Signalling and Metabolic Regulation

What is the structure of G-protein coupled receptors (GPCRs)?

They are 7-transmembrane domain receptors.

What occurs to the GPCR when an extracellular ligand binds?

The receptor undergoes a conformational change.

Which protein is activated by the conformational change of a GPCR?

A heterotrimeric G protein.

What is the inactive state of a heterotrimeric G protein?

$\alpha$ bound to GDP and $\beta\gamma$ tightly associated.

What characterizes the active state of a heterotrimeric G protein?

$\alpha$ exchanges GDP for GTP and becomes $\alpha$-GTP active.

What happens to the G protein components after the $\alpha$ subunit exchanges GDP for GTP?

Both $\alpha$-GTP and $\beta\gamma$ interact with downstream effectors.

What determines the specific effect on downstream signalling in GPCR pathways?

The type of $G\alpha$ subunit activated.

What is the function of the $G\alpha_{s}$ subunit?

It stimulates adenylyl cyclase, increasing [cAMP].

What is the function of the $G\alpha_{i}$ subunit?

It inhibits adenylyl cyclase, decreasing [cAMP].

What pathways are linked to subunits like $G\alpha_{q}$ and $G\alpha_{12/13}$?

Phospholipase C and cytoskeletal changes.

What is biased signalling in GPCRs?

When specific downstream pathways are preferentially activated by different receptor conformations.

What is cAMP produced from?

ATP.

Which enzyme produces cAMP from ATP?

Adenylyl cyclase.

What causes cAMP levels to increase rapidly in neurones?

Serotonin binding to a GPCR activating $G\alpha_{s}$ and then adenylyl cyclase.

Which enzyme hydrolyses cAMP to 5'-AMP to degrade it?

Phosphodiesterases (PDEs).

Why is the degradation of cAMP by PDEs important?

It ensures transient signalling and prevents continuous activation.

Name three features of a good second messenger.

Low basal levels, rapid transient upregulation, and fast termination.

What response does cAMP stimulate in the Thyroid Gland?

Thyroid hormone synthesis.

What response does cAMP stimulate in the Adrenal Cortex?

Cortisol secretion.

What response does cAMP stimulate in the Ovary?

Progesterone secretion.

What response does cAMP stimulate in Muscle?

Activation of glycogenolysis.

What is the tetrameric structure of Protein Kinase A (PKA)?

2 Regulatory Subunits (R) and 2 Catalytic Subunits (C).

How is PKA kept inactive in the resting state?

The R subunits bind to the C subunits, preventing phosphorylation activity.

How many cAMP molecules are required to activate one PKA tetramer?

4 total (2 cAMP per R subunit).

What happens after cAMP binds to the PKA regulatory subunits?

A conformational change releases active C subunits.

Which residues do PKA catalytic subunits phosphorylate?

Serine or threonine residues on target proteins.

What characterizes the short-term effects of PKA activation?

Phosphorylation of cytosolic proteins leading to rapid functional changes.

What characterizes the long-term effects of PKA activation?

C-subunits translocate to the nucleus to activate gene transcription.

What is the role of A-Kinase Anchoring Proteins (AKAPs)?

They localize PKA to specific subcellular compartments by binding R subunits.

To which structures can AKAPs bind?

Organellar membranes, cytoskeletal elements (actin/microtubules), and other signalling molecules.

What is the functional significance of localized signalling via AKAPs?

It ensures a rapid and precise cellular response.

What is the specific role of M-AKAP?

Anchors PKA and PDE to the nuclear envelope for a short, tightly regulated pulse of activation.

What does PKA phosphorylate in muscle cells to trigger glycogen breakdown?

Glycogen phosphorylase kinase.

In olfactory neurones, which G protein is stimulated by an odourant binding to a GPCR?

$G\alpha_{olf}$.

What happens in olfactory neurones after cAMP rises due to an odourant?

cAMP opens CNG channels, causing an influx of sodium and calcium.

In olfactory signalling, what leads to local depolarisation after sodium/calcium influx?

$Ca^{2+}$-gated $Cl^{-}$ channels causing an efflux of $Cl^{-}$.

What nuclear protein is phosphorylated by PKA catalytic subunits for long-term effects?

CREB (cAMP response element-binding protein).

At which residue is CREB phosphorylated by PKA?

Serine-133.

What does phosphorylated CREB bind to on DNA?

CRE (cAMP response element).

What co-activator is recruited by phosphorylated CREB?

CBP (CREB-binding protein).

In olfactory neurones, what gene is transcribed via long-term CREB activation for axon targeting?

Neuropilin (an adhesion molecule).

What accelerates cAMP degradation as a feedback mechanism after PKA activation?

PKA phosphorylates neighbouring PDE.

What does Phospholipase C-beta (PLC$\beta$) hydrolyse?

$PIP_{2}$ (phosphatidylinositol 4,5-bisphosphate).

What are the two second messengers produced from the cleavage of $PIP_{2}$?

Diacylglycerol (DAG) and Inositol 1,4,5-trisphosphate ($IP_{3}$).

Which $PIP_{2}$ derivative remains tethered in the plasma membrane?

Diacylglycerol (DAG).

Which $PIP_{2}$ derivative diffuses into the cytosol to target calcium stores?

Inositol 1,4,5-trisphosphate ($IP_{3}$).

Where are the $IP_{3}$-gated calcium release channels located?

On the membrane of the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR).

Binding of $IP_{3}$ to its receptor triggers the release of which ion into the cytosol?

Calcium ($Ca^{2+}$).

What is the primary role of Diacylglycerol (DAG) at the plasma membrane?

Activation of Protein Kinase C (PKC).

How are cytosolic calcium levels in a resting cell compared to the ER/SR lumen?

Relatively low ($100\,nM$) compared to millimolar levels in the lumen.

Name three components required for the full activation of Protein Kinase C (PKC).

Elevated cytosolic $Ca^{2+}$, Diacylglycerol (DAG), and Phosphatidylserine.

How does phosphatidylserine contribute to PKC activation?

It is a negatively charged phospholipid that stabilizes PKC's active conformation at the membrane.

Which specific residues are phosphorylated by activated PKC?

Serine and threonine residues.

What is the effect of PKC activation in the Liver?

It promotes glycogenolysis, releasing glucose into the blood.

What is the effect of PKC activation in Pancreatic acinar cells?

It stimulates amylase secretion.

What effect can PKC have on Smooth Muscle units?

Induce muscle contraction via actin-myosin interactions.

What biological process in blood platelets involves PKC activation?

Platelet aggregation.

Name a rapid effect of calcium ions (milliseconds).

Neurotransmitter release or muscle contraction.

Name a delayed effect of calcium ions (hours to days).

Modulation of gene transcription.

What is the 'calcium signalling toolkit'?

The conserved set of molecular components (channels, pumps, sensors, buffers) governing calcium dynamics.

What triggers Voltage-Operated Channels (VOCs)?

Changes in membrane potential.

Name an example of a Receptor-Operated Channel (ROC).

NMDA receptor.

What triggers Second Messenger-Operated Channels (SMOCs)?

Intracellular secondary messengers like $IP_{3}$.

What is the role of Plasma Membrane Calcium ATPases (PMCAs)?

Active transport extruding $Ca^{2+}$ from the cytosol to the extracellular space.

What is the role of SERCAs?

Active transport of $Ca^{2+}$ from the cytosol back into the ER/SR stores.

What are calcium buffers?

Proteins that reversibly bind $Ca^{2+}$ to regulate concentration and diffusion.

What characterizes the C2 domain family of calcium sensors?

They interact with cell membranes in a $Ca^{2+}-dependent$ manner (e.g., PKC).

What characterizes the EF-hand family of calcium sensors?

A conserved helix-loop-helix motif that directly binds $Ca^{2+}$ ions.

What is the typical concentration of cytosolic free calcium in resting cells?

$100\,nM$.

By how much can cytosolic calcium levels increase upon signal induction?

10-20 fold ($500\,nM$ to a few $\mu M$).

Which G protein type leads to the activation of PLC$\beta$?

$G_{q}$ G proteins.

Where are Ryanodine Receptors (RyRs) located?

On the ER/SR membrane.

What is Calcium-Induced Calcium Release (CICR)?

A positive feedback process where elevated cytosolic calcium stimulates RyRs to release more calcium.

What mechanism eventually halts calcium release as levels rise very high?

High concentrations of cytosolic $Ca^{2+}$ paradoxically inhibit $IP_{3}Rs$ and RyRs (negative feedback).

What is the stoichiometry of the Sodium-Calcium Exchanger (NCX)?

3 $Na^{+}$ in for 1 $Ca^{2+}$ out.

Is the Sodium-Calcium Exchanger (NCX) electrogenic?

Yes.

Which organelle acts as a temporary high-capacity calcium buffer near open channels?

Mitochondria.

Which transporter allows mitochondria to take up $Ca^{2+}$?

Mitochondrial calcium uniporter (MCU).

What is Calmodulin (CaM)?

A ubiquitous, highly conserved EF-hand protein that binds four $Ca^{2+}$ ions.

What happens to Calmodulin after binding four calcium ions?

It undergoes a conformational change exposing hydrophobic surfaces to bind target proteins.

Name two types of enzymes activated by Calmodulin.

CaM kinases and phosphatases (e.g., calcineurin).

What is a post-translational modification (PTM)?

A covalent modification of a protein after it has been translated.

Name four types of PTMs.

Phosphorylation, Methylation, Acetylation, Ubiquitylation.

How does alternative splicing and PTMs impact proteomic diversity?

Increases diversity from ~20,000 genes to >1,000,000 protein species.

What enzymes catalyze the addition of phosphate groups?

Kinases.

What enzymes catalyze the removal of phosphate groups?

Phosphatases.

What amino acid residues are typically phosphorylated in eukaryotes?

Serine (Ser), Threonine (Thr), and Tyrosine (Tyr).

What is the biological role of phosphorylation-dephosphorylation cycles?

They act as a reversible molecular on/off switch.

In the GTP-binding protein analogy, which regulatory protein in-activates the switch?

GAPs (GTPase-activating proteins).

In the GTP-binding protein analogy, which regulatory protein activates the switch?

GEFs (guanine nucleotide exchange factors).

Phosphorylation of CREB at Serine-133 recruits which coactivators?

CBP/p300.

If CREB is not phosphorylated, can it bind effectively to DNA?

No.

Approximately how many kinases are in the human kinome?

500.

What is the specific total of Tyrosine kinases in humans?

~90.

Name three conserved domains found in kinases derived from a common vertical ancestor.

ATP-binding site, Catalytic loop, and Activation loop.

What is mTOR and why is it therapeutically relevant in cancer?

A central regulator of cell growth, often hyperactivated in cancer and inhibited by rapamycin.

Which second messenger activates CaM Kinase II?

$Ca^{2+}$/calmodulin complex.

Approximately how many phosphatases have been identified in the human genome?

147.

Why is the phylogenetic relationship of phosphatases represented as a 'wheel'?

They are structurally diverse and lack a single ancestral gene.

What is the catalytic mechanism used by all phosphatases?

Hydrolytic removal of phosphate using water.