cell bio exam 2

What is the general sequence of a cell-signaling pathway?

Ligand → receptor → signaling molecules → effector → response.

In cell signaling, what is a ligand?

A molecule that carries information to a target cell.

What does a receptor do in cell signaling?

Binds a ligand and initiates intracellular signaling.

What structural change follows ligand-receptor binding?

An allosteric conformational change.

What does allostery mean in receptor activation?

Binding at one site changes another, often distant, site.

What do intracellular signaling molecules do?

Relay information from activated receptors to effectors.

What is an effector in a signaling pathway?

A molecule that directly changes cellular activity.

What are two common types of signaling effectors?

Enzymes and transcription factors.

What are two broad cellular responses to signaling?

Modify existing proteins or change gene expression.

Why can modification of existing proteins be rapid?

It does not require new transcription or translation.

What is endocrine signaling?

Long-distance signaling to distant target cells.

Why is insulin an endocrine signal?

It travels through circulation to distant target tissues.

What is paracrine signaling?

Local signaling within the same tissue or neighborhood.

What is juxtacrine signaling?

Direct-contact signaling between adjacent cells.

What is autocrine signaling?

A cell releases a signal that acts on itself.

Where is autocrine signaling especially common in this lecture?

Tumor cells.

Order endocrine, paracrine, juxtacrine by distance.

Endocrine → paracrine → juxtacrine.

How do hydrophobic signals generally reach their receptors?

Cross the membrane and bind intracellular receptors.

What signaling molecules exemplify hydrophobic signals?

Steroid hormones.

What receptor exemplified hydrophobic signaling?

The glucocorticoid receptor.

Where do hydrophilic signals generally bind?

Cell-surface receptors.

What does receptor-ligand binding affinity describe?

How tightly receptor and ligand bind.

What quantity measures receptor-ligand affinity?

The dissociation constant, Kd.

What is the Kd-affinity relationship?

Lower Kd means higher affinity.

What does lower Kd mean for 50% receptor occupancy?

A lower ligand concentration is required.

Why aren't all receptors needed for a strong response?

Signal amplification magnifies downstream effects.

What is signal amplification?

A small initial signal produces a much larger downstream response.

What second messenger appeared in the amplification example?

cAMP.

What determines a cell's sensitivity to a signal?

Receptor-ligand affinity and receptor number.

How does more receptor affect signaling sensitivity?

The cell can respond to lower ligand concentrations.

Why can receptor overexpression matter in cancer?

It can make cells abnormally sensitive to signals.

What mainly controls receptor protein abundance?

Transcription.

How can receptor endocytosis reduce receptor levels?

Receptors are internalized and can be lysosomally degraded.

What does a protein kinase do?

Phosphorylates target proteins.

What does a protein phosphatase do?

Removes phosphate groups from proteins.

How can phosphorylation activate a kinase?

It can favor ATP and target-protein binding.

What is transphosphorylation?

One protein molecule phosphorylates another in a complex.

Why use a phospho-specific antibody?

To specifically detect a phosphorylated protein form.

What does a total-protein antibody detect?

The protein regardless of phosphorylation state.

Constant total protein but more phospho-protein means what?

Phosphorylation increased without increased protein abundance.

Why is one phosphate hard to detect by SDS-PAGE size?

It adds only about 80 Da.

Which monomeric GTPase state is active?

The GTP-bound state.

Which monomeric GTPase state is inactive?

The GDP-bound state.

What does GTP's gamma phosphate do in a GTPase?

Stabilizes the active Switch I/II conformation.

What happens when a GTPase hydrolyzes GTP to GDP?

Its switch regions change to the inactive conformation.

Do Ras, Ran, and Rac bind receptors directly?

No; they function downstream as signaling molecules.

What does a GEF do to a monomeric GTPase?

Promotes GDP-GTP exchange and activation.

What does the PAK1-PBD pull-down detect?

Active Rac-GTP.

What did increased Rac pull-down after PDGF indicate?

PDGF increased active Rac-GTP.

What regulator should increase Rac-GTP downstream of a signal?

A GEF.

Are all intracellular signaling molecules proteins?

No; second messengers can be nonproteins.

Which four second messengers were shown in this lecture?

cAMP, cGMP, DAG, and IP3.

What is a loading control in a Western blot used to verify?

That comparable amounts of total protein were loaded into the lanes.

Why was beta-actin used as a loading control in the Rac-GTP experiment?

Beta-actin is abundant and relatively consistently expressed, so similar bands indicate similar total protein loading.

Why is a loading control needed when comparing Western blot bands?

A weak or missing experimental band could otherwise result from unequal protein loading rather than a biological difference.

What type of receptor is a G-protein-coupled receptor (GPCR)?

A seven-pass transmembrane receptor.

How many times does a GPCR cross the plasma membrane?

Seven times.

Where is the N-terminus of the GPCR structure discussed in class?

On the extracellular side of the membrane.

Where is the C-terminus of the GPCR structure discussed in class?

On the cytoplasmic side of the membrane.

Approximately how many functional GPCRs are present in the human genome?

About 800.

In GPCR pharmacology, what does an agonist do?

Mimics the signal and activates the receptor.

In GPCR pharmacology, what does an antagonist do?

Blocks receptor activation.

In GPCR signaling, what does the term "G protein" refer to?

A GTPase coupled to the receptor.

Why is a heterotrimeric G protein called heterotrimeric?

It contains three different subunits: Gα, Gβ, and Gγ.

Which heterotrimeric G-protein subunit binds GDP and GTP?

Gα.

What nucleotide is bound to Gα when the heterotrimeric G protein is inactive?

GDP.

What nucleotide is bound to Gα when Gα is active?

GTP.

Which heterotrimeric G-protein subunits are lipidated in the class diagram?

Gα and Gγ.

What happens to a GPCR when its extracellular ligand binds?

The receptor undergoes an allosteric conformational change.

What allows an activated GPCR to activate its heterotrimeric G protein?

The receptor's active conformation allows it to interact with Gα.

What role does an activated GPCR play in nucleotide exchange on Gα?

The GPCR acts as a GEF for Gα.

What does a GPCR acting as a GEF cause Gα to do?

Release GDP and bind GTP.

Does a GPCR directly convert GDP into GTP on Gα?

No; it promotes nucleotide exchange so GDP leaves and GTP binds.

What happens to Gα after it exchanges GDP for GTP?

Gα becomes active and dissociates from Gβγ.

What can active Gα-GTP do after separating from Gβγ?

Bind downstream effector proteins and activate or inhibit their activity.

Can Gβγ affect downstream signaling after Gα dissociates?

Yes; Gβγ can interact with membrane proteins and alter their activity.

How can one activated GPCR contribute to signal amplification?

One active receptor can activate multiple heterotrimeric G proteins.

What molecular event turns off active Gα-GTP?

Hydrolysis of GTP to GDP.

What is intrinsic GTPase activity of Gα?

Gα's ability to hydrolyze its own bound GTP to GDP.

What happens to Gα after GTP is hydrolyzed to GDP?

Gα becomes inactive and reassociates with Gβγ.

Why does GTP hydrolysis downregulate GPCR signaling?

It converts active Gα-GTP into inactive Gα-GDP.

Why does GPCR signaling usually produce rapid, short-term effects?

It often changes the activity of existing enzymes or ion channels.

What does FRET stand for?

Förster Resonance Energy Transfer.

What type of biological interaction can FRET detect?

Very close molecular proximity or protein complexes in living cells.

In the CFP/YFP FRET pair, which fluorophore is the donor?

CFP.

In the CFP/YFP FRET pair, which fluorophore is the acceptor?

YFP.

What happens during FRET between CFP and YFP?

Excited CFP transfers energy to nearby YFP, causing YFP emission.

What must be true about two fluorescent tags for a FRET signal to occur?

They must be very close together.

In the class GPCR FRET experiment, when is the FRET signal high?

When Gα and Gβγ are together in the inactive heterotrimeric complex.

In the class GPCR FRET experiment, why does FRET decrease after signal addition?

Activated Gα dissociates from Gβγ, separating the fluorescent tags.

What does decreased FRET after GPCR activation indicate in the class experiment?

Dissociation and activation of the heterotrimeric G protein.

Why can FRET detect interactions that co-immunoprecipitation may miss?

FRET can examine close, dynamic interactions in living cells without lysing them.

Why does a negative co-immunoprecipitation result not prove that two proteins never interact?

Cell lysis or transient interactions can prevent an actual interaction from being detected.

Which heterotrimeric G-protein subunit provides much of GPCR pathway specificity?

Gα.

Why can different Gα proteins produce different signaling outcomes?

Different Gα proteins interact with different downstream effectors.

What ligand activates the β2-adrenergic receptor in the pathway studied in this lecture?

Epinephrine, also called adrenaline.

What type of receptor is the β2-adrenergic receptor?

A G-protein-coupled receptor (GPCR).

What downstream effector enzyme is activated in the epinephrine GPCR pathway studied in class?

Adenylyl cyclase.

What reaction does adenylyl cyclase catalyze in GPCR signaling?

It converts ATP into cAMP.

What is cAMP in the epinephrine GPCR pathway?

An intracellular second messenger.