Molecular Biology · End-of-chapter questions below · Part 1 of 2 · 10 questions per part
Part 1 of 2
Signal Molecules, Cell-Surface Receptors, and Second Messengers
From a muscle contracting to a tumor growing uncontrollably, virtually every cell behavior is governed by molecular signals. Understanding how cells receive, transduce, and respond to these signals is foundational to understanding physiology and the molecular basis of disease.
In Part 1, you will explore:
Classes of extracellular signal molecules: hormones, local mediators, neurotransmitters
Cells communicate by releasing signal molecules that bind specific receptor proteins on or in target cells. Signal molecules fall into three broad classes: endocrine hormones (secreted into the bloodstream and act on distant cells, e.g., insulin, cortisol), local mediators (act on nearby cells, e.g., histamine, growth factors, nitric oxide), and neurotransmitters (released at synapses, e.g., acetylcholine, glutamate). Hydrophilic signals bind cell-surface receptors; lipophilic signals (steroids, thyroid hormone) cross the plasma membrane and bind intracellular receptors.
Key term
GPCR (G protein-coupled receptor)
A seven-transmembrane-helix receptor that, upon ligand binding, activates a heterotrimeric G protein (Gα, Gβ, Gγ); the largest family of cell-surface receptors in the human genome (>800 members), including receptors for hormones, odorants, and light.
15.2 G Protein-Coupled Receptors and cAMP Signaling
When a ligand binds a GPCR, the receptor undergoes a conformational change that allows it to act as a GEF for the Gα subunit—exchanging GDP for GTP and dissociating Gα from Gβγ. Stimulatory Gαs activates adenylyl cyclase, which converts ATP to cyclic AMP (cAMP). cAMP activates protein kinase A (PKA) by binding and displacing regulatory subunits from catalytic subunits. PKA then phosphorylates serine/threonine residues on target proteins, altering their activity. The pathway is terminated when Gα hydrolyzes GTP to GDP (intrinsic GTPase activity) and phosphodiesterase degrades cAMP to 5'-AMP.
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Pause & Recall
How does cholera toxin cause persistent cAMP elevation in intestinal cells?
Cholera toxin ADP-ribosylates Gαs, locking it in the GTP-bound (active) state by preventing GTP hydrolysis. Gαs therefore continuously activates adenylyl cyclase, producing massive cAMP accumulation in gut epithelial cells. This maximally activates PKA, which phosphorylates chloride channels (CFTR) and inhibits Na⁺/Cl⁻ absorption, causing the profuse watery diarrhea characteristic of cholera.
15.3 IP₃/DAG and Calcium Signaling
Ligand binding to some GPCRs (coupled to Gαq) or receptor tyrosine kinases activates phospholipase C (PLC), which cleaves the membrane phospholipid PIP₂ into two second messengers: IP₃ (inositol trisphosphate) and DAG (diacylglycerol). IP₃ diffuses to the ER and binds IP₃ receptors (ligand-gated Ca²⁺ channels), releasing Ca²⁺ from the ER lumen into the cytosol. The rise in cytosolic Ca²⁺ together with DAG activates protein kinase C (PKC) at the plasma membrane. Ca²⁺ alone also activates calmodulin (CaM), which in turn activates CaM-dependent kinases (CaMKII) and other targets including myosin light-chain kinase and calcineurin phosphatase.
Key term
IP₃ (inositol trisphosphate)
A second messenger generated by phospholipase C cleavage of PIP₂; it binds IP₃ receptors on the ER membrane, opening Ca²⁺ channels and raising cytosolic calcium concentration.
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Pause & Recall
Why does phorbol ester (a DAG mimic) cause constitutive PKC activation and promote tumorigenesis?
Phorbol esters bind the DAG-binding C1 domain of PKC with high affinity and cannot be hydrolyzed like DAG, so they lock PKC in an active membrane-bound state indefinitely. Persistent PKC activity drives proliferative gene expression, bypassing normal growth-factor control. Phorbol esters are classic tumor promoters—they do not initiate mutation but amplify growth signals in cells that have already acquired a mutation.
Practice questions — Part 1Score: 0 / 10
1. Which intracellular second messenger is produced by adenylyl cyclase activation?
Adenylyl cyclase converts ATP to cyclic AMP (cAMP), the second messenger that activates protein kinase A (PKA). DAG and IP₃ are produced by phospholipase C from PIP₂, a different branch of signaling downstream of Gαq-coupled receptors.
2. How many transmembrane helices does a canonical GPCR contain?
GPCRs (also called 7-TM receptors or serpentine receptors) have seven hydrophobic transmembrane helices that snake back and forth through the plasma membrane. The ligand binds within the extracellular face or the transmembrane bundle, and the cytoplasmic face couples to heterotrimeric G proteins.
3. When stimulatory Gαs binds GTP and dissociates from Gβγ, what enzyme does it directly activate?
Gαs-GTP directly binds and activates adenylyl cyclase, a membrane enzyme that catalyzes conversion of ATP to cAMP. PKA is activated downstream by cAMP; PLC is activated by Gαq, not Gαs; phosphodiesterase degrades cAMP and is not activated by Gαs.
4. IP₃ releases Ca²⁺ from which intracellular store?
IP₃ diffuses from the plasma membrane to the ER, where it binds IP₃ receptors (IP₃R)—ligand-gated Ca²⁺ channels in the ER membrane. Ca²⁺ is maintained at high concentration (~0.5 mM) in the ER lumen by SERCA pumps; IP₃R opening releases this store into the cytosol.
5. Which enzyme degrades cAMP to terminate PKA signaling?
Phosphodiesterase (PDE) hydrolyzes the 3'-phosphodiester bond of cAMP, converting it to inactive 5'-AMP. Caffeine and theophylline inhibit PDE, prolonging cAMP elevation. Some PDE isoforms are themselves activated by PKA-mediated phosphorylation—a negative-feedback loop that limits signaling duration.
6. Pertussis toxin (whooping cough) ADP-ribosylates Gαi, locking it in the GDP-bound inactive state. What is the signaling consequence?
Gαi normally inhibits adenylyl cyclase, reducing cAMP. Pertussis toxin locks Gαi in the inactive (GDP-bound) state, so it cannot inhibit adenylyl cyclase even when its GPCR is stimulated. The result is loss of inhibitory regulation of cAMP, leading to elevated cAMP levels in affected cells (including immune cells and airway epithelia).
7. What two products does phospholipase C generate from PIP₂?
Phospholipase C-β (activated by Gαq) or PLC-γ (activated by RTKs) cleaves PIP₂ (phosphatidylinositol 4,5-bisphosphate) into IP₃ (soluble, diffuses to ER) and DAG (remains in the membrane). Together they activate PKC; IP₃ alone triggers Ca²⁺ release from the ER.
8. Calmodulin (CaM) is a Ca²⁺-binding protein. Which of the following is directly activated by the Ca²⁺-calmodulin complex?
Ca²⁺-calmodulin binds and allosterically activates several target proteins including CaMKII (important in synaptic plasticity and learning), myosin light-chain kinase (smooth muscle contraction), calcineurin phosphatase (T-cell activation; target of cyclosporin), and nitric oxide synthase.
9. Which of the following signal molecules crosses the plasma membrane and binds an intracellular receptor?
Steroid hormones (cortisol, estradiol, testosterone, aldosterone) and thyroid hormone are lipophilic and freely cross the plasma membrane. They bind intracellular receptors (nuclear receptors) in the cytoplasm or nucleus. The hormone–receptor complex acts directly as a transcription factor. Epinephrine, glucagon, and insulin are hydrophilic and cannot cross the membrane; they signal through cell-surface receptors.
10. What is the intrinsic activity that terminates Gα signaling after receptor stimulation ends?
Gα subunits have an intrinsic, slow GTPase activity that hydrolyzes bound GTP to GDP, returning Gα to the inactive state and allowing reassociation with Gβγ. RGS (Regulators of G protein Signaling) proteins stimulate this GTPase activity, accelerating signal termination. This built-in off switch limits the duration of Gα signaling without requiring an additional off signal.
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Part 1 complete
Part 1 → 2
The GPCR/second-messenger pathways regulate mainly metabolic and rapid responses. Part 2 examines receptor tyrosine kinases and the downstream MAP kinase and PI3K/Akt cascades that control cell growth and survival, plus nuclear receptors, signal amplification, and feedback regulation including receptor desensitization.
Part 2 of 2
RTKs, Growth Factor Signaling, and Signal Regulation
15.4 Receptor Tyrosine Kinases (RTKs)
Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors with an extracellular ligand-binding domain and an intracellular tyrosine kinase domain. Examples include the EGF receptor (EGFR), PDGF receptor, and insulin receptor. Ligand binding induces receptor dimerization, which brings the two kinase domains together, enabling trans-autophosphorylation of specific cytoplasmic tyrosine residues. These phosphotyrosines become docking sites for SH2-domain-containing adaptor proteins (e.g., Grb2), initiating multiple downstream signaling cascades simultaneously.
15.5 The MAP Kinase Cascade
The Ras → Raf → MEK → ERK (MAP kinase) cascade is a prototypical growth-factor signaling pathway. Activated EGFR recruits Grb2 (via SH2 domain), which brings the GEF SOS to the membrane. SOS activates Ras by exchanging GDP for GTP. Ras-GTP activates Raf (a MAPKKK), which phosphorylates and activates MEK (a MAPKK), which phosphorylates and activates ERK (MAPK). Active ERK translocates to the nucleus and phosphorylates transcription factors (e.g., Elk-1, Fos), driving proliferative gene expression. Ras is inactivated by its intrinsic GTPase activity, accelerated by RasGAP. Activating RAS mutations are present in ~30% of all human cancers.
Key term
MAP kinase (ERK) cascade
A sequential kinase relay (Ras→Raf→MEK→ERK) downstream of RTKs that amplifies growth-factor signals and activates nuclear transcription factors to drive cell proliferation and differentiation.
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Pause & Recall
Why does a single activating RAS mutation transform cells, even though Ras GTPase activity is normally slow?
Common oncogenic RAS mutations (G12V, G12D, Q61L) impair the intrinsic GTPase activity of Ras and prevent GAP-stimulated GTP hydrolysis. With GTPase activity abolished, Ras remains locked in the GTP-bound (active) conformation, constitutively activating Raf and the ERK cascade regardless of whether growth factors are present—driving continuous cell proliferation.
15.6 The PI3-Kinase/Akt Pathway
RTKs and Ras also activate PI3-kinase (PI3K), which phosphorylates PIP₂ to produce PIP₃ at the inner leaflet of the plasma membrane. PIP₃ recruits the kinase PDK1 and the proto-oncogene kinase Akt (PKB) to the membrane; PDK1 phosphorylates and activates Akt. Akt promotes cell survival (inhibiting pro-apoptotic proteins like Bad and activating anti-apoptotic Bcl-2), stimulates protein synthesis (via mTOR), and promotes glucose uptake and glycolysis. The phosphatase PTEN reverses PIP₃ production; PTEN is a major tumor suppressor. Loss of PTEN leads to constitutive Akt activity and is among the most frequent events in human cancer.
15.7 Nuclear Receptors and Signal Amplification
Nuclear receptors (e.g., glucocorticoid receptor, estrogen receptor, thyroid hormone receptor) are ligand-activated transcription factors. In the absence of hormone, many are bound to chaperones (Hsp90) and held in the cytoplasm; hormone binding causes conformational change, dissociation from Hsp90, nuclear translocation, and dimerization. In the nucleus, the receptor dimer binds specific DNA response elements and recruits co-activator complexes that remodel chromatin and recruit RNA polymerase II.
Signal amplification occurs because a single activated receptor can activate many G protein molecules, each activating many effector molecules, each producing many second-messenger molecules, each activating many kinase molecules. This kinase cascade architecture allows a tiny hormonal signal to orchestrate large cellular responses. Receptor desensitization limits prolonged stimulation: agonist-bound GPCRs are phosphorylated by GRKs (GPCR kinases), which recruit β-arrestin, sterically blocking G protein coupling and targeting the receptor for endocytosis and degradation.
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Pause & Recall
How does β-arrestin recruitment to an agonist-bound GPCR lead to receptor desensitization?
GRK (GPCR kinase) phosphorylates multiple serine/threonine residues on the C-terminal tail and third intracellular loop of the agonist-bound receptor. β-arrestin binds these phosphorylated residues with high affinity, physically occluding the G protein-binding site (steric desensitization) and simultaneously serving as an adaptor for clathrin/AP2, triggering receptor internalization. The internalized receptor is either dephosphorylated and recycled (resensitization) or sorted to lysosomes for degradation (downregulation).
Practice questions — Part 2Score: 0 / 10
1. What event directly activates Ras after EGF binds its receptor?
Autophosphorylated EGFR recruits Grb2 (via its SH2 domain). Grb2's SH3 domains constitutively bind the proline-rich region of SOS (a RasGEF). Membrane-localized SOS then catalyzes exchange of GDP for GTP on Ras, activating it. Ras activation is NOT mediated by phosphorylation, cAMP, or PTEN (which is a phosphatase acting on PIP₃).
2. What lipid product of PI3-kinase recruits Akt to the plasma membrane?
PI3-kinase phosphorylates the 3-OH position of PIP₂ to produce PIP₃ (phosphatidylinositol 3,4,5-trisphosphate). PIP₃ is recognized by the PH (pleckstrin homology) domain of Akt, anchoring it to the membrane where PDK1 can phosphorylate and activate it. PTEN reverses this by dephosphorylating PIP₃ back to PIP₂.
3. PTEN is described as a tumor suppressor because it opposes which survival pathway?
PTEN (Phosphatase and TENsin homolog) is a phospholipid phosphatase that converts PIP₃ back to PIP₂, opposing PI3K. By reducing PIP₃ levels, PTEN reduces Akt activity and promotes apoptosis. PTEN loss/mutation occurs in ~40% of human cancers, constitutively activating Akt and suppressing apoptosis—driving tumor cell survival.
4. In the MAP kinase cascade, which protein kinase directly phosphorylates and activates ERK?
The MAP kinase relay is Ras→Raf (MAPKKK)→MEK (MAPKK)→ERK (MAPK). MEK is a dual-specificity kinase that phosphorylates both a threonine and a tyrosine residue in the ERK activation loop. This dual phosphorylation is required for full ERK activation. Grb2 and SOS are upstream adaptors for Ras activation.
5. How do nuclear receptors (e.g., the glucocorticoid receptor) become active transcription factors?
Unliganded nuclear receptors are often retained in the cytoplasm by chaperone complexes containing Hsp90, which masks the nuclear localization signal. Steroid binding causes a conformational change that releases the receptor from Hsp90, exposes the NLS, allows nuclear import, homodimerization, binding to specific response elements (e.g., GREs for the glucocorticoid receptor), and recruitment of co-activators to regulate transcription.
6. RTK dimerization upon ligand binding triggers which catalytic event between the two receptor cytoplasmic domains?
When ligand bridges two RTK monomers or induces conformational changes that force dimerization, the two intracellular kinase domains phosphorylate each other (trans-autophosphorylation) on specific tyrosine residues. These phosphotyrosines create docking sites for SH2-domain-containing proteins, amplifying signal transduction into multiple intracellular cascades simultaneously.
7. Which mechanism limits the duration of GPCR signaling after sustained agonist exposure?
Receptor desensitization: GRKs (GPCR kinases) preferentially phosphorylate agonist-occupied GPCRs on C-terminal serine/threonine residues. β-arrestins bind phosphorylated receptors, sterically block G protein coupling (desensitization), and recruit the clathrin/AP2 endocytic machinery. Internalized receptors are dephosphorylated and recycled (resensitization) or degraded (downregulation depending on signaling context).
8. Signal amplification in the GPCR→cAMP→PKA pathway means that:
Amplification occurs at each step: one receptor activates many G proteins (receptor is a catalyst); each Gα activates one adenylyl cyclase that produces many cAMP molecules; each cAMP activates PKA catalytic subunits that phosphorylate many substrate proteins. The cascade multiplies the signal at each enzymatic step, so a femtomolar concentration of hormone can trigger a response involving millions of substrate phosphorylations.
9. mTOR is a kinase activated by Akt. Which of the following best describes one of mTOR's downstream effects?
mTORC1 (mTOR complex 1) activated by Akt phosphorylates S6 kinase 1 (activating ribosome biogenesis and translation elongation) and 4EBP1 (releasing it from eIF4E, promoting cap-dependent translation initiation). Together these stimulate protein synthesis and cell growth. mTOR also suppresses autophagy—consistent with its role as a nutrient sensor that promotes anabolism when nutrients are available.
10. Imatinib (Gleevec) treats chronic myelogenous leukemia (CML) by inhibiting the BCR-Abl tyrosine kinase. This is an example of which therapeutic strategy?
BCR-Abl is a constitutively active tyrosine kinase produced by the Philadelphia chromosome translocation, the driver mutation in CML. Imatinib occupies the ATP-binding pocket of Abl kinase, blocking phosphoryl transfer and thereby switching off the constitutive proliferative signal. This "targeted therapy" revolutionized CML treatment, converting it from a fatal disease to a manageable chronic condition in most patients.
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Part 2 complete
Chapter 15 takeaways
GPCRs activate heterotrimeric G proteins; Gαs→adenylyl cyclase→cAMP→PKA; Gαq→PLC→IP₃+DAG→Ca²⁺/PKC.
G protein signaling is terminated by Gα GTPase activity and cAMP degradation by phosphodiesterase.
RTKs dimerize upon ligand binding, trans-autophosphorylate, and recruit SH2-domain adaptors to activate Ras→MAP kinase and PI3K→Akt cascades.
Oncogenic RAS mutations lock Ras-GTP on; PTEN loss locks PI3K/Akt on—both are common in cancer.
Nuclear receptors are cytoplasmic transcription factors activated by lipophilic hormones that directly cross the membrane.
Receptor desensitization: GRK phosphorylation + β-arrestin recruitment → internalization, limiting signal duration and enabling crosstalk.
End-of-chapter questions
Type your answer, then click Check answer for feedback and a sample answer.
Section B — Recall Questions
B1
Describe the sequence of molecular events from GPCR ligand binding to PKA activation, including how the signal is terminated.
Sample answer: Ligand binds GPCR → receptor acts as GEF for Gαs, swapping GDP for GTP → Gαs-GTP dissociates from Gβγ and activates adenylyl cyclase → adenylyl cyclase converts ATP to cAMP → cAMP binds regulatory subunits of PKA, releasing active catalytic subunits → PKA phosphorylates substrates. Termination: Gα hydrolyzes GTP→GDP (intrinsic GTPase, accelerated by RGS proteins); phosphodiesterase degrades cAMP to 5'-AMP.
B2
Explain how PLC activation generates two second messengers and what downstream targets each activates.
Sample answer: PLC cleaves PIP₂ → IP₃ (soluble) + DAG (membrane-bound). IP₃ binds IP₃R on ER → Ca²⁺ release → activates calmodulin/CaMKII, calcineurin, and other Ca²⁺-sensitive proteins. DAG remains in membrane + elevated Ca²⁺ together → recruit and activate PKC, which phosphorylates many substrates driving proliferation and secretion.
B3
Outline the MAP kinase cascade from Ras activation to nuclear transcription factor phosphorylation.
Sample answer: Ras-GTP activates Raf (MAPKKK) → Raf phosphorylates/activates MEK (MAPKK) → MEK dual-phosphorylates ERK (MAPK) → active ERK translocates to nucleus → phosphorylates transcription factors (Elk-1, Fos) → drives expression of proliferative genes (e.g., cyclin D1, Myc). Ras is inactivated by its GTPase (accelerated by RasGAP).
B4
Describe how RTK activation leads to Akt activation via PI3K and explain the tumor-suppressive function of PTEN in this pathway.
Sample answer: RTK→PI3K activated (directly or via Ras) → PI3K phosphorylates PIP₂→PIP₃ at membrane → PIP₃ recruits Akt and PDK1 (via PH domains) → PDK1 phosphorylates Akt Thr308 → active Akt promotes survival (Bad phosphorylation, Bcl-2 promotion), protein synthesis (mTOR activation), glucose uptake. PTEN is a PIP₃ phosphatase converting PIP₃ back to PIP₂, opposing Akt activation. Loss of PTEN → constitutive Akt → cell survival without growth factor signals → tumorigenesis.
B5
Explain how the glucocorticoid receptor transitions from a cytoplasmic chaperone complex to a nuclear transcription factor upon cortisol binding.
Sample answer: Unliganded GR is held in cytoplasm by Hsp90/Hsp70 chaperone complex that masks the NLS. Cortisol (lipophilic) diffuses across plasma membrane → binds GR ligand-binding domain → conformational change → Hsp90 releases GR → NLS exposed → nuclear import via importins → GR homodimerizes → binds GREs in promoters of target genes → recruits co-activators (SRC-1, p300) → chromatin remodeling and RNA Pol II recruitment → gene transcription.
B6
Describe GPCR desensitization and the roles of GRKs and β-arrestins.
Sample answer: Sustained agonist → GRKs phosphorylate serine/threonine residues on agonist-occupied receptor (C-tail, ICL3) → β-arrestin 1/2 bind phosphorylated receptor with high affinity → steric block of G protein coupling (desensitization) → β-arrestin recruits clathrin + AP2 → receptor internalized in clathrin-coated vesicle → dephosphorylated and recycled (resensitization) or degraded in lysosome (downregulation). β-arrestin also scaffolds its own signaling complexes (e.g., ERK activation independent of G proteins).
B7
Explain the molecular mechanism by which cholera toxin causes watery diarrhea.
Sample answer: Cholera toxin (CT) catalytic A subunit enters intestinal epithelial cells and ADP-ribosylates Gαs at Arg201, which prevents GTP hydrolysis. Gαs is therefore locked in the active (GTP-bound) state and continuously activates adenylyl cyclase → massively elevated cAMP → maximal PKA activation → PKA phosphorylates CFTR (open, Cl⁻ secretion) and inhibits apical Na⁺/H⁺ and Na⁺/Cl⁻ cotransporters → net secretion of NaCl and water into gut lumen → rice-water diarrhea and dehydration.
B8
What is the function of SH2 domains, and why is trans-autophosphorylation of RTKs important for their role as signaling platforms?
Sample answer: SH2 (Src Homology 2) domains bind phosphotyrosine-containing sequences with high specificity; different SH2 domains recognize different phosphotyrosine contexts. Trans-autophosphorylated RTKs display multiple distinct phosphotyrosines that serve as docking sites for different SH2-containing proteins (Grb2, PI3K p85, PLCγ, Src, SHP2)—simultaneously recruiting many signaling molecules. This allows a single RTK to activate multiple downstream pathways in parallel, enormously amplifying and diversifying the cellular response to a single growth factor.
B9
What is signal crosstalk, and give one example of how the cAMP and MAP kinase pathways might interact.
Sample answer: Crosstalk is the modulation of one signaling pathway by components of another, allowing cells to integrate multiple inputs. Example: PKA (activated by cAMP) can phosphorylate Raf in some cell types, inhibiting Raf activity and thus reducing ERK activation. This means a Gs-coupled receptor signal can dampen a growth-factor-driven MAP kinase signal. Conversely, in other cell contexts PKA activates Rap1, a Ras-like GTPase that activates B-Raf, stimulating ERK. The direction of crosstalk is cell-type and context dependent.
B10
Describe how nitric oxide (NO) acts as a local signaling molecule to cause smooth muscle relaxation and vasodilation.
Sample answer: Acetylcholine or shear stress → endothelial cells → Ca²⁺/CaM → eNOS (endothelial nitric oxide synthase) → produces NO from arginine. NO is a gas that diffuses into adjacent smooth muscle cells → binds and activates soluble guanylyl cyclase → converts GTP to cGMP → cGMP activates PKG → PKG phosphorylates MLCK (inhibiting it) and SERCA (stimulating Ca²⁺ reuptake) → reduced myosin light-chain phosphorylation → smooth muscle relaxation → vasodilation. Sildenafil (Viagra) inhibits PDE5 (which degrades cGMP), prolonging vasodilation in penile vasculature. Nitroglycerin is a NO donor used to treat angina.
Section C — Critical Thinking
C1
Explain why activating mutations in RAS and inactivating mutations in PTEN both produce similar outcomes (constitutive cell proliferation and survival), even though Ras and PTEN act at different points in the signaling network.
Sample answer: Both mutations constitutively activate overlapping downstream effectors. Ras-GTP activates both Raf→ERK (proliferation) and PI3K→Akt (survival). Active PTEN loss removes the brake on PI3K, constitutively elevating PIP₃ and Akt. Even though Ras also feeds PI3K, PTEN loss specifically locks on the Akt arm. Both ultimately drive: (1) ERK-dependent expression of cyclin D1 and Myc (G1/S progression), and (2) Akt-dependent suppression of apoptosis (Bad phosphorylation, mTOR activation). The convergence on these two proliferative/survival nodes explains why different genetic lesions produce similar cancer phenotypes and why inhibiting either node (with Ras or PI3K/Akt inhibitors) can be therapeutically beneficial.
C2
Opioid tolerance involves desensitization and downregulation of mu-opioid receptors (GPCRs). Analyze how the molecular mechanisms of GPCR desensitization explain why patients requiring chronic opioid therapy need escalating doses.
Sample answer: Opioids bind mu-opioid receptors (MOR), activating Gi (reducing cAMP) and other pathways to produce analgesia. Sustained opioid exposure → GRK phosphorylation of MOR → β-arrestin recruitment → acute desensitization (G protein uncoupling). Continued exposure → receptor internalization → some receptors are degraded (downregulation), reducing total surface receptor number. With fewer functional MORs producing less Gi signaling per dose, a higher opioid concentration is needed to achieve the same analgesic effect. Tolerance is also influenced by upregulation of adenylyl cyclase (cAMP superactivation) as a homeostatic counter-adaptation. Understanding this molecular tolerance provides rationale for analgesic rotating, dose holidays, and development of biased agonists that engage G proteins but not β-arrestin.
C3
Imatinib was a breakthrough in CML treatment, but patients sometimes develop resistance. Propose two molecular mechanisms by which CML cells could become resistant to imatinib without losing BCR-Abl.
Sample answer: 1) Point mutations in the BCR-Abl kinase domain (e.g., T315I "gatekeeper" mutation) alter the ATP-binding pocket so that imatinib no longer binds with sufficient affinity while ATP and substrate binding are retained; this is the most common resistance mechanism. 2) Amplification of BCR-Abl gene → overexpression of BCR-Abl protein → more total kinase activity than imatinib can inhibit at therapeutic concentrations. Other mechanisms: activation of alternative survival kinases (Src kinases, Lyn) that bypass BCR-Abl dependence; upregulation of drug efflux pumps (P-glycoprotein). Second/third-generation inhibitors (dasatinib, nilotinib, ponatinib) were designed to overcome specific resistance mutations.
C4
Cyclosporin A is an immunosuppressant that inhibits calcineurin. Trace the Ca²⁺ signaling pathway in T cells that cyclosporin blocks, and explain why this prevents T-cell activation.
Sample answer: TCR antigen recognition → PLC-γ activation → IP₃ → Ca²⁺ release from ER + CRAC-channel–mediated store-operated Ca²⁺ entry → sustained elevated cytosolic Ca²⁺ → Ca²⁺-CaM → activates calcineurin phosphatase → calcineurin dephosphorylates NFAT (nuclear factor of activated T cells) → NFAT translocates to nucleus → drives IL-2 transcription → IL-2 drives T-cell clonal expansion and activation of immune response. Cyclosporin binds cyclophilin; cyclophilin-cyclosporin complex inhibits calcineurin → NFAT remains phosphorylated and cytoplasmic → IL-2 gene not transcribed → no T-cell proliferation → immunosuppression. This is exploited to prevent transplant rejection but increases infection risk.
C5
Analyze how negative feedback within and between signaling pathways prevents runaway cell proliferation in normal cells, and why loss of these feedbacks is a hallmark of cancer.
Sample answer: Multiple interlocking negative feedbacks dampen mitogenic signals: (1) Active ERK phosphorylates SOS, dissociating it from Grb2, reducing Ras activation. (2) Active Akt phosphorylates and activates PDE-3, reducing cAMP. (3) PKA phosphorylates Raf (some contexts), reducing ERK activity. (4) mTORC1 phosphorylates IRS-1 (insulin receptor substrate), reducing PI3K activation downstream of the insulin receptor. (5) DUSP (dual-specificity phosphatases) are induced by ERK and dephosphorylate ERK itself. Together these ensure signals are transient and dose-proportional. Cancer cells disable these feedbacks: RAS mutations bypass GAP-mediated inactivation; PTEN loss removes the PIP₃ brake; ERK target transcription factors like Sprouty (normally negative regulators of RTK signaling) may be lost. Without these brakes, pro-growth signals become constitutive, satisfying Hanahan and Weinberg's hallmark of "sustaining proliferative signaling."
Section D — Interactive Questions
D1
What second messenger is produced when adenylyl cyclase is activated? (abbreviation)
D2
Which small GTPase links RTK activation to the MAP kinase cascade? (one word)
D3
Which phosphatase opposes PI3-kinase by converting PIP₃ back to PIP₂? (four letters)
D4
IP₃ triggers Ca²⁺ release from which organelle? (two words)
D5
Which enzyme directly phosphorylates and activates ERK in the MAP kinase cascade? (three letters)