Chapter 15 - Dr.Siebler PDF

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Dr.Siebler...

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4Outline - Part 1 Principles of Cell Signaling 

Cells respond to each other through signaling

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o o High specificity between signal and receptor o Some signals can cross the membrane, but majority can not  Most signals are polar because that is the kind of environment found in cells  Because they are polar, they can not cross the membrane o There are receptors that are transmembrane proteins that bind to signal on one side and causes the change of conformation on the other side  This activates downstream pathways o Effector proteins result in the final action  Alter metabolism  Alter gene expression  Alter cell shape or movement Forms of intercellular signaling

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o Contact Dependent o Must touch other cell for target cell to recognize the signal cell

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Paracrine

Ex: Immune system: Dendritic cells will pick up something and bring it to T-cell to see there is a problem  They must touch  Like a hug

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o o Sends signal to local cells  Short distance signaling  Signal doesn’t last very long  Ex. Blood clot wound: not all blood should clot, only clot blood cells that are near wound Synaptic o Cells of nervous system or sensory organ system

o o Can be very long distance, but only as long as axon o Long distance signaling o Releases signal into very small synapse  Only sends signal to target Endocrine o Most long distance signaling o Sends signal into bloodstream  Usually hormones  Affects any and every cell that has the receptor for signal

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o Receptors o

Only will open if binds to signal molecule  The channel is not the ion that goes through  The signal is different than the substrate or solute that goes through  Note color change o Cells receive signals through receptors o Complex structures shaped to recognize the signal molecule with high specificity  Want cell to respond to correct signal! o Three classes of transmembrane receptors  Ion channel coupled receptors  G-protein coupled receptors  Enzyme coupled receptors Cell-surface and intracellular receptors 

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o o Second type of receptors: intracellular receptors o Must be nonpolar signal to go through Molecular Switches

o o Kinase adds phosphate to proteins to turn on or turn off  Phosphate comes from ATP to form ADP  Some proteins are activated by phosphorylation, other proteins are inactivated by phosphorylation o G-proteins  Only Off in the GDP state; only On in the GTP state  GDP is removed and GTP is added to activate protein  Done by GEF  GTP is hydrolyzed  Done by GAPs o Phosphatase removes phosphate group Complexity (specific and precise!) o Nice to think of one ligand, one receptor, one effector o Is biology ever that simple?? o Need:  Be able to have large number of specific and efficient responses o How?  Through cell-cell communication  Make some sense out of signals

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Signaling features that differ between systems for regulation o Response timing – Once signal is received; how long does it take to get response? o Sensitivity – how much signal do you need? o Dynamic range – over what range of signal concentration do we get activation? o Persistence – how long does the response last? o Signal processing – on/off? Oscillatory?  Is it on and off self regulation o Integration – need multiple inputs/diff. signals?  How many signals to one response? o Coordination – multiple responses to one signal  How many responses to one signal? Cells respond to combinations of signals

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o o Some responses require multiple signals  The combination of signals dictates what will happen  Each signal does not have a specific function, it is the combination of signals o What if the combination is incomplete?  It will die One signal, different responses: Acetylcholine

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o Multiple responses to one signal type o Acetylcholine can have different effects on different cell types  Receptors have same binding site but they are all different receptors  There can also be two cell types with the same receptor but the downstream signaling is different  Different responses to same signal at organismal level or cellular level Always activation? Double negatives

o o Not all signals turn on pathways  Some turn off pathways o Negative means something is being turned off o Positive is something is being turned on o Double negatives means that the gene expression is turned on  The inhibitor protein is being turned off so there is no inhibitor and thus gene expression is on o Example:  Protein kinase turns off inhibitor protein.  The inhibitor protein usually blocks the transcription regulator

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o Since inhibitor protein is off, the transcription regulator is on  Results in gene expression Many signals affect a cascade of proteins Signaling complex 1

o o Preformed complex  Bound to a scaffold protein, they are all gathered at the receptor waiting  Starts going as soon as signal binds to receptor o Everything is preassembled, and the signal activates the receptor then affects protein 1, then 2, then 3  Don’t have to look through protein soup o Makes sure everything is in close vicinity Many signals affect a cascade of proteins Signaling complex 2

o o When inactive, the receptor is not bound to anything  Assemble all proteins to receptor tail when receptor is activated

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 When active, it gathers all the proteins to the tail o Benefit is nothing is activated until signal binds  Other pathways may need the enzyme so its better to not have it blocked off New binding sites created by phosphorylation are specific o Do not bind until tail is phosphorylated o Phosphates creates new binding sites

o o 2 pockets  One pocket says which receptor to bind  One is used to make sure binds to correct receptor only when receptor tail is phosphorylated  One site for phosphorylated amino acids o Tyrosine (major), serine, or threonine Many signals affect a cascade of proteins: Signaling complex 3

o o Tether everything to the membrane o When there is no signal, the complex does not form o When there is a signal bound to the receptor

PIPS become hyperphosphorylated  The proteins require all three binding sites o The PIPS will be hyperphosphorylated  Brings the Proteins  Brings it to the membrane Speed of response? Depends on turnover rate of effectors o Turnover – balance between synthesis and degradation  How fast it will be made and its half life o Lot of factors how fast a pathway will become active o Fast  Signal binds to receptor and modifies function of protein to turn on  Left pathway o Slow  Needs to make something new  Last step in pathway is transcription factor  Send TF to nucleus,  Wait for genes to be transcribed o RNA has to be exported  Wait for protein to be synthesized by ribosomes o Basically, pathway will be faster if there does not have to be transcription/translation  Right pathway 

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o Signal Processing

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o All -or-none response  Respond or don’t respond  There is a threshold concentration it takes for response to occur  Once threshold is hit, there is a 100% response o Hyperbolic response  Signal and response are linear until max response o Sigmoidal response  There is a threshold to make the response happen  But as signal increases it will keep increasing amount of signal Positive feedback control

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Positive feedback  Something downstream in a pathway favors synthesis of something upstream or activates something upstream  Cells with positive feedback have a sustained signal  Once active, the pathway stays active longer than the signal is present If signal activates kinase and turns on  If product can activate more substrate, then it will keep activating more pathway until it is inhibited by the inhibiter

o “I” stands for inhibitor o

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No feedback  If no signal, there is not response  If there is signal, it will respond

 Negative feedback o Something downstream in the pathway turns the pathway enzymes off o Speed game of how quick the signal can turn the pathway off  Vs how quick the pathway can create the enzyme

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Short Delay  The speed of signal being made and enzyme turning off are the same  Taking turns making enzyme and turning off Long delay  It takes a while for enzyme to build up to turn off  Oscillatory response o Activate them all then turn them all off

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o o The enzyme goes back and shuts off the enzymes around it All or none response determination

o o Sigmoidal response  After hitting threshold, they will all respond more and more  Gradual increase o All or none  Some cells are completely on and others are completely off  This graph shows the average of the cells  We have to think how the experiment was done to see if all the cells were responding at the same time or one at a time Desensitization of cells to signals (adaptation) o Respond to changes in concentration of signal!

o o Receptor sequestration

Bring in receptor  Get rid of ligand in endosome o Bring receptor back Receptor downregulation  Bring receptor in  Receptor and signal gets degraded by lysosome Receptor inactivation  Block receptor inside of cell with inhibitor Last three utilize an inhibitor  Just inhibits different steps in the pathway Downregulation used for  To turn off pathway  Desensitize Desensitization  Cells can have sensory overload  Respond to it at first but then adapt o Only respond if signal is increased 

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Outline - Part 2 G-protein coupled receptors Over 80% of drugs block this pathway See smell and taste use this pathway 

G-protein-coupled receptors

o o Structure  Downstream is a trimeric G-protein  3 subunits o Receptor, G-protein, enzyme downstream o Steps:  When signal binds to receptor  Recruits G-protein o Usually causes it to exchange GDP for GTP and becomes active  Splits into 2 halves  Activates downstream enzymes o Receptor – G protein - effector

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 Trimeric G-protein G-protein-coupled receptors (GPCRs)

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o o These pathways have very similar receptors  Called GCPR’s  G-protein coupled receptors  All have 7 transmembrane domains  Different amino acids but similar structure Inactive trimeric G protein

o o Three domains of trimeric G-protein  Not a Ras protein  The domain is only named because it looks like it  Alpha domain  Has a nucleotide domain o GTP or GDP bound  Lipid tail anchor  Beta domain  Stays wherever gamma is  Gamma domain  Lipid tail anchor

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o When protein splits, the halves will stay in the membrane because of lipid tails Activation of G proteins

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 Activated GPCR acts as GEF o Steps  When signal binds GPCR changes conformation and becomes active GEF  Exchanges GDP for GTP to become catalytically active o Alpha and beta-gamma region split o Alpha or beta-gamma may activate the pathway downstream Second Messengers – cAMP o Second messengers spread and amplify the signal o Small molecules  Single nucleotide, single ion, not larger than a disaccharide

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Production of cAMP

o o Cyclic AMP is a nucleotide o Formed by enzyme adenyl cyclase from ATP  Remove two phosphate and forms cyclic bond to make cAMP o Cyclic AMP phosphodiesterase  Breaks cyclic bond to make AMP (adenosine monophosphate) Signals can alter cAMP levels through stimulatory and inhibitory G proteins o DON’T NEED TO MEMORIZE o Cholera toxin  Enzyme that transfers ADP ribose from NAD+ to Gs alpha  Gs can no longer hydrolyze GTP, so is always active  Too much cAMP causes Cl- and water efflux to gut, causing severe diarrhea associated with the disease o Pertussis toxin  Catalyzes ADP ribosylation of Gi alpha  Prevents Gi from binding to receptor so no response 1. PKA: cAMP activates protein kinase A (PKA)

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o o PKA is a hetero tetramer in inactive state  Two domains  Regulatory subunit  Inhibitor (inactive catalytic subunit) o Protein Kinase A is activated by cyclic AMP  cAMP Binds to regulatory subunits and release the active catalytic subunits Concentration of cAMP alters gene transcription

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o 1. Signal binds to receptor a. Will act as GEF 2. Receptor is now activated a. Activate Gs-protein 3. Activated receptor results in active G protein a. Activates downstream enzyme 4. G protein activates adenylyl cyclase 5. Adenylyl cyclase cleaves ATP to make cAMP 6. cAMP binds to regulatory subunits of PKA and they dissociate 7. Now PKA is active 8. Goes through the NPC into nucleus a. PKA activates CREB by phosphorylating 9. CREB is activated and binds to cyclic AMP response element (CRE) which is a transcription factor for gene transcription 10. Gene transcribes

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o DO not need to know, these are just possible responses 2. PLCβ Signaling through phospholipids o Players  PLCβ – phospholipase C-β  PI(4,5)P2 – phosphatidylinositol 4,5-bisphosphate  IP3 – inositol 1,4,5-trisphosphate  Diacylglycerol  PKC – protein kinase C Hydrolysis of PI(4,5)P2 by PLCβ

o o PLCβ substrate is PI(4,5)P2  Splits PIP2 into two molecules  Diacylglycerol o Stays in membrane

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 IP3 o Split into diacylglycerol and inositol 1,4,5-triphosphate How GPCRs raise Ca2+ and activate PKC

o Signal comes in and activates GPCR o Becomes an active GEF Activate alpha subunit of G-protein Beta-gamma will be activated by alpha and split off o Beta-gamma will regulate downstream effects Activated PLCβ by beta-gamma subunit PLCβ will cleave PIP2 into diacylglycerol and IP3 IP3 is an example of a small molecule o Nonprotein mediator o Ligand for a ligand gated calcium channel IP3 comes to the membrane of the ER and binds to the IP3 gated Calcium channel to open it o Calcium is very high in the ER Once channel is open, calcium flows out of the ER down its concentration gradient Calcium activates PKC but it must be tethered to the membrane done by diacylglycerol o Ca 2+ is a ubiquitous intracellular mediator! o Used for many pathways Functions

o o Downstream of PKC pathway

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3. Phototransduction: Vision depends on GPCRs o Visual transduction o Fastest G-protein response in vertebrates o Rod receptors o Utilizes cGMP

o o Cell type specific  Only has one outcome o Just black and white vision  Used in low light o Synaptic region looks like neuron Rod receptors respond to light o Signal that activates rhodopsin?

o o Transmitters inhibit retinal neurons: No inhibition of retinal neurons o Rhodopsin activates the GPCR o If completely dark, rhodopsin is inactive  Channels are open if Rhodopsin is off  Black dots are rhodopsin o 1 photon of light will turn Rhodopsin on  Closes the channels  Very negative inside than outside

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o cGMP turned to GMP so it can not hold channels open any more o 1. Light hits rhodopsin o 2. o 3. o 4. Transducisn activates o 5. PDE6B converts o 6. Channels close o 7. Because of close, neurotransmitter doesn’t release o 8. Allows to see dim light Steps: Rhodopsin has 11-cis-retinal that reacts with light and turns into 11- trans-retinal Change of conformation activates next step of pathway Activates rhodopsin which activates the GPCR called transducin Once GPCR transducin is active, alpha domain will activate PDE6B PDE6B converts cGMP into GMP cGMP keeps CNGA1and CNGB1 which is a ligand gated channel open but without it, the channel is closed because GMP can’t do that anymore Cell becomes hyperpolarized and neurotransmitters have low release See dim light Rod cell releases glutamate which activates bipolar cells Bipolar cells give GABA which shuts off the optic nerve

Signal amplification by second messenger cGMP o

o o One rhodopsin can activate 500 transducins o There is a lot of amplication Outline - Part 3 Enzyme coupled receptors 

Enzyme-coupled receptors

o o They all require dimer formation to be active  When bound to signal it forms a dimer  Signal allows them to autoactivate each other  Like IRE1 pathway  Monomer before signal  The signal can be a dimer itself or the signal binds to both domains together  In these pathways, receptor is enzyme itself or is tightly bound to enzymes

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 To be an enzyme, it has to change substrate to a product o Receptor is an enzyme or activates an enzyme Receptor tyrosine kinases (RTKs) o Many signal proteins act through these receptors o Don’t need to know table

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o Receptors are single pass, but diverse

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o o Don’t memorize this, just appreciate diversity General steps for RTKs

o o Dimerization o Steps:

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1. When bound to signal, it causes the receptor to form a dimer 2. They become close enough to phosphorylate each other’s kinase domains  Without this, they are a little bit active but after phosphorylation they are very active  Add phosphate to tyrosine kinase domain 3. Once active, they can phosphorylate each other at other sites as well  Trans auto phosphorylation 4. Phosphates cause recruitment of additional downstream proteins 1. Ras Activation of Ras by an RTK o Ras pathway is cell growth and proliferation pathways mainly  Mutated in 1/3 of all human cancer patients  Inhibiting Ras is hard to do

o o Steps 1. Signal binds to receptor and forms dimer 2. The monomers phosphorylate each other and then again along length of tail 3. Once receptor is active, it recruits an adaptor protein called Grb2  Grb2 is not an enzyme but it is an adaptor protein  Has a SH2 domain that bind to the phosphorylated region of the receptor o Really high affinity for receptors with phosphorylated tyrosine on it 4. Grb2 recruits Sos which is a Ras-Gef 5. Exchanges Ras GDP with GTP 6. Gives off active Ras which activates downstream signals  Monomeric G-protein o Just because Ras is a G protein does not mean that this is a G-protein receptor coupled signaling! Ras activates a kinase cascade

o o Ras attempts to achieve some amplification o Activates a string of kinases called a kinase cascade o Ras activates Raf through phosphorylation  Activates Mek  Activates Erk o Phosphorylates downstream proteins  Both fast and slow pathways are turned on 

2. PI3K-Akt PI3-kinase creates phosphoinositide docking sites

o o 3rd carbon have phosphate added by PI3 Kinase

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1. Receptor binds to signal, forms a dimer, phosphorylate each other 2. Activated receptor phosphorylates and activates PI3 Kinase 3. PI3 Kinase adds a phosphate to carbon 3 and converts PI(4,5)P2 to PI(3,4,5)P3 4. PIP3 Recruits PDK1 and Akt to membrane 5. mTOR is recruited 6. Activation of Akt a. Before activation, PDK1 and mTOR bind to PIPs b. mTOR gives phosphate to Akt c. Akt gets other phosphate from PDK1 d. This all needs to happen to activate Akt 7. Akt is now activated and leaves the membrane a. Goes to protein Bad and phosphorylates Bad to inactivate it 8. Bad Protein inhibits an inhibitor of cell death a. So if there is Bad, they will undergo apoptosis b. Phosphorylated Bad is inactivated and releases the ...