Intracellular Signaling Molecules

What is the largest class of molecular switches in cells?

Proteins controlled by phosphorylation.

How are many intracellular signaling proteins turned on or off?

By phosphorylation or dephosphorylation.

What do kinases do to signaling proteins?

Phosphorylate them.

What do phosphatases do to signaling proteins?

De-phosphorylate them.

Approximately what percentage of human proteins are phosphorylated?

About 30-50%.

How are protein kinases themselves often regulated?

By phosphorylation.

Do all signaling proteins activate by phosphorylation?

No, some are activated by dephosphorylation.

What class of molecular switches are G-proteins?

GTPases (GTP-binding proteins).

When are G-proteins active?

When GTP-bound.

When are G-proteins inactive?

When GDP-bound.

How are G-proteins activated?

By exchanging GDP for GTP.

How are G-proteins inactivated?

By GTP hydrolysis.

What are GEFs?

Guanine nucleotide exchange factors that activate G-proteins.

What are GAPs?

GTPase-activating proteins that inactivate G-proteins.

How many subunits make up heterotrimeric G-proteins?

Three.

Which subunit of heterotrimeric G-proteins has the GTPase activity?

The α subunit.

How are the β and γ subunits arranged?

They form a single functional unit anchored via the γ subunit.

When is a heterotrimeric G-protein inactive?

When GDP-bound and associated with the βγ subunit.

What acts as a GEF for heterotrimeric G-proteins?

An activated GPCR.

How does an activated GPCR amplify a signal?

One GPCR can activate multiple α and βγ subunits.

Which G-protein subunits can signal downstream?

Both the activated α and the βγ subunits.

Why are GPCR pathways medically important?

They are the targets of half of all known drugs.

What are second messengers?

Small intracellular molecules that relay signals from outside the cell to the inside.

What enzyme synthesizes cAMP from ATP?

Adenylyl cyclase.

How do activated G proteins affect adenylyl cyclase?

They stimulate its activity, increasing cAMP production.

What happens to ATP when converted to cAMP?

The phosphate becomes linked to both the 5' carbon and the 3' OH.

How fast can extracellular signals increase cAMP levels?

By twenty-fold in a few seconds.

How was cAMP visualized during serotonin stimulation?

Using a fluorescent protein that changes fluorescence when it binds cAMP.

What breaks down cAMP?

Cyclic AMP phosphodiesterase.

How does cyclic AMP phosphodiesterase deactivate cAMP?

It hydrolyzes cAMP into AMP by breaking the phosphate-sugar bond.

What is the major effector of cAMP in animal cells?

Protein kinase A (PKA).

How is PKA activated?

By increases in cAMP concentration.

What does PKA do?

Phosphorylates specific target proteins, including intracellular signaling and effector proteins.

Which enzyme hydrolyzes PI(4,5)P2?

Phospholipase C-β.

What activates phospholipase C-β?

GPCRs and their G proteins.

What second messengers are produced from PI(4,5)P2 hydrolysis?

DAG and IP3.

Where does DAG remain?

In the membrane (lipid soluble).

What does IP3 bind to?

A Ca2+ channel on the smooth ER.

What does IP3-triggered Ca2+ release help activate?

Protein kinase C (PKC).

Why is Ca2+ considered a ubiquitous second messenger?

It is used widely in signaling due to steep electrochemical gradients.

Why do free Ca2+ ions not travel far inside cells?

They are quickly pumped out or bound by Ca2+-binding proteins.

What is an example of a Ca2+ signal causing a large cellular effect?

The Ca2+ wave triggered by fertilization of an egg.

What does the fertilization-induced Ca2+ wave do?

Blocks entry of additional sperm and initiates embryonic development.

What is the most important protein in relaying Ca2+ signals?

Calmodulin.

How abundant is calmodulin in cells?

It can make up as much as 1% of a cell's total protein mass.

What type of protein is calmodulin?

A multi-purpose intracellular Ca2+ receptor.

What does calmodulin govern?

Many Ca2+-regulated processes.

What does Ca2+ binding do to calmodulin?

Induces dramatic conformational changes.

What does Ca2+-calmodulin activate?

Many other proteins, including CaM-kinase II.

How abundant is CaM-kinase II in some brain regions?

Up to 2% of total protein mass.

What activates CaM-kinase?

Ca2+/calmodulin binding to the inhibitory domain.

How does CaM-kinase become fully active?

Through auto-phosphorylation (self-phosphorylation).

How is CaM-kinase inactivated?

A protein phosphatase removes the phosphate.

How does a signaling complex assemble on an activated receptor?

It assembles transiently only after the receptor binds its extracellular signal molecule.

How do phosphoinositides contribute to signaling complex assembly?

Specific PIPs recruit and dock intracellular signaling proteins.

How do adaptor proteins help assemble signaling complexes?

They act as scaffolds linking two or more proteins in a pathway.

What opposes the action of protein kinases?

Protein phosphatases.

What do heterotrimeric G proteins relay signals from?

GPCRs.

What do some G proteins regulate?

Production of cyclic AMP.

How do other G proteins signal?

Via phospholipids such as IP3.

What are examples of second messengers?

cAMP, lipid-derived messengers, Ca2+.

What is the function of second messengers?

They rapidly amplify signals and activate many molecules like kinases.

Where do intracellular signaling complexes form?

At cell-surface receptors.

What brings intracellular proteins together using phosphorylation of lipids?

Hyperphosphorylated phosphoinositides (PIPs).

What do scaffold/adaptor proteins do?

Recruit intracellular proteins into signaling complexes.