Key Cell Signaling Pathways How They Shape Survival, Growth, and Health

What type of ligands do GPCRs with small extracellular domains bind?

Small ligands like epinephrine.

Where do small ligands bind in GPCRs?

Deep within the plasma membrane in a pocket formed by transmembrane segments.

What type of GPCRs have large extracellular domains?

Those that bind protein ligands.

How many transmembrane helices are found in a typical GPCR?

Seven.

Which GPCR binds epinephrine?

The β2-adrenergic receptor.

What happens when a ligand binds to a GPCR?

It causes a conformational change that promotes G-protein activation.

Which parts of a G protein are lipid-anchored to the plasma membrane?

The α and γ subunits.

What is bound to the α subunit of an inactive G protein?

GDP.

What are the two main subdomains of the α subunit's GTPase domain?

Ras domain and α-helical (AH) domain.

What is the function of the Ras domain in a G protein?

It helps form the GTP-binding pocket.

What does the AH domain do in a G protein?

It clamps GTP in place.

How is a G protein activated by a GPCR?

Ligand binding to GPCR causes a conformational change that opens the GTP-binding site on the G protein.

What happens after GDP is released from the Gα subunit?

GTP binds, causing the α subunit to dissociate from the receptor and the βγ complex.

What do the activated Gα and βγ subunits do?

Regulate downstream signaling pathways.

How long can a GPCR activate G proteins?

As long as the extracellular ligand is bound.

What does Gs protein stimulate?

Adenylyl cyclase.

What is the result of Gs protein activation?

Increased cAMP, leading to activation of cellular responses.

Which receptors are associated with Gs proteins?

β-adrenergic receptors.

What does Gi protein inhibit?

Adenylyl cyclase.

What is the result of Gi protein activation?

Decreased cAMP, leading to dampened cellular activity.

Which receptors are associated with Gi proteins?

α₂-adrenergic receptors.

What does Gq protein activate?

Phospholipase C.

What is the result of Gq protein activation?

It triggers alternative signaling cascades.

Which receptors are associated with Gq proteins?

α₁-adrenergic receptors.

How does serotonin affect cAMP levels in Aplysia sensory nerve cells?

It binds to a GPCR, increasing intracellular cAMP levels.

What does a fluorescent protein do in the cAMP experiment with Aplysia cells?

It changes fluorescence when binding to cAMP, allowing monitoring of cAMP levels.

What does blue fluorescence indicate in the cAMP experiment?

Low levels of cAMP.

What does yellow fluorescence indicate in the cAMP experiment?

Intermediate levels of cAMP.

What does red fluorescence indicate in the cAMP experiment?

High levels of cAMP.

What catalyzes the synthesis of cyclic AMP (cAMP)?

Adenylyl cyclase.

From which molecule is cAMP synthesized?

ATP.

What does the reaction of adenylyl cyclase release during cAMP synthesis?

Pyrophosphate (PP).

What enzyme degrades cAMP?

Phosphodiesterase.

What does cAMP degrade to?

5′-AMP.

What happens when cAMP binds to the regulatory subunits of PKA?

It induces a conformational change that causes the regulatory subunits to dissociate from the catalytic subunits, activating the kinase.

How many cAMP molecules are needed to release the catalytic subunits of PKA?

More than two cAMP molecules are required.

What does the dissociation of regulatory subunits from catalytic subunits of PKA lead to?

Activation of PKA's kinase activity.

How does an increase in intracellular cAMP alter gene transcription?

It activates PKA, which enters the nucleus and phosphorylates the transcription regulatory protein CREB, leading to gene transcription.

What does the phosphorylation of CREB recruit?

The coactivator CBP (CREB-binding protein).

What role does CBP play in gene transcription?

It stimulates gene transcription by interacting with phosphorylated CREB.

What class of receptors activates phospholipase C-β (PLCβ)?

GPCRs (G-protein-coupled receptors).

What happens when phospholipase C-β hydrolyzes PI(4,5)P2?

It produces two second messengers

What is the function of IP3 produced by phospholipase C-β?

It diffuses through the cytosol and releases Ca²⁺ from the endoplasmic reticulum.

What is the function of diacylglycerol (DAG) produced by phospholipase C-β?

It remains in the membrane and helps activate protein kinase C (PKC).

What other class of phospholipase C is activated by receptor tyrosine kinases (RTKs)?

Phospholipase C-γ.

How do GPCRs increase cytosolic Ca²⁺ levels?

By activating phospholipase C-β (PLCβ) through a G protein called Gq, which hydrolyzes PI(4,5)P2 into IP3 and diacylglycerol.

What G-protein subunit is involved in activating PLCβ in response to a GPCR?

Both the α subunit and the βγ complex of Gq are involved.

What second messengers are produced when PLCβ hydrolyzes PI(4,5)P2?

Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).

How does IP3 increase cytosolic Ca²⁺?

IP3 diffuses through the cytosol and binds to IP3-gated Ca²⁺ release channels on the ER membrane, causing Ca²⁺ to be released into the cytosol.

What role does diacylglycerol (DAG) play in PKC activation?

DAG, along with phosphatidylserine and Ca²⁺, activates protein kinase C (PKC), recruiting it to the plasma membrane.

How is protein kinase C (PKC) activated?

PKC is activated when DAG and Ca²⁺ bind to it, bringing PKC from the cytosol to the cytosolic face of the plasma membrane.

How many isoforms of PKC are present in humans, and how many can be activated by DAG?

Humans have at least 10 distinct isoforms of PKC, and at least 4 of them can be activated by DAG.

What mechanism propagates calcium waves?

Calcium waves propagate via a "domino effect," where each release of Ca²⁺ triggers adjacent receptors.

How does regenerative feedback affect calcium signaling?

Regenerative feedback maintains signal strength by preventing the dilution of the signal, unlike simple diffusion.

What is the purpose of a refractory period in calcium signaling?

The refractory period temporarily inhibits receptors, allowing for rhythmic calcium oscillations.

How does oscillation frequency influence cellular signaling?

The frequency of calcium oscillations can encode distinct cellular messages, conveying different signals depending on the oscillation pattern.

How do cells use calcium oscillations in response coordination?

Calcium oscillations help coordinate complex responses across various regions of the cell by providing long-distance intracellular signaling.

What is the structure of Ca²⁺/calmodulin?

Ca²⁺/calmodulin has a dumbbell shape with two globular ends connected by a long, exposed α helix, and each globular head contains two Ca²⁺-binding sites.

How does Ca²⁺/calmodulin change when binding to a target protein?

Upon binding to a target protein, Ca²⁺/calmodulin folds to surround the peptide, adopting a conformation specific to the target protein.

What happens in the activation of CaM-kinase II?

When calcium levels rise, calcium/calmodulin complexes activate CaM-kinase II by binding to the kinase domains and releasing inhibition.

How does CaM-kinase II spread activation after binding Ca²⁺/calmodulin?

Once activated, a kinase domain can phosphorylate neighboring domains, spreading activation through a molecular domino effect.

What are the two major effects of CaM-kinase II activation?

1) The calmodulin remains bound, delaying its release. 2) The kinase becomes calcium-independent and stays active after the calcium signal fades.

What is the role of CaM-kinase II in memory?

CaM-kinase II creates a molecular memory by converting brief calcium spikes into long-lasting cellular responses that persist after the signal fades.

How does CaM-kinase II respond to calcium oscillation patterns in neurons?

Different isoforms of CaM-kinase II are fine-tuned to respond to specific calcium oscillation patterns, encoding different types of experiences as distinct biochemical memories.

What is the desensitization mechanism of GPCR signaling?

Upon activation by an extracellular signal, the GPCR recruits GPCR kinase (GRK), which phosphorylates serine and threonine residues on the receptor's cytoplasmic tail. This phosphorylation creates binding sites for arrestin.

How does arrestin contribute to GPCR desensitization?

Arrestin binds to the phosphorylated GPCR, blocking further interaction between the receptor and G proteins. Arrestin binding also promotes receptor internalization through clathrin-coated pits, silencing the signal.

What role does receptor internalization play in GPCR desensitization?

Internalization through clathrin-coated pits helps further silence the signal and targets the receptor for recycling or degradation, ensuring that the cell can adapt to constant stimulation.

Why is GPCR desensitization important for cellular function?

Desensitization mechanisms help prevent prolonged or excessive signaling, allowing cells to maintain sensitivity to changes in the external environment.

What happens when a GPCR is activated by an extracellular signal?

The GPCR undergoes conformational changes that activate heterotrimeric G proteins, initiating downstream signaling cascades.

What role does GPCR kinase (GRK) play in GPCR activation?

GRK phosphorylates specific serine and threonine residues on the receptor's cytoplasmic tail, creating binding sites for arrestin.

What is the function of arrestin in GPCR desensitization?

Arrestin binds to phosphorylated GPCR, blocking further interaction with G proteins and promoting receptor internalization.

How does β-arrestin contribute to receptor internalization?

β-arrestin recruits clathrin and adaptor protein-2 (AP-2), which are involved in the formation of the endocytic complex and vesicle internalization.

What happens during the process of receptor sequestration?

Activated receptors are internalized into endosomes, temporarily removing them from the cell surface, and may be recycled back to the membrane.

What occurs during receptor down-regulation?

Internalized receptors are sorted to lysosomes for degradation, reducing total receptor numbers in the cell.

What is receptor inactivation?

Receptor inactivation involves direct modification, like phosphorylation, to prevent signal transmission even with the signal molecule bound.

How does inactivation of signaling proteins regulate GPCR signaling?

Signaling proteins like G proteins are inhibited by GTPase-activating proteins, phosphodiesterases, and phosphatases to stop the signal.

How does the production of inhibitory proteins affect receptor signaling?

Inhibitory proteins like RGS or SOCS create negative feedback loops that provide delayed but sustained signal attenuation.

How does an RTK activate Ras?

Upon ligand binding, the RTK becomes autophosphorylated on tyrosine residues. The adaptor protein Grb2 binds to a phosphotyrosine via its SH2 domain and recruits Sos, a Ras GEF, through its SH3 domains. Sos facilitates the exchange of GDP for GTP on Ras, activating it.

What is the role of Grb2 in RTK signaling?

Grb2 binds to phosphorylated tyrosines on the RTK via its SH2 domain and recruits Sos via its SH3 domains, activating Ras.

How does Sos activate Ras?

Sos exchanges GDP for GTP on inactive Ras, converting it into its active form, which can then propagate downstream signaling.

What is the MAP kinase module?

The MAP kinase module consists of a series of three kinases—Raf, Mek, and Erk—that transmit signals from the plasma membrane into the cytoplasm and nucleus.

How does Ras activate Raf in the MAP kinase module?

Ras, bound to GTP, recruits Raf to the plasma membrane, activating Raf, a MAP kinase kinase kinase.

How does Raf activate Mek?

Raf phosphorylates and activates Mek, a MAP kinase kinase, using ATP.

How does Mek activate Erk?

Mek phosphorylates Erk (MAP kinase) on two residues, which is necessary for Erk's activation.

What happens after Erk is activated in the MAP kinase pathway?

Erk phosphorylates various downstream targets, including cytoplasmic proteins and transcription factors in the nucleus, leading to changes in protein activity and gene expression.

What are the outcomes of the MAP kinase cascade?

The pathway leads to short-term changes through protein activity and long-term effects through gene transcription, influencing cell processes like proliferation, differentiation, and survival.

Why is the MAP kinase cascade important?

This cascade allows for signal amplification, regulation at multiple steps, and precise control of cellular responses.

How does normal Ras function as a molecular switch?

In normal cells, Ras alternates between an inactive GDP-bound state and an active GTP-bound state in response to upstream signals. This switching ensures tight regulation of Ras activity, promoting growth, proliferation, and migration only when needed.

What happens in oncogenic Ras?

Mutations in Ras (Ras*) prevent it from hydrolyzing GTP to GDP, causing Ras to remain locked in the active GTP-bound state. This leads to continuous signaling to downstream effectors, driving uncontrolled cell growth, which is characteristic of many cancers.

How does Ras regulate cellular processes?

Normal Ras functions as a molecular switch that controls growth and migration by toggling between an inactive and active state. When mutated, it leads to unchecked signaling, driving cancerous cell behavior

What is the role of GLUT1 in Ras-driven cancers?

In Ras-driven cancers, increased GLUT1 expression enhances glucose uptake, facilitating the Warburg effect. This process diverts glucose toward glycolysis for rapid ATP production and the pentose phosphate pathway for nucleotide synthesis instead of the TCA cycle.

How does Ras influence glutamine metabolism?

Glutamine becomes essential for cancer cell survival, serving both as a carbon source and for maintaining redox balance. Ras upregulates enzymes like GOT1, promoting the rerouting of glutamine into pathways that produce NADPH to counteract oxidative stress, creating a dependency on glutamine (glutamine addiction).

What is macropinocytosis, and how does it aid Ras-driven cancers?

Macropinocytosis allows Ras-driven cancer cells to scavenge proteins, amino acids, and lipids from the extracellular environment. This reduces their dependence on de novo synthesis and provides metabolic flexibility, particularly in nutrient-limited conditions.

How does autophagy contribute to Ras-driven cancers?

Ras-driven cancers rely on autophagy for recycling cellular components, particularly for mitochondrial function and nucleotide synthesis. This dependency represents a therapeutic vulnerability, as autophagy cannot be easily bypassed, even in metabolically flexible cancer cells.

What are the metabolic vulnerabilities in Ras-driven cancers?

The metabolic adaptations in Ras-driven cancers, such as increased GLUT1 expression, glutamine addiction, macropinocytosis, and autophagy, support their rapid growth. These processes create potential therapeutic targets for disrupting the metabolic networks that fuel cancer cell survival and proliferation.

How does RAS help tumors evade immune recognition?

Oncogenic RAS reduces MHC I expression on cancer cells, making it harder for immune cells like T-cells to recognize and attack the tumor. Additionally, RAS increases PD-L1 levels, which bind to PD-1 on T-cells and inhibit their immune response, allowing the tumor to escape destruction.

How does RAS shape the immune environment around tumors?

RAS-driven tumors recruit immunosuppressive macrophages while blocking the entry of beneficial immune cells such as T-cells and NK cells, creating a protective immune environment that supports tumor growth.

How does RAS contribute to chronic inflammation in tumors?

RAS increases the release of cytokines like IL-8, which attracts neutrophils and promotes inflammation. This chronic inflammation supports tumor growth and survival.

What role does RAS play in tumor blood vessel formation?

Oncogenic RAS stimulates the production of VEGF, which promotes the growth of new blood vessels to supply the tumor with oxygen and nutrients, aiding in tumor growth and metastasis.

Direct Molecular Inhibitors of RAS

Some drugs trap RAS in its inactive GDP-bound form, preventing its activation and signaling that drives cancer growth. Small interfering RNAs (siRNAs) delivered via liposomes or exosomes can lower RAS levels by targeting its mRNA before it is translated into protein.