MCB 2000 Chapter 13

why cells need signaling

• the membrane is a barrier. cells still have to respond and coordinate

• the membrane is selective

  • most signaling molecules can’t simply cross the PM. the message has to be received at the surface and relayed inward

• the environment changes

  • glucose rises, glucose falls, a predator appears. cells need to detect those changes and adjust their internal chemistry

• the body must coordinate

  • liver, muscle, brain. hormones let one tissue tell another what to do, so the whole organism acts in concert

the five-step framework

• signal → reception → transduction → response → termination

• hormone arrives

• receptor binds it

• message relayed

• cell changes

• signal off

without termination, the signal runs forever. cholera is exactly this failure modes.

amplification

• one hormone molecule, three enzymatic steps, a thousandfold response

• each step is catalytic, the receptor, the G- protein-activated enzyme, and pKa all keep working until something turns them off

why amplification matters

• three reasons it shows up everywhere in physiology

• Speed

• economy

• sensitivity

that’s why “all of the above” is the right answer to the flight-or-flight question. speed, economy and sensitivity all matter

speed

• a flight-or-flight response can’t wait minutes for the cell to make enough downstream protein

• amplification lets tiny signal produce a massive response in seconds

economy

• hormones are expensive for the body to synthesize

• amplification means you can release them at a very low concentrations and still hit every target cell

sensitivity

• cells need to detect signals that are present at nanomolar or even picomolar concentrations

• without amplification, those signals would be lost in the noise

GPCR

• G-protein coupled receptors

• Glucagon & epinephrine

  • glucagon released when blood glucose is low

  • epinephrine for fight or flight

• fast, seconds

• “release glucose, NOW”

follows 5-step framework

RTK

• receptor tyrosine kinase

• Insulin

• slower, minutes

• “store that glucose”

follow 5-step framework

what a GPCR looks like

• 7 transmembrane helices and a heterotrimeric G-protein on the inside

• 7 α-helices weave back and forth across the membrane

• the hormone-binding site sits among them on the extracellular side

• on the cytoplasmic side, the heterotrimer αβγ is tethered to the receptor.

  • its α subunit carries the GDP/GTP switch — binds GTP/GDP and that nucleotide shape determines whether the cascade is on or off

• when epinephrine binds, the receptor changes shape and tells α to swap its GDP for GTP

GDP vs GTP

• GDP = off

• GTP = on

• the alpha subunit is a molecular switch with two states

every G-protein in the cell uses this same trick. GDP is the resting nucleotide, GTP is the signal

• when epinephrine binds the receptor, the receptor changes shape and acts like a GEF (guanine nucleotide exchange factor)

• Helps GDP leave the alpha subunit so that GTP can bind

• the extra phosphate on GTP changes the shape of the G-alpha subunit

the full β-adrenergic cascade

1. epinephrine is released from the adrenal medulla. arrives to the liver cell and binds beta-adrenergic receptor

2. receptor acts as a GEF so Gαs swaps GDP for GTP. active Gαs protein dissociates from the beta-gamma subunits

3. Gαs activates adenylate cyclase at the interface of the plasma membrane

4. adenylate cyclase converts ATP into cAMP (secondary messenger)

5. cAMP activates PKA

6. PKA phosphorylates downstream target proteins. In the liver, that cascade activates glycogen breakdown to release glucose into the blood

how cAMP activates PKA

• resting PKA is a R2C2 tetramer, cAMP pulls it apart

• R holds C off

  • in the resting tetramer, R subunits are inhibitors, they bind and block the catalytic Cs

• cAMP binds R

  • 2 cAMP per R subunit, four total. the Rs change shape and release the Cs

• free C is active kinase

  • it diffuses through the cytoplasm phosphorylating serine and threonine residues on target proteins

4 ways tun the signal off

• GTPase activity

• phosphodiesterase

• protein phosphatases

• ligand dissociation

GTPase activity

• Gα has slow intrinsic GTPase activity

• it hydrolyzes its own bound GTP back to GDP, returning to the off state and releasing adenylate cyclase

phosphodiesterase

• PDEs hydrolyze cAMP to plain AMP

• with cAMP gone, R rebinds C and PKA shuts off

protein phosphatases

• phosphatases remove the phosphate groups that PKA installed

• targets return to their unphosphorylated, baseline state

ligand dissociation

• eventually, epinephrine unbinds from the receptor

• without ligand, the receptor stops acting as a GEF, and Gα isn’t being reactivated

why termination matters

• a signal that won’t shut off is a disease in waiting

• Normal: pulse, then silence

  • epinephrine arrives. PKA fires, glucose pours out

  • seconds late, all four shutoff mechanisms kick in, the signal decays and the cell returns to rest

  • response is matched to need

• broken: pulse, then stuck on

  • same hormone arrives, PKA fires.

  • but one of the shutoff mechanisms is broken, Gα can’t hydrolyze its GTP, or cAMP can’t be degraded

  • PKA keeps firing. the cell keeps responding. this is exactly the cholera mechanism

caffeine

• caffeine inhibits phosphodiesterase

• phosphodiesterase is one of the four shutoff mechanisms. it hydrolyzes cAMP to AMP. block it, and cAMP isn’t being degraded

• result: cAMP accumulates. PKA stays active longer and downstream phosphorylation increases

pathway logic: blocking cAMP breakdown prolongs PKA signaling. Note: caffeine’s alertness mostly comes from adenosine receptor blockade

cholera: termination broken

• choleragen locks Gαs in the active GTP-bound form

• what happens in the gut:

  • Vibrio cholerae releases a toxin, choleragen, that chemically modifies Gαs (by ADP ribosylating an arginine) so it cannot hydrolyze GTP

  • adenylyl cyclase keeps making cAMP so,

  • PKA fires continuously in intestinal epithelial cells. it phosphorylates and opens a chlordie channel (CFTR)

  • Cl- pours into the gut lumen, Na+ follows for electroneutrality, water follows by osmosis

  • Result: watery diarrhea up to a liter per hour. untreated cholera can kill in 24 hours from dehydration

not every GPCR uses cAMP

• a second second-messenger system, same 5-step framework

• The Gαq/ PLC pathway

  • some GPCRs don’t use Gαs or cAMP. they use Gαq, which activates phospholipase C (PLC) at the inner membrane

  • PLC cleaves PIP2 into two second messengers: IP3 and DAG. IP3 diffuses to the ER and releases Ca 2+. DAG stays in the membrane. together they activate PKC

  • the point: second messengers are a strategy, not a single molecule. cAMP, IP3/DAG, Ca2+. the receptor decides which one fires

the insulin receptor: an α₂β₂ tyrosine kinase

• two α subunits outside. the two β subunits crossing the membrane, with kinase domains built in

• 
  

• the receptor is its own kinase. pre-built as a dimer, with tow ligand-binding domains outside and two kinase domains inside

a pre-formed dimer, activated by rearrangement

• insulin doesn’t assemble the receptor. it changes the shape of one already there

• the dimer is already built. insulin binding repositions the kinase domains so they can activate each other

autophosphorylation

• trans-autophosphorylation creates docking sites

• what P-TYR does

  • each phosphorylation tyrosine is a docking site. cytoplasmic proteins with the right biding pocket recognize phospho-tyrosine and sticks to it

  • this is how the receptor recruits its downstream cascade. no diffusible messenger like cAMP is needed; the receptor displays docking sites

  • its a totally different signal-transduction logic from GPCRs

adapter protein dock and propagate

• phospho-Tyr is the start of a cascade, not the end

• the insulin cascade

  • phospho-Tyr sites recruit IRS-1, which itself gets phosphorylates and recruits PI3K

  • PI3K coverts PIP2 to PIP3 in the membrane, PIP3 recruits PDK1 and Akt. PDK1 phosphorylates and activates Akt

  • activated Akt drives GLUT4 translocation to the cell surface. RTKs use docking sites plus membrane lipids such as PIP3, rather than cAMP-style diffusion

the end of the cascade: GLUT4 at the surface

• glucose finally enters the cell

• insulin’s job: store glucose . the Atk cascade moves GLUT4 from inside the cell to the surface

termination, RTK style

phosphatases and dissociation, plus internalization

• protein phosphatases: tyrosine phosphatases remove the phospho-Tyr marks on the receptor. adapter protein falls off. the cascade quiets

• lipid phosphatases: PTEN converts PIP3 back to PIP2. Akt loses its docking sits at the membrane and goes idle

• ligand dissociation: insulin unbinds from the receptor. without insulin, the dimer relaxes back to its inactive conformation and the kinase domains stop trans-phosphorylating

• receptor internalization: the receptor itself gets endocytosed and either recycled or degraded. the cell can also down-regulate how many receptors are at the surface

signaling breakpoints

type 2 diabetes: receptor binds, signal stalls

• what insulin resistance means:

  • hormone is fine. receptor binds

  • something in the IRS-1 → PI3K→ Akt chain is impaired

  • Phospho-Tyr signals arrive but don’t propagate

  • GLUT4 stays in vesicles. without GLUT4 at the surface, muscle and fat cells can’t take up glucose efficiently and blood glucose stays high

• what contributes:

  • excess intracellular lipids and inflammatory signals can activate stress kinases that interfere with IRS1 function

  • chronic high insulin contributes to receptor downregulation and feedback inhibition

  • result= blunted signaling through the IRS1 pathway because AKT signaling is what drives GLUT 4 translocation, fewer GLUT 4 transporters reach the surface of muscle and fat cells so glucose uptake is impaired and glucose stays elevated

the compensation spiral

• the pancreas tries harder. eventually it burns out

two diseases, two break points

• break at transduction:

  • receptor binds insulin normally, but IRS/ Akt signaling is impaired. GLUT4 doesn’t reach the surface. glucose stays high

  • chronic, slow onset. treated with sensitizers or exogenous insulin

• Break at termination

  • cascade fires correctly, but Gαs can no longer hydrolyze GTP. PKA stays on, chloride flows out, water follows

  • acute, can kill in hours. treated with aggressive rehydration

the whole story in one picture