Module 6: Signal Transduction

What is Dictyostelium discoideum and how does it respond to resource scarcity?

• Eukaryotic slime mold with both unicellular and multicellular stages

• Transitions:

  • From single amoebae to multicellular slug

  • Then to a fruiting body

• During food scarcity:

  • Cells aggregate to form a slug

  • Slug migrates toward heat, light, and humidity to find food

  • Differentiates into:

    • Anterior → prestalk cells

    • Posterior → prespore cells

• In suitable environment:

  • Anterior forms stalk

  • Posterior forms spores of the fruiting body (~2 mm tall)

What triggers aggregation in Dictyostelium and what is the outcome?

• Vegetative Growth Phase: Food Abundant

  • Feed on bacteria (E.coli)

  • Amoeba divide by mitosis

• Trigger: Starvation

• Signal: Cyclic AMP (cAMP)

  • Secreted by starved cells

  • Attracts other amoebae

• Outcome:

  • Aggregation into slug

  • In a nutrient rich environment, differentiation into stalk and spores

  • Spores have hard cell walls (dormant survival)

• Spore germination occurs when food returns and new single-celled amoebae form

How do Dictyostelium cells detect and respond to cyclic AMP (cAMP) during aggregation?

• cAMP acts as aggregation signal

• Detected by GPCR which binds cAMP on extracellular domain

• Triggers actin cytoskeleton reorganization

• Allows cells to move toward cAMP source

• Example: cells in dish move toward pipette releasing cAMP

How do Dictyostelium cells physically respond to cAMP?

• Extend filopodia toward signal

• Undergo actin reorganization:

  • Nucleation

  • Polymerization

  • Depolymerization

• Dynamic movement is directed toward cAMP source

How does a clathrin heavy chain mutation affect Dictyostelium’s response to cAMP?

• Mutation blocks vesicle formation → no protein transport to membrane

• GPCR (receptor for cAMP) can't reach cell surface

• cAMP still present (signal source unchanged)

• Cells can form filopodia but show no net movement

• Reason: no surface GPCR → no cAMP detection → no directed movement

How do neutrophils in humans respond to bacterial infections?

• Detect chemical signals from bacteria

• Move towards signal source (chemotaxis)

• Process:

  • Surface receptors bind signal

  • Internal signaling cascade activates

  • Leads to movement and endocytosis of bacteria

How do neutrophils detect and respond to bacterial signals?

• Neutrophils = type of white blood cell

• Detect chemical signals from bacteria

• Surface receptors bind bacterial signal

• Triggers internal signaling cascade

• Leads to directed movement toward bacteria (chemotaxis)

• Eventually engulf bacteria via endocytosis

What signal do neutrophils detect to find bacteria, and how do they respond?

• Bacteria unintentionally release fMLP (formylated-Met-Leu-Phe peptide)

• fMLP = bacterial protein fragment

• Neutrophils have GPCR on surface that binds fMLP

• GPCR activation → intracellular signaling cascade

• Neutrophil moves toward fMLP source

What is cell signaling and what are its key steps?

• Cell signaling = transmission of info from one cell to another

• Must lead to change in behavior in receiving cell

• Signal alone is not useful without a response

• Key steps:

  • Production & release of signal

  • Perception by receptor (usually on cell surface)

  • Interpretation via intracellular pathways

  • Cellular response (e.g., movement, division, secretion)

Outcomes of STP

• Signal transduction pathway (STP): Series of chemical events inside cell

• Leads to changes in target cell behavior, e.g.:

  • Gene transcription

  • Cell movement/growth

  • Cell differentiation

  • Metabolic changes (enzyme activation/inactivation)

• Signal must be removed to stop response

• Only cells with the correct receptor can respond to the signal

What determines specificity in signal-receptor interactions?

• Based on molecular complementarity

• Factors:

  • Shape and fit of interacting surfaces

  • Non-covalent interactions (e.g., hydrogen bonds)

• Specific amino acid residues in:

  • Signal molecule

  • Receptor

• Single amino acid changes can disrupt binding

• Receptors usually bind only one natural ligand or closely related molecules

• Receptor binds signal:

  • Conformational change in receptors intracellular domain → triggers signal transduction cascade → cellular response

How is specificity of the signal response achieved in cells?

• First level: specificity of ligand-receptor binding

  • Only cells with the matching receptor respond

• Second level: specificity of intracellular response

  • Same signal can activate different proteins in different cells

  • Leads to varied outcomes (e.g., transcription factor activation, movement, metabolism)

• Signal transduction pathway translates extracellular signal into specific cellular response

• Specificity determined by internal signal transduction pathway

What distinguishes fast and slow cellular responses to signals?

• Fast response:

  • Signal binds membrane receptor

  • Activates cytosolic enzyme by modification (phosphorylation, methylation, acetylation)

  • Changes activity of existing proteins

  • Rapid response

• Slow response:

  • Signal passes through membrane to soluble cytosolic receptor

  • Receptor moves to nucleus

  • Acts as transcriptional activator → mRNA produced → protein synthesis

  • Requires transcription, translation, folding, modifications

  • Slower response

• Same signal & receptor types can trigger either response depending on cell type

How can receptor-signal binding affinity and cellular physiological response be measured, and what does their comparison reveal?

• Measure receptor affinity by plotting ligand concentration vs. fraction of receptors bound

  • Kd = ligand concentration at half-maximal binding (rep receptor-signal affinity)

• Measure physiological response by plotting ligand concentration vs. fraction of cells responding

  • Half-maximal response concentration often lower than Kd

• Indicates signal amplification inside the cell

• Small amounts of signal can trigger a large cellular response

What are the main differences between endocrine and paracrine intercellular signaling?

• Endocrine signaling:

  • Signals (usually hormones) secreted into circulatory system

  • Signal reaches distant target cells throughout body

  • Many different tissues can respond simultaneously

  • Signaling and target cells are far apart

• Paracrine signaling:

  • Signals released into extracellular space

  • Affect nearby neighboring cells

  • Common signals: growth factors, neurotransmitters

  • Signaling and target cells are close together

What is proximal signaling and how do cells communicate through direct contact or cytosolic connections?

• Proximal signaling: signal and receptor are transmembrane proteins on adjacent cells

• Requires direct cell-cell contact via cell integral membrane proteins

• Plants: use plasmodesmata — channels connecting cytoplasm across cell walls, allowing fast cytosolic messenger movement

  • Forms vascular system to transfer signals from root → leaves

• Animals: use gap junctions — cytoplasmic channels for diffusion of small molecules/secondary messengers

• Allows coordination of responses by sharing internal signals between neighboring cells

What is autocrine signaling and give an example?

• Cell signals to itself (signaling cell = target cell)

• Produces and responds to its own secreted signal

• Example: growth factors that regulate cell division (promote or inhibit)

• Allows a cell to self-regulate based on internal and external conditions

What are the 3/7 major types of cell surface receptors focused on in this module, and what signaling pathways do they involve?

• Cytokine receptors: involved in JAK/STAT pathway, regulate red blood cell production

• Receptor Tyrosine Kinases (RTKs): linked to phosphorylation cascade via small G-protein Ras, regulate gene expression

• G-Protein Coupled Receptors (GPCRs): activate effector proteins to produce second messenger cAMP, regulate cell metabolism

How does erythropoietin (Epo) regulate red blood cell production?

• Adult human body produces ~2 million new erythrocytes (red blood cells) per second

• Develop in bone marrow, circulate ~4 months, then recycled by macrophages

• Red blood cells replaced when pluripotent stem cells (progenitor cells) stop dividing and differentiate

• Erythropoietin (Epo) is the key cytokine signal triggering erythrocyte maturation

• Epo production regulated by an oxygen-binding transcription factor in kidney cells

• Epo is secreted into the circulatory system (public signal)

• Only erythrocyte progenitor cells express the erythropoietin receptor (EpoR), a cytokine receptor

• Binding of Epo to EpoR activates the JAK-STAT signal transduction pathway

• JAK-STAT activation causes:

  • Inhibition of apoptosis (cell death) in progenitor cells

  • Changes in gene expression patterns supporting maturation

  • Commitment of progenitors to differentiate into mature erythrocytes

How is the EpoR activated?

• Inactive state:

  • EpoR is a monomeric, single-pass transmembrane protein, inactive alone

• Signal binding:

  • One Epo molecule binds two EpoR monomers, causing receptor dimerization

  • This brings cytosolic domains close together, initiating intracellular signaling

What happens when erythropoietin binds to its receptor?

• Epo receptor has 3 domains:

  • Extracellular domain (binds Epo)

  • Transmembrane alpha-helix

  • Cytosolic domain (binds JAK kinase)

• Each receptor is associated with a JAK kinase (inactive by default)

• Epo binding → Receptor dimerization

• Dimerization brings JAKs close → Autophosphorylation

  • JAKs phosphorylate each other on activation lip

  • Activates kinase activity

• Activated JAKs → Phosphorylate tyrosine residues on receptor’s intracellular domain

• JAK = tyrosine kinase (targets tyrosine residues only)

How does activation of the erythropoietin receptor lead to STAT activation and gene expression?

• Phosphorylated tyrosines on EpoR = docking sites for other proteins

• STAT transcription factors bind using SH2 domain (recognizes phosphotyrosine)

• Binding brings STAT near JAK kinase

• JAK phosphorylates STAT → activates it

• Phosphorylated STAT dimerizes (activation step)

• Dimerization exposes nuclear localization sequence

• STAT dimer enters nucleus → activates transcription of target genes

What is the function of the SH2 domain in cytokine signaling?

• Protein-protein interaction domain (no enzymatic function)

• Binds to specific phosphotyrosine-containing sequences

• Binding is high affinity when tyrosine is phosphorylated

• Used for:

  • Re-localizing proteins (e.g., STAT to receptor)

  • Linking proteins in a signaling pathway

• Example binding sequence: Pro-Asn-pTyr-Glu-Glu-Ile-Pro

• Binding is reversible depending on phosphorylation state

What are different types of protein-protein interaction domains, and how do they differ?

• All link proteins together

• Some require reversible modifications (like phosphorylation):

  • SH2, PTB, 14-3-3: bind phosphorylated tyrosine

  • Allow reversible binding

• Others bind unmodified sequences:

  • PDZ: binds C-terminal hydrophobic residues

  • SH3 & WW: bind proline-rich sequences

  • Binding is not reversible

How does the cytokine/JAK-STAT pathway regulate red blood cell production (erythrogenesis)?

• Signal: Erythropoietin (Epo)

• Receptor: Epo receptor (activates JAK-STAT pathway)

• STAT5: Key transcription factor activated

• Target gene: Bcl-xL (codes for Bcl-XL protein)

  • Bcl-XL inhibits apoptosis

  • Allows erythroid progenitor cells to survive & differentiate

• Erythrogenesis sites:

  • Bone marrow (primary source)

  • Fetal liver (during development)

• Mutation impacts: Defects in Epo, EpoR, JAK, STAT5, or Bcl-xL → no red blood cells produced

• Example assay:

  • WT embryo: red fetal liver (active erythrogenesis)

  • EpoR knockout: pale liver (no RBC production)

Why and how is the cytokine/JAK-STAT pathway turned off, and what are the risks of failure to regulate it?

• Why regulation is critical:

  • Too little signaling → no red blood cell production → lethal

  • Too much signaling → overproduction of RBCs → elevated hematocrit

    • Increases blood viscosity

    • Risk of capillary blockages, stroke, heart attack

• Example:

  • Athletes may use Epo doping (external Epo) to boost oxygen capacity

  • Increases endurance but is dangerous & potentially lethal

• How the pathway is turned off:

  • Short-term inactivation via dephosphorylation

    • Protein: SHP1 phosphatase

    • Has two SH2 domains → binds to same sites as STAT

    • Once bound, SHP1 dephosphorylates JAK kinase → turns off signaling

    • Can be quickly reversed when SHP1 detaches

What are the long-term mechanisms that turn off erythrogenesis via the JAK-STAT pathway, and what happens when this regulation fails?

• SOCS Protein (Suppressor of Cytokine Signaling):

  • Binds phosphorylated docking sites via SH2 domain

  • Expressed in response to high O2 levels

  • Blocks STAT binding

  • Acts as an E3 ubiquitin ligase:

    • Targets JAK kinase for ubiquitination

    • Leads to proteasomal degradation of JAK

    • Long-term shutdown (JAK must be re-synthesized to reactivate)

• Gene Expression Regulation:

  • SOCS is upregulated when oxygen levels are high

  • Acts as a negative feedback loop to stop red blood cell production

• Mutations in EpoR gene:

  • Truncated receptors → shorter docking sites

  • Decreased sensitivity to SHP1 and SOCS

  • Results in elevated hematocrit (mimics Epo doping)

  • Seen naturally in some individuals (not doping)

• Receptor recycling:

  • Endocytosis removes receptor from surface

  • Ligand (Epo) dissociates → signal ends

  • Receptor may be recycled but won’t be reactivated unless Epo is present

What are Receptor Tyrosine Kinases (RTKs) and what do they do?

• RTKs = cell surface receptors with intrinsic tyrosine kinase activity

• Activated by hormones: NGF, PDGF, EGF, insulin

• Functions: cell differentiation, survival, proliferation, metabolism

• Mechanism:

  • Hormone binds → receptor dimerizes

  • Activates kinase → phosphorylates tyrosines

  • Triggers downstream signaling (e.g., Ras G-protein activation)

• Example: NGF → neuron axon growth

How do Receptor Tyrosine Kinases (RTKs) activate cell signaling pathways?

• RTKs have:

  • Extracellular signal-binding domain

  • Single transmembrane domain

  • Intrinsic kinase activity on cytoplasmic side

• Ligand binding → dimerization → autophosphorylation

• Activates downstream signaling via Ras G-protein (intracellular, membrane-anchored protein)

• Key players that regulate and link Ras to the activated RTK:

  • GRB2 (adaptor protein)

  • Ras effectors

    • GEF (activates Ras)

    • GAP (inactivates Ras)

• Activated Ras → MAP kinase cascade

• MAP kinase → phosphorylates transcription factors → changes gene expression

How is RTK activation similar to cytokine receptor activation?

• Ligand binding (e.g., EGF) initiates activation

• Causes conformational change in extracellular domain

• Leads to dimerization of two RTK molecules

• Dimerization is necessary for activation of kinase activity

How does dimerization activate Receptor Tyrosine Kinases (RTKs)?

• RTK monomers have low intrinsic kinase activity

• Dimerization brings kinase domains close together

• Enables autophosphorylation of each other's activation lip

• Phosphorylation of activation lip increases kinase activity

• Activated RTKs phosphorylate tyrosine residues on intracellular domains

• These phosphorylated sites serve as docking sites for proteins with SH2 or PTB domains

What is the role of adaptor proteins in RTK signaling pathways?

• Adaptor proteins have multiple protein interaction domains

• Link activated RTK receptors to downstream signaling proteins

• Bind phosphorylated docking sites on RTKs and other signaling proteins

• Help bridge signaling components without enzymatic activity

• Scaffold proteins are larger adaptor proteins that organize multiple signaling proteins into a structured sequence

What is GRB2 and how does it function in RTK signaling?

• GRB2 is an adaptor protein in RTK signaling

• Has 3 domains:

  • 1 SH2 domain → binds phosphorylated tyrosine on RTK docking site (reversible)

  • 2 SH3 domains → bind proline-rich sequences (like in SOS)

• Links RTK receptor to SOS, a guanine nucleotide exchange factor (GEF)

• Facilitates activation of Ras by recruiting SOS to the membrane

What is the role of the SH3 domain in RTK signaling?

• SH3 domain is a protein-protein interaction domain

• Binds to proline-rich sequences on target proteins (e.g., SOS)

• Interaction is based on molecular complementarity:

  • SH3 domain (red) fits into clefts of proline-rich peptide (green)

• Binding is specific and stable, not dependent on phosphorylation

• Enables adaptor proteins (like GRB2) to link RTKs to downstream signaling components

How does the Ras G-protein cycle between active and inactive states?

• Ras is a GTPase switch protein: active when bound to GTP, inactive when bound to GDP

• GTP-bound ("ON" state):

  • GTP’s terminal phosphate interacts with glycine and threonine residues on the switch regions

  • Switches are pulled together, stabilizing the active conformation

• Intrinsic GTPase activity:

  • Always active but not regulated ON/OFF

  • Hydrolyzes GTP to GDP + Pi, turning Ras "OFF"

• GDP-bound ("OFF" state):

  • Loss of terminal phosphate causes switches to fold out

  • Glycine and threonine residues no longer interact with GDP

• GDP has low affinity and dissociates, allowing new GTP to bind and reactivate Ras

• This cycle regulates Ras activity in RTK signaling pathways

What proteins regulate the activation and inactivation of G-proteins?

• GEF (Guanine Nucleotide Exchange Factor):

  • Promotes GDP release

  • Facilitates GTP binding → activates G-protein

• GAP (GTPase Activating Protein):

  • Enhances intrinsic GTPase activity (up to 100x)

  • Accelerates GTP hydrolysis → inactivates G-protein

• GDI (Guanine Nucleotide Dissociation Inhibitor):

  • Increases GDP affinity to nucleotide-binding pocket

  • Keeps G-protein in inactive “OFF” state

How is Ras G-protein activity regulated in the RTK pathway?

• OFF = GDP-bound (inactive), stabilized by GDI

• GEF triggers GDP release → GTP binds → ON (active) state → G-protein interacts with target protein

• GAP accelerates GTP hydrolysis → returns to OFF state → can’t interact with target protein

• Duration of ON state controlled by GAP → affects signaling length

How is Ras G-protein activated by SOS in the RTK pathway?

• Activated RTK recruits adaptor protein GRB2

• GRB2’s SH3 domains bind SOS, bringing it to the membrane

• SOS (a GEF) interacts with membrane-bound Ras

• SOS promotes GDP release from Ras → GTP binds

• Ras becomes activated (ON state)

What are the three conformational states of Ras protein?

• Inactive: Ras bound to GDP

• Intermediate: SOS binds Ras, displacing GDP

• Active: Ras bound to GTP

What is the role of NF1 in regulating Ras activity, and what happens when NF1 is mutated?

• NF1 is a GAP protein that enhances Ras’s intrinsic GTPase activity, speeding up GTP hydrolysis and inactivating Ras.

• Presence of NF1 shortens Ras active time, turning signaling off faster.

• Loss of NF1 function removes GAP activity, causing Ras to stay active longer.

• Prolonged Ras activity leads to excessive signaling, increasing cell division rates.

• NF1 mutations are linked to tumor formation in neural tissue (neurofibromatosis 1).

How does Ras activation relate to RTK signaling and cell division?

• RTK activation is upstream of Ras activation

• Ras activation occurs after RTK is activated by ligand (e.g., EGF)

• Ras links RTK signaling to changes in gene expression via downstream pathways

Key experiment findings:

• EGF only → RTK activated → cell division (control)

• Anti-Ras antibody + EGF → Ras blocked → no cell division

  • Shows Ras is required downstream of RTK

• Ras-D (always active) + no EGF → cell division

  • Shows Ras can bypass RTK if always active

Conclusion:

• Ras is downstream of RTK

• Ras activation alone can trigger cell division

• Constitutively active Ras (Ras-D) mimics tumor-like overproliferation

• Dominant Ras mutations are linked to cancer in various cell types

How do mutations in the RTK-Ras pathway lead to cancer?

• Constitutively active Ras → continuous signaling → uncontrolled cell division

  • Example: loss of glycine in Ras blocks GAP binding, so Ras stays ON

• Her2 mutant (RTK) → always dimerized → active without EGF

  • Leads to persistent signaling → hereditary breast cancer

• NF1 loss-of-function → no GAP activity → Ras not turned OFF → overproliferation

What happens after Ras is activated in the RTK signaling pathway?

• Activated Ras binds and activates Raf (a serine/threonine kinase)

• Raf is normally inhibited by binding to 14-3-3 adaptor protein

• Ras binding causes release of 14-3-3 → Raf activation

• Raf activation begins the MAP kinase cascade, leading to changes in gene expression and cell behavior

What is the kinase cascade activated by Ras in the RTK pathway?

• Ras activates Raf (MAP kinase kinase kinase)

• Raf phosphorylates MEK (MAP kinase kinase)

• MEK phosphorylates MAP kinase at 2 residues, tyrosine & threonine

• Activated MAP kinase:

  • Dimerizes

  • Moves to nucleus

  • Phosphorylates transcription factors → changes in gene expression

• Scaffold proteins help organize and stabilize the kinase cascade

How is MAP kinase activated in Ras-linked RTK signaling pathways?

• Activated by MEK, a dual-specificity kinase (targets threonine & tyrosine)

• Phosphorylation occurs on the activation lip of MAP kinase

• Conformational change exposes ATP & substrate binding pockets

• Similar activation mechanism as seen in other kinases

• MAP kinase = final downstream target in Ras-linked RTK pathways

What does activated MAP kinase do in the Ras-RTK pathway?

• Phosphorylates P90 RSK in cytoplasm → RSK moves into nucleus

• MAP kinase also enters nucleus

• In nucleus:

  • MAP kinase phosphorylates TCF (ternary complex factor)

  • P90 RSK phosphorylates SRF (serum response factor)

• TCF + SRF bind to SRE (Serum Response Element) on DNA

• This activates transcription of genes like C-fos

• C-fos promotes transcription of cell cycle genes

What is the final outcome of RTK signaling and how does signal amplification work?

• Final outcome: Activation of gene transcription → cell division, differentiation, other behaviors

• Signal amplification:

  • Each kinase can activate many target proteins

  • Example: One EGF molecule → millions of active MAP kinases (ERK1/2)

  • Leads to millions of copies of proteins needed for cell division

• Sensitivity: Pathway responds to very low hormone levels (nanomolar or 10^-9 M range)

What are GPCRs and why are they important in medicine?

• GPCRs = large family of receptors in humans

• Involved in almost every physiological process

• Many diseases linked to GPCR dysfunction

• Most pharmaceuticals target GPCRs to treat diseases

What is the common structure of GPCRs and what do their segments do?

• 7 transmembrane alpha helices

• 4 extracellular loops (E1–E4) form the signal-binding domain

• 4 cytoplasmic loops (C1–C4) interact with trimeric G protein

• Examples: stress receptors, rhodopsins (vision), odorant receptors, hormone/neurotransmitter receptors, plant growth hormone receptors, yeast glucose sensor

• ~800 GPCR genes in humans (~4% of protein-coding genome)

• Many diseases linked to GPCR dysfunction

What role do GPCRs play in the mammalian stress response involving catecholamines?

• Catecholamines (water-soluble signals that circulate bloodstream): epinephrine, norepinephrine, dopamine (fight-or-flight hormones)

• Catecholamine receptors are GPCRs

• Activation triggers trimeric G-protein → activates adenylyl cyclase (effector protein)

• Adenylyl cyclase increases cAMP (secondary messenger)

• Increased cAMP triggers:

  • Fast response: enzyme activation (quick energy release)

  • Slow response: gene transcription activation

How do alpha-2 and beta adrenergic receptors respond to epinephrine in different tissues?

• Beta adrenergic receptors (stimulatory):

  • Liver & adipose: stimulate glycolysis & lipolysis (fuel mobilization)

  • Heart muscle: increase contraction (more blood supply)

  • Intestinal smooth muscle: cause relaxation (reduce energy use in digestion so it can be focused on on locomotory muscles)

• Alpha-2 adrenergic receptors (inhibitory):

  • Found in blood vessels in skin, kidneys, smooth muscles of intestines

    • Cause artery constriction and reduce blood flow to periphery

• Overall: Coordinated responses increase energy supply to stress-response tissues.

Where do catecholamines like epinephrine and norepinephrine come from, and what roles do they play in the stress response?

• Catecholamines come from:

  • Adrenal glands (small endocrine glands that sit on top of kidneys) produce epinephrine (adrenaline) and steroids like aldosterone & cortisol.

  • Norepinephrine is secreted by nerve cells and acts as a neurotransmitter.

• Function of epinephrine:

  • Binds both beta and alpha-2 adrenergic receptors, causing different responses depending on receptor type.

  • Triggers stress response by increasing ATP supply through breakdown of energy stores, glycogen and triacylglycerol:

    • Glycolysis in liver (breaking down glycogen to glucose)

    • Lipolysis in adipose tissue (breaking down fatty acids).

How is a GPCR signaling pathway activated, and what role does the trimeric G-protein play?

• Inactive GPCR: Not bound to signal; not associated with trimeric G-protein.

• Trimeric G-protein: Lipid-anchored, with 3 subunits (α, β, γ);

  • Inactive when bound to GDP

  • Active when bound to GTP

• Activation:

  • Signal binds to GPCR → GPCR undergoes conformational change

  • GPCR activates the G-protein by promoting GDP → GTP exchange

  • Activated G-protein then interacts with effector protein to trigger downstream effects.

How is a GPCR activated and how does it activate the trimeric G-protein?

• Signal binds GPCR → conformational change in intracellular domain

• GPCR binds trimeric G-protein with high affinity

• This causes GDP to dissociate from the G-protein

• GTP binds in place of GDP → G-protein becomes active

• `Activated G-protein can now initiate downstream signaling

What happens after GPCR activates the trimeric G-protein, and how is the signal turned off?

• GTP-bound G-alpha subunit dissociates from beta & gamma

• G-alpha activates effector enzyme (e.g., adenylyl cyclase)

• Effector stays active while G-alpha is bound with GTP

• Intrinsic GTPase of G-alpha hydrolyzes GTP → GDP

• G-alpha becomes inactive, releases effector

• G-alpha rejoins beta/gamma subunits → signal is off

How do beta-adrenergic receptors activate cAMP production through Gs protein?

• Beta-adrenergic receptors activate Gs protein (stimulatory G protein)

• Gs alpha subunit (GTP-bound) activates adenylyl cyclase

• Adenylyl cyclase increases cAMP (secondary messenger)

• GTP → GDP hydrolysis inactivates Gs alpha → stops adenylyl cyclase

• cAMP levels drop without continuous receptor activation

How do alpha2-adrenergic receptors inhibit cAMP production via Gi protein?

• Alpha2-adrenergic receptors activate Gi protein

• Gi has the same beta & gamma subunits as Gs, but a different alpha subunit (Gi alpha)

• Gi alpha (GTP-bound) interacts with different region and inhibits adenylyl cyclase catalytic domain

• This prevents cAMP production

• Result: No increase in cellular energy signal (cAMP stays low)

How do β-adrenergic and α2-adrenergic receptors differently regulate cAMP levels?

• β-adrenergic receptor → activates Gsα → stimulates adenylyl cyclase → ↑ cAMP

• α2-adrenergic receptor → activates Giα → inhibits adenylyl cyclase → ↓ cAMP

• Epinephrine can bind both receptors to create diverse, tissue-specific responses

How does adenylyl cyclase produce cyclic AMP (cAMP)?

• Converts ATP → cAMP

• Releases diphosphate (PPi) as a byproduct

• Active adenylyl cyclase = high intracellular cAMP levels

What role does phosphodiesterase play in cAMP regulation?

• Breaks down cAMP → 5’AMP

• Constitutively active, making cAMP unstable

• If adenylyl cyclase is inhibited, cAMP levels drop quickly due to phosphodiesterase

What does cyclic AMP (cAMP) do in GPCR signaling?

• Acts as a secondary messenger

• Concentration reflects GPCR activity

• Modulates target protein activity, especially Protein Kinase A (PKA)

• PKA = serine/threonine kinase → phosphorylates many target proteins

How is Protein Kinase A (PKA) activated by cAMP?

• Inactive PKA = tetramer (2 regulatory + 2 catalytic subunits)

• Low cAMP: regulatory subunits block catalytic activity

• High cAMP: cAMP binds regulatory subunits → conformational change

• Catalytic subunits released → active PKA

What role does the pseudo-substrate domain play in PKA regulation?

• Pseudo-substrate domain (red) on regulatory subunit blocks catalytic site in inactive PKA

• With cAMP bound: pseudo-substrate retracts, freeing catalytic subunit → PKA active

• Without cAMP: pseudo-substrate extends, blocking substrate binding → PKA inactive

How does Protein Kinase A (PKA) regulate energy supply during the stress response?

• PKA increases glucose supply by regulating glycogen metabolism

• Inhibits glycogen synthase (stops glycogen formation)

• Activates glycogen phosphorylase (promotes glycogen breakdown to glucose)

• Released glucose feeds glycolysis → pyruvate + NADH → ATP production for energy during stress

How does PKA regulate glycogen metabolism differently in muscle and liver during the stress response?

• Muscle:

  • Glycogen → glucose-6-phosphate → glycolysis → ATP for skeletal and cardiac muscle contraction

• Liver:

  • PKA phosphorylates phosphorylase kinase → activates glycogen phosphorylase → glycogen breakdown

  • PKA phosphorylates/inactivates glycogen synthase → stops glycogen synthesis

  • Liver releases free glucose into bloodstream for other tissues

• This is a fast, short-term response through enzyme modification, not gene expression

What is the long-term role of activated PKA in the nucleus during the stress response?

• PKA catalytic subunit translocates to nucleus

• Phosphorylates transcription factor CREB

• CREB binds cAMP response element (CRE) on DNA

• Initiates transcription of genes (e.g., phosphorylase kinase, glycogen phosphorylase)

• Leads to increased production of enzymes for glucose production

• Represents a slow, long-term response to stress signaling

Why is signal amplification important in cell signaling pathways like the stress response?

• Amplifies small external signals to large internal responses

• Example: 10⁻¹⁰ M epinephrine triggers 10⁸-fold amplification

• Amplification mainly occurs at enzyme activation steps

• One adenylyl cyclase enzyme can produce ~100 cAMP molecules

• Ensures sensitive and widespread cellular responses to low hormone levels

Applied Lecture

GPCRs and Disease

What are the key features of cell signaling via GPCRs, what types of signals exist, and which responses are fast vs. slow?

• Receptor specificity: only cells with the matching receptor respond

• Signal types:

  • Physical stimulus (e.g., light, touch)

  • Chemical molecules:

    • Small molecules (e.g., ions, gases)

    • Peptides or soluble proteins (e.g., hormones)

    • Proteins bound to cell surface or ECM

• Signal transduction outcomes:

  • Fast responses:

    • Alteration of existing proteins (e.g., enzyme activation, second-messenger cascades)

    • Occur within seconds–minutes

  • Slow responses:

    • Changes in gene expression (e.g., transcription, new protein synthesis)

    • Occur over hours–days

How does ligand binding to a receptor lead to a cellular response?

• Receptor location: Found on or within target cells

• Specificity: Each receptor binds a specific ligand or related molecules

• Binding mechanism: Weak, noncovalent interactions (ionic, van der Waals, hydrophobic) and structural complementarity

• Effect of binding: Triggers a conformational change in the receptor

• Outcome: Initiates a signaling cascade, leading to a specific cellular response

How do GTP-binding proteins function as on/off switches in signaling pathways?

• GTP-binding proteins act as molecular switches that toggle between active and inactive states.

• Activation: A GEF (Guanine nucleotide Exchange Factor) promotes exchange of GDP for GTP, turning the switch ON.

• Inactivation: GTP hydrolysis (via intrinsic GTPase activity) converts GTP to GDP, turning the switch OFF.

• Examples:

  • Trimeric G proteins (receptor-bound)

  • Monomeric G proteins like Ras (not receptor-bound)

What are intracellular second messengers and what is their role in signaling?

• Ligands (first messengers) trigger changes in levels of low-molecular-weight second messengers

• Second messengers are small molecules (e.g., Ca²⁺, cAMP, cGMP, DAG, IP₃)

• They bind to proteins and alter their activity to propagate the signal

What is the role of protein kinases and phosphatases in cell signaling?

• Kinases add phosphate groups (to serine, threonine, or tyrosine)

  • Can be cytosolic or receptor-associated

• Phosphatases remove phosphate groups

• Phosphorylation often enables protein-protein interactions

• Human genome: ~600 kinases, ~100 phosphatases

How does signal transduction enable signal amplification?

• One ligand binding can activate a cascade

• Each step can activate multiple downstream molecules

• Results in hundreds of thousands of proteins activated from a single signal

What maintains water homeostasis under normal conditions?

• Output of water in our body comes from sensible (sweat, feces, urine) and insensible (skin and lungs) losses

• Normal water intake is balanced by normal water loss

• This balance maintains proper hydration and homeostasis

How do the kidneys maintain water balance and filter blood through nephrons?

• Kidneys maintain water balance, crucial for cytoplasm, tissues, and blood plasma

• Water loss occurs mainly via urine

• Renal arteries bring blood to kidneys for filtration; renal veins return filtered blood

• Nephron = functional unit of the kidney (millions in renal pyramids)

• Blood flows: Renal artery → Afferent arteriole → Glomerulus → Bowman's capsule

• Only small molecules (water, salts, ions, urea) pass into nephron; large ones (proteins, RBCs/WBCs) don’t

• Fluid travels: Bowman's capsule → Proximal tubule → Loop of Henle → Distal tubule → Collecting duct

• Reabsorption of water, salts, ions occurs along nephron via peritubular capillaries

• Excess fluid becomes urine → bladder → excretion

• Kidneys fine-tune reabsorption to maintain homeostasis

What happens in the descending and ascending limbs of the nephron during osmoregulation?

• Descending limb of loop of Henle:

  • Water reabsorption via transporters

  • Water moves into interstitial space → peritubular capillaries

  • Urine volume decreases

  • Urine becomes more concentrated

• Ascending limb of loop of Henle:

  • Impermeable to water

  • Ions reabsorbed (Na⁺, Cl⁻, K⁺)

  • Urine becomes more dilute

• Collecting duct:

  • Final water reabsorption

  • Urine concentration increases

  • Remaining fluid → bladder → excretion

  • Uses gradient to adjust urine concentration

• Osmotic gradient:

  • Increases from cortex to medulla in interstitial space

  • Drives passive water diffusion in descending limb & collecting duct

How does the body use ADH to respond to dehydration?

• Trigger:

  • Low blood pressure

  • High plasma osmolarity

• Sensors:

  • Pressure receptors in vasculature

  • Osmoreceptors in hypothalamus

• Signal Pathway:

  • Hypothalamus signals posterior pituitary to release ADH

• ADH Action:

  • Increases water reabsorption in collecting duct

  • Promotes water reabsorption from urine back into bloodstream

  • Helps restore blood pressure

  • Decreases plasma osmolarity

• Mechanism:

  • Part of negative feedback loop to counter dehydration

What roles do ADH receptors and Aquaporin 2 channels play in the collecting duct?

• ADH receptors (on basolateral membrane) bind ADH hormone

• Trigger insertion of Aquaporin 2 channels on luminal membrane

• Aquaporin 2 channels increase water permeability of principal cells

• This allows water reabsorption from urine into the body

What is the function and selectivity of aquaporins in cell membranes?

• Aquaporins are protein channels that allow rapid water transport

• They are exclusively permeable to water (no ions or solutes)

• Water moves through aquaporins by osmosis, following concentration gradients

How does ADH increase water permeability in the kidney's collecting duct?

• ADH activates its receptor → activates Protein Kinase A (PKA)

• PKA phosphorylates AQP-2 channels

• Phosphorylation increases AQP-2 vesicle fusion to the luminal membrane and reduces endocytosis

• Result: More AQP-2 channels are inserted and remain on luminal surface → increased water permeability

• AQP-3 & AQP-4 are on basolateral membrane; AQP-2 constantly cycles to luminal membrane

How does ADH regulate water reabsorption in the collecting duct via principal cells?

• Trigger: Dehydration → low BP & high plasma osmolarity

• ADH release: From posterior pituitary → enters circulation

• Target: Principal cells in collecting duct (only cells w/ ADH receptors)

• ADH binds: To GPCR on basolateral membrane

• Signal cascade:

  • Activates adenylate cyclase → ↑ cAMP

  • Activates PKA

  • Leads to phosphorylation of proteins

• Effect:

  • AQP2-containing vesicles fuse to luminal membrane

  • Water enters via AQP2, exits to interstitium via AQP3/4 (always present)

• Regulation:

  • AQP2 insertion is ADH-dependent → prevents constant water reabsorption

• Outcome:

  • Water reabsorbed into blood

  • ↑ Blood pressure, ↓ plasma osmolarity

  • Negative feedback loop controls water homeostasis

How do ADH receptors (GPCRs) regulate water reabsorption in the kidney collecting duct?

• ADH binds to GPCR on basolateral (non-lumen) side → activates Gαs subunit

• Gαs activates adenylyl cyclase → converts ATP to cAMP

• cAMP activates Protein Kinase A (PKA)

• PKA promotes exocytic insertion of AQP-2 channels to the luminal membrane

• AQP-2 channels increase water reabsorption from collecting duct into blood

Describe the detailed signaling pathway activated by ADH in principal cells of the collecting duct.

• ADH binds to GPCR (7 transmembrane receptor) on basolateral membrane of principal cells

• GPCR functions as GEF (guanine nucleotide exchange factor)

  • Activates Gαs subunit → GDP swapped for GTP

• Activated Gαs → stimulates adenylyl cyclase

  • Converts ATP → cyclic AMP (cAMP)

• cAMP activates PKA

  • Binds to regulatory subunits → releases catalytic subunits

• PKA phosphorylates proteins involved in vesicle trafficking

  • Promotes exocytic insertion of AQP2-containing vesicles into luminal membrane

  • AQP2 allows water to enter cell from nephron

• AQP3 & AQP4 are always present on basolateral membrane → water exits into interstitial fluid

• Inactivation of the pathway:

  • Phosphodiesterases (PDEs) degrade cAMP → reduces PKA activity

  • Without PKA, AQP2 insertion ceases

• Outcome: Regulated water reabsorption based on ADH levels

How is GPCR activity regulated after persistent stimulation?

• Persistent agonist binding → GPCR phosphorylated by GPCR kinases (GRK)

• Arrestins bind phosphorylated GPCRs

• Arrestins recruit clathrin and adaptor protein AP2

• GPCRs internalized into endosomes

• GPCRs either degraded in lysosomes or dephosphorylated and recycled to the membrane

How is ADH GPCR signaling regulated at the basolateral membrane of principal cells?

• GPCRs are not permanently present on the membrane; they undergo dynamic regulation

• Agonist binding (e.g., ADH) → activates G-protein signaling

• Persistent stimulation → receptor desensitization

  • Mediated by GPCR kinases (GRKs) → phosphorylation of GPCR

  • Phosphorylated GPCR binds to arrestin

• Arrestin recruits clathrin & AP-2 adaptor protein
  → Leads to endocytosis of GPCR into an endosome

• Endosome fate:

  • Lysosomal degradation (receptor destruction)

  • Dephosphorylation & recycling back to basolateral membrane

• Net effect:

  • Controls sensitivity of cells to ADH

  • Regulates intensity and duration of water reabsorption response

What causes Congenital Nephrogenic Diabetes Insipidus (XNDI) and what are its main symptoms?

• Caused by mutations in the ADH receptor (GPCR) — X-linked recessive inheritance

• Mainly affects males; females are carriers

• Symptoms: excessive thirst, large volume of hypotonic/dilute urine (>3L/day, <250 mmol/kg)

• Leads to dehydration, fatigue, seizures due to electrolyte imbalance, enlarged bladder

• Nephrons are insensitive to ADH → impaired water reabsorption

What are the four classes of GPCR defects and their main characteristics?

• Class 1: Loss of transcription

• Class 2: mRNA instability, improper translation, retention in the ER

• Class 3: Retention in the Golgi

• Class 4: Altered ligand binding, altered signal transduction, altered internalization

What causes nephrogenic diabetes insipidus (XNDI) at the molecular level?

• Inactivating mutations (mostly Class II) in the ADH receptor gene

  • 221 currently known ADH receptor gene mutations cause XNDI

• Misfolded ADH receptors trapped in the endoplasmic reticulum

• Loss of ADH signaling → no AQP2 expression or insertion into the luminal membrane

• Results in impaired water reabsorption in the collecting duct

What is the mechanism of nephrogenic diabetes insipidus related to ADH receptor mutations?

• Mutations cause absence of ADH receptors in the basolateral membrane

• Leads to lack of aquaporin-2 (AQP2) channels in the luminal membrane

• Result: impaired water reabsorption in the collecting duct causing excessive urine output, irritation and damage to urinary bladder

How do Class II ADH receptor mutants behave in cells compared to wild-type receptors?

• Wild-type ADH receptors localize to the plasma membrane.

• Class II mutant ADH receptors (e.g., L62P, Y205C) are retained inside the secretory pathway, trapped in the ER and Golgi.

• This retention prevents receptor trafficking to the membrane, impairing function.

What are pharmacological chaperones and how can they help with Class II ADH receptor defects?

• Synthetic small molecules that assist in proper protein folding

• Bind target proteins via non-covalent interactions (van der Waals, hydrogen bonds)

• Stabilize native protein structure and prevent aggregation

• Allow misfolded ADH receptors to fold correctly and traffic properly in the cell

How do pharmacological chaperones help correct Class II ADH receptor folding defects?

1. Misfolded proteins are normally retained in ER and degraded

2. Pharmacochaperones enter the cell and bind misfolded proteins

3. They assist correct folding of proteins

4. Properly folded proteins are routed to the Golgi

5. Mature proteins reach the cell surface

6. Pharmacochaperones dissociate

7. Receptors can now bind their ligand and function

How can pharmacological chaperones like SR121463A help mutant ADH receptors in HEK (human embryonic kidney) cells?

Rescue receptors by:

• Promote proper folding of mutant ADH receptors

• Increase receptor localization to the cell membrane

• Restore responsiveness to ADH

What effects were observed in adult males with X-linked nephrogenic diabetes insipidus after treatment with pharmacological chaperones?

• Decreased urine volume

• Increased urine osmolarity

• Stable blood plasma osmolarity

• Decreased water intake (less signals for thirst)

Most promising treatment to date.