Changes in signal transduction

Two ways to break a switch

Every protein in a signalling pathway is a switch that turns on when the signal arrives and off when it goes away. A mutation or a toxin can change that switch in one of two ways.

• A loss-of-function change stops the protein working. The switch is stuck off. The pathway can't be turned on, even when the signal is present.
• A gain-of-function change makes the protein work when it shouldn't, or more than it should. The switch is stuck on. The pathway keeps signalling even when the signal has gone. A protein that's always active is called constitutively active.

Predicting the effect, step by step

Take the adrenaline pathway in a liver cell: adrenaline → GPCR → Gs → adenylyl cyclase → cAMP → PKA → glycogen breakdown. Here's how to predict what a defect at each point does.

• Receptor can't bind adrenaline (loss): no response to adrenaline at all. The rest of the pathway is fine, so a drug that raises cAMP directly would still work.
• Receptor active without adrenaline (gain): glycogen breakdown all the time.
• Gs can't bind GTP (loss): no signal passes the G protein, however much adrenaline arrives.
• Gs can't hydrolyse GTP (gain): once switched on, it stays on. cAMP keeps being made after the hormone has gone.
• Phosphodiesterase missing (loss of an off-switch): cAMP builds up and lingers, so the response is stronger and longer. Losing an inhibitor has the same effect as gaining an activator.
• PKA missing (loss): no glycogen breakdown, even though cAMP rises normally.

Notice the pattern. A defect's effect shows up at its own step and everything after it. Components upstream of the defect still behave normally: in the "PKA missing" case, cAMP still rises.

Cholera toxin: a G protein that can't switch off

The toxin De found is made of two parts. A ring of five B subunits binds a lipid called GM1 on the surface of intestinal cells, and the cell takes the toxin in. The A subunit is an enzyme that then attaches a chemical group (an ADP-ribose) to the α subunit of Gs.

1. 1The modified Gα can't hydrolyse its GTP, so it's locked in the active state.
2. 2Adenylyl cyclase is switched on continuously, and cAMP levels in the intestinal cells rise and stay high.
3. 3cAMP activates PKA, which phosphorylates and opens a chloride channel called CFTR in the membrane facing the gut.
4. 4Chloride ions (Cl⁻) pour out of the cells into the gut. Sodium ions follow, attracted by the negative charge.
5. 5The salt in the gut draws water after it by osmosis. Huge volumes of water are lost as diarrhoea, and without treatment the dehydration can kill.

Cholera is a clear example of a gain-of-function change caused by a toxin rather than a mutation. No hormone is needed: the pathway is stuck at the G protein, so everything after it is on.

Ras, growth signals and cancer

Recall that growth factors act through receptor tyrosine kinases, then Ras, then the MAP kinase cascade, ending with genes for cell division. Normally Ras switches itself off by hydrolysing GTP, helped by proteins called GAPs.

Certain mutations, often changing amino acid 12, 13 or 61 (for example, G12V swaps a glycine for a valine at position 12), stop Ras hydrolysing its GTP. The mutant Ras stays on, and the cell receives a constant "divide" message even with no growth factor present. Permanently active Ras is found in a large share of human tumours, roughly a fifth to a quarter of them by recent estimates, and in most pancreatic cancers.

The receptor itself can also be the problem. In roughly one in five breast cancers, cells make far too much of an RTK called HER2, so they're over-sensitive to growth signals. The antibody drug Herceptin (trastuzumab) targets HER2 and has improved survival in these patients.

Many everyday drugs work by deliberately disrupting a pathway step. Beta blockers block one type of adrenaline receptor on the heart, so adrenaline raises the heart rate less. The same logic of "which step is changed, and what happens downstream" explains them all.

Using mutants to put a pathway in order

Before biologists could see the molecules in a pathway, they often worked out the order of steps using mutants. The method is called epistasis analysis. "Epistasis" means that one gene's mutation masks the effect of another's.

The idea is to combine two mutations with opposite effects in one organism: one that switches the pathway on and one that switches it off. Then you ask which effect wins. For a simple linear switch pathway, the double mutant looks like the mutant in the downstream gene, because the downstream gene is closer to the output and has the last word.

Think of it with Ras. A stuck-on receptor plus a broken Ras gives no signal: the receptor is shouting, but the message dies at Ras. A broken receptor plus a stuck-on Ras gives constant signal: Ras doesn't need the receptor any more. Either way, the phenotype follows Ras, so Ras must act after the receptor.

Key terms

Loss-of-function

A change that reduces or abolishes a protein's activity.

Gain-of-function

A change that makes a protein active when it shouldn't be, or more active than normal.

Constitutively active

Switched on all the time, regardless of the signal.

Cholera toxin

A bacterial toxin that locks Gαs in the active state, raising cAMP in gut cells.

CFTR

A chloride channel in gut and airway cells that opens when PKA phosphorylates it.

Oncogenic Ras

A mutant Ras that can't hydrolyse GTP, so it sends a constant signal to divide.

Epistasis analysis

Ordering genes in a pathway by seeing which single-mutant phenotype a double mutant shows.

Check yourself

Try answering in your head before you open each answer.

• 1.A patient's cells lack a working phosphodiesterase in the intestine. Would you expect their symptoms to resemble cholera, in a milder form? Explain.Show answerHide

  Yes, possibly. Without phosphodiesterase, cAMP made after normal signals isn't broken down, so it stays high, PKA keeps CFTR open, and Cl⁻ and water are secreted into the gut. It would likely be milder than cholera, because adenylyl cyclase is still switched off normally when the signal ends; only the clean-up is missing.

• 2.A tumour has a Ras mutation that locks Ras in the GTP-bound state. A drug blocks the growth factor receptor upstream. Will the drug stop the tumour cells dividing? What kind of drug might?Show answerHide

  No. Ras is already on and doesn't need the receptor, so blocking the receptor has no effect on the signal. A drug would need to act at Ras or downstream of it, for example an inhibitor of one of the MAP kinases.

• 3.In cells treated with cholera toxin, what happens to cAMP levels if you add a drug that blocks the adrenaline receptor? Explain.Show answerHide

  cAMP stays high. The toxin acts on Gαs, below the receptor, so Gαs is active whether or not the receptor is stimulated. Blocking a step upstream of a locked-on component can't switch the pathway off.