor for wnt is called frizzled
    2. the protein dishevelled is inactive
    3. complex of 4 proteins: Axin, CK1, GSK3, and APC form a binding pocket for the protein Beta-catenin, and it gets phosphorylated and destroyed
  2. on
    1. wnt binds and frizzled is activated
    2. frizzled recruits dishevelled and activates it
    3. dishevelled busts up the complex of 4 proteins that destroy beta-catenin
    4. beta-catenin is stable and goes into the nucleus and kicks groucho off of DNA and acts as a co-activator for TFC LEF