MODULE 5

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Questions and Answers

Describe the process of desensitization of the β-adrenergic receptor.

Desensitization of the β-adrenergic receptor involves phosphorylation of the receptor by GPCR kinases, leading to binding of arrestin proteins and internalization of the receptor.

How does the GTPase activity of G proteins regulate signal transduction?

The GTPase activity of G proteins hydrolyzes GTP to GDP, which inactivates the G protein and terminates the signal transduction.

What is the role of the stimulatory G protein (Gs) in GPCR signaling?

The stimulatory G protein (Gs) activates adenylyl cyclase to produce cAMP from ATP, amplifying the signaling cascade.

How does the inhibitory G protein (Gi) affect GPCR signaling?

The inhibitory G protein (Gi) inhibits adenylyl cyclase activity, reducing cAMP levels and signaling through the GPCR. Signup and view all the answers

What happens to the Gα subunit during the G-protein activation cycle?

Binding of a GPCR-ligand complex causes the Gα subunit to exchange GDP for GTP, leading to activation of the G protein. Signup and view all the answers

How is cAMP hydrolyzed to 5′ AMP?

By cyclic nucleotide phosphodiesterase Signup and view all the answers

What activates adenylyl cyclase?

Activated Stimulatory G alpha subunit (active Gs-alpha) Signup and view all the answers

What is the function of β-arrestin in desensitization of the β-adrenergic receptor?

Blocks receptor sites that interact with the G protein Signup and view all the answers

What is the role of adaptor proteins in cell signaling?

To hold together protein molecules that function in concert so that signals can be targeted to a specific region of the cell Signup and view all the answers

How does GTPase activator proteins (GAPs) affect G proteins? (GAPS: GTP -----> GDP)

Increase the GTPase activity of G proteins by about 10^5-fold Signup and view all the answers

What happens to the G protein Ras when GTP is hydrolyzed?

Turns to its inactive conformation Signup and view all the answers

How does Cholera Toxin block Gs activity?

By blocking the GTPase activity of Gsα leaving it active Signup and view all the answers

What is the consequence of activating mutations in Gα proteins?

Lead to a continuously elevated [cAMP] Signup and view all the answers

How does Gq signaling pathway lead to the production of second messengers?

Activates phospholipase C to cleave PIP2 Signup and view all the answers

Explain how cAMP activates cAMP-dependent protein kinase (PKA).

cAMP binds regulatory region of PKA and activates cAMP-dependent protein kinase (PKA). Signup and view all the answers

What is the role of the autoinhibitory domain in the R2C2 complex of PKA?

The autoinhibitory domain of each R subunit occupies the substrate-binding cleft of each C subunit, making the complex catalytically inactive. Signup and view all the answers

How does FRET help measure PKA activity?

FRET measures the nonradiative transfer of energy between fluorescent probes attached to proteins. When PKA is active, FRET is observed. Signup and view all the answers

What causes the termination of the β-adrenergic response?

<ul> <li>a drop in epinephrine concentration below the Kd for its receptor - Gsα intrinsic GTPase activity. - cAMP hydrolysis</li> </ul> Signup and view all the answers

Describe the role of 14-3-3 in PKA activity.

When PKA is active, 14-3-3 binds the phosphorylated Ser residue. Signup and view all the answers

Describe the three general mechanisms by which signaling molecules are released.

<ol> <li>Exocytosis (e.g. insulin stored in secretory vesicles) 2. Cleavage (e.g. epidermal growth factor) 3. ABC transporter (e.g. prostaglandins)</li> </ol> Signup and view all the answers

What is the main route for signaling molecule release in eukaryotes?

Exocytosis Signup and view all the answers

In multicellular animals, what is the largest group of plasma membrane receptors?

G-Protein Coupled Receptors (GPCRs) Signup and view all the answers

What is the function of G-Protein Coupled Receptors (GPCRs) in signaling?

To initiate signaling cascades in response to extracellular stimuli Signup and view all the answers

What are the 3 components that make up a GPCR?

receptor with 7 transmembrane helices, heterotrimeric G protein, enzyme in the plasma membrane that generates a 2nd messenger Signup and view all the answers

What are the second messengers generated by the effecter enzyme associated with GPCRs?

cAMP, cGMP, inositol 1,4,5-trisphosphate (PIP3) Signup and view all the answers

What is the role of EL2 in GPCR architecture?

EL2 (the gate) controls access to the ligand binding site Signup and view all the answers

Describe the structural characteristics of GPCR TM6 upon ligand binding.

TM6 moves 14 Å and bends at a conserved Pro kink Signup and view all the answers

What is the significance of the ligand binding site in GPCR architecture?

The ligand binding site is located in the outer leaflet portion and is diverse between families Signup and view all the answers

How do large conformational changes convey signals in GPCRs?

Large conformational changes occur in the inner leaflet portion, which forms a signaling module Signup and view all the answers

What connects the ELs

Disulfide bonds Signup and view all the answers

Sometimes, there's an additional helix; where is it found, and what is its property?

Along the intracellular membrane surface and is amphipathic and lipidated. Signup and view all the answers

What do adrenergic receptors bind to, and how many types are there?

Epinephrine and 4 types (2alpha, 2beta) Signup and view all the answers

What Do beta-blocker drugs target?

they target the GPCRs of β-adrenergic receptors Signup and view all the answers

Explain the mechanism by which sulfonylurea drugs stimulate insulin release in pancreatic β cells.

Sulfonylurea drugs bind to the SUR1 subunits of ATP-gated K+ channels, closing the channels and stimulating insulin release. Signup and view all the answers

Describe the role of tyrosine phosphatases in the regulation of RTK

Tyrosine phosphatases dephosphorylates pTyr and other targets, turning off RTKs. Signup and view all the answers

Explain how cholera toxin affects G protein signaling.

Cholera toxin ADP-ribosylates the Gsa , inactivating its gtpase activity, leading to continuous stimulation of adenylate cyclase. Signup and view all the answers

What is the sequence of events that leads to insulin release by pancreatic β cells in response to changes in blood glucose?

Glucose entry via GLUT2 increases ATP production, closing K+ channels, depolarizing the membrane, opening Ca2+ channels, triggering insulin release. Signup and view all the answers

How does the insulin receptor serve as a prototype for receptor tyrosine kinases?

The insulin receptor, like other RTKs, possesses cytoplasmic Tyr kinase domains and variable extracellular domains specific to different growth factors. Signup and view all the answers

What is the role of the enzyme phosphoinositide 3kinase (PI3K) in insulin signaling?

Activate PI3K catalyses the phosphorylation of PIP2 to PIP3 Signup and view all the answers

How does autophosphorylation contribute to the activation of tyrosine kinase receptors?

Each β subunit phosphorylates three essential Tyr residues near the C-terminus of the other β subunit. Signup and view all the answers

What specific domain binds phosphorylated tyrosine residues in a protein partner during insulin signaling?

SH2 domain Signup and view all the answers

How does the movement of GLUT4 to the plasma membrane get initiated in the insulin signaling pathway?

Activation of PKB starts a cascade of events initiating the movement of GLUT4 to the plasma membrane. Signup and view all the answers

What is the function of insulin receptor substrate 1 (IRS1) in insulin signaling?

Becomes the point of nucleation for a complex of proteins that carry the message from the receptor to end targets. Signup and view all the answers

Describe the mechanism of action of Cholera Toxin.

Cholera Toxin catalyzes transfer of ADP ribose from NAD+ to Gsα, blocking the GTPase activity of Gsα and leading to high intracellular cAMP levels. Signup and view all the answers

Explain the process of activation of Tyrosine Kinase in the Insulin Receptor.

One molecule of insulin binds between the two subunits of the Insulin Receptor, leading to autophosphorylation of the Tyrosine Kinase domains on the intracellular β subunits. Signup and view all the answers

Describe the activation of the IP3-Gated Ca2+ Channel.

IP3 binds to the IP3-Gated Ca2+ Channel, causing it to open and release sequestered Ca2+ into the cytosol. Signup and view all the answers

How does Gq signaling pathway lead to the production of second messengers?

Gq activates Phospholipase C, which cleaves PIP2 into DAG and IP3, serving as potent second messengers. Signup and view all the answers

Study Notes

Desensitization of β-adrenergic Receptor

The β-adrenergic receptor undergoes desensitization following prolonged stimulation, reducing responsiveness to ligands.
β-arrestin binds to phosphorylated receptors, blocking further G protein coupling, promoting receptor internalization.

GTPase Activity of G Proteins

GTPase activity hydrolyzes GTP to GDP, terminating signaling and resetting the G protein for future activation.
Regulates signal transduction by controlling the duration and intensity of the signal.

Role of Stimulatory G Protein (Gs)

Gs activates adenylyl cyclase, increasing intracellular cAMP levels, which modulate various signaling pathways.

Effect of Inhibitory G Protein (Gi)

Gi inhibits adenylyl cyclase activity, decreasing cAMP levels and countering effects of stimulatory pathways.

Gα Subunit during Activation Cycle

Upon activation, Gα subunit exchanges GDP for GTP, dissociates from β and γ subunits, and interacts with downstream effectors.

Hydrolysis of cAMP to 5′ AMP

cAMP is hydrolyzed by phosphodiesterase enzymes, converting it to 5′ AMP, which inactivates signaling pathways initiated by cAMP.

Activation of Adenylyl Cyclase

Stimulated by Gs protein binding to its receptor, enhancing the enzyme's chemistry that converts ATP to cAMP.

Function of β-arrestin

β-arrestin serves as a scaffold for signaling and mediates receptor internalization, playing a critical role in receptor desensitization.

Role of Adaptor Proteins

Adaptor proteins facilitate interactions between signaling molecules, organizing complexes for efficient signal transduction.

GTPase Activator Proteins (GAPs)

GAPs accelerate GTP hydrolysis on G proteins, promoting the return to the inactive GDP-bound state.

Behavior of Ras Protein Post-GTP Hydrolysis

When GTP on Ras is hydrolyzed to GDP, Ras becomes inactive, leading to termination of downstream signaling pathways.

Cholera Toxin's Effect on Gs Activity

Cholera toxin modifies Gs proteins, preventing GTP hydrolysis, resulting in prolonged activation and excessive cAMP production.

Consequences of Mutations in Gα Proteins

Activating mutations lead to constitutive signaling, contributing to cancer proliferation and other pathologies.

Gq Signaling Pathway and Second Messengers

Gq activates phospholipase C, generating inositol trisphosphate (IP3) and diacylglycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC).

Activation of cAMP-dependent Protein Kinase (PKA)

cAMP binds to the regulatory subunits of PKA, causing dissociation from the catalytic subunits, which then activate target proteins.

Role of Autoinhibitory Domain in PKA Complex

The autoinhibitory domain maintains PKA's inactive state, preventing unwarranted phosphorylation prior to cAMP binding.

FRET in Measuring PKA Activity

Förster resonance energy transfer (FRET) visualizes PKA activation in live cells by detecting conformational changes in PKA's proximity to energy donor and acceptor molecules.

Termination of β-adrenergic Response

Termination occurs via receptor phosphorylation, β-arrestin binding, and eventual receptor internalization, reducing available receptors for ligand binding.

Role of 14-3-3 in PKA Activity

14-3-3 proteins bind phosphorylated serine residues on PKA, aiding in maintaining the active conformation required for signaling.

Mechanisms of Signaling Molecule Release

Release can occur via exocytosis, diffusion through membranes, or active transport mechanisms.

Main Route for Signaling Molecule Release in Eukaryotes

Exocytosis is the predominant mechanism for secreting signaling molecules from cells.

Largest Group of Plasma Membrane Receptors in Multicellular Animals

G-Protein Coupled Receptors (GPCRs) represent the largest group, involved in numerous signaling pathways.

Function of GPCRs in Signaling

GPCRs transduce extracellular signals through conformational changes, activating intracellular G proteins.

Components of GPCRs

Comprised of an extracellular ligand-binding domain, seven transmembrane alpha helices, and intracellular G protein coupling regions.

Second Messengers from GPCR Effecter Enzymes

cAMP, inositol trisphosphate (IP3), and diacylglycerol (DAG) are generated by GPCR-associated enzymes.

Role of Extracellular Loop 2 (EL2) in GPCR Architecture

EL2 connects various structural elements and influences ligand binding specificity and receptor activation.

Structural Characteristics of GPCR TM6 upon Ligand Binding

TM6 undergoes conformational changes that facilitate G protein interaction and activation, crucial for signal transduction.

Significance of Ligand Binding Site in GPCRs

The ligand binding site utilizes specificity and affinity, determining receptor activation and downstream effects.

Conveying Signals through Conformational Changes in GPCRs

Ligand binding induces large scale changes in GPCR conformation, relaying the signal to intracellular partners.

Connection of Extracellular Loops (ELs)

ELs can interact with each other or with ligands, influencing receptor conformation and function.

Additional Helix in GPCRs

Sometimes present in the C-terminal region, it stabilizes the receptor structure or serves as a regulatory element.

Adrenergic Receptors and their Types

Adrenergic receptors primarily bind catecholamines (epinephrine and norepinephrine), categorized into α and β types.

Target of Beta-blocker Drugs

Beta-blockers specifically target β-adrenergic receptors, inhibiting norepinephrine action to reduce blood pressure and heart rate.

Mechanism of Sulfonylurea in Insulin Release

Sulfonylurea drugs close ATP-sensitive potassium channels in pancreatic β cells, leading to depolarization and calcium influx, stimulating insulin secretion.

Role of Tyrosine Phosphatases in RTK Regulation

Tyrosine phosphatases dephosphorylate activated receptors, acting as a counterbalance to tyrosine kinases and regulating signaling intensity.

Cholera Toxin Impact on G Protein Signaling

By preventing GTP hydrolysis, cholera toxin maintains G proteins in their active GTP-bound state, leading to excessive cAMP levels and overstimulation.

Sequence of Events for Insulin Release in Pancreatic β Cells

Increased blood glucose → ATP rises → K+ channels close → Membrane depolarization → Ca2+ influx → Insulin granules release.

Insulin Receptor as Prototype for Receptor Tyrosine Kinases

Activates via autophosphorylation upon ligand binding, initiating signaling cascades related to glucose uptake and metabolism.

Role of Phosphoinositide 3-kinase (PI3K) in Insulin Signaling

PI3K is recruited by phosphorylated IRS proteins, generating lipid products that further propagate insulin signaling.

Contribution of Autophosphorylation in Tyrosine Kinase Activation

Autophosphorylation amplifies the receptor's signaling capacity, creating docking sites for downstream signaling molecules.

Binding Domain for Phosphorylated Tyrosine in Insulin Signaling

The SH2 domain specifically binds to phosphorylated tyrosines, facilitating interactions with signaling partners.

Initiation of GLUT4 Movement in Insulin Signaling Pathway

Insulin binding stimulates signaling cascades culminating in GLUT4 translocation to the plasma membrane, enhancing glucose uptake.

Function of Insulin Receptor Substrate 1 (IRS1)

Acts as a primary signaling mediator, linking insulin receptor activation to downstream signaling pathways related to metabolism.

Mechanism of Action for Cholera Toxin

Cholera toxin catalyzes ADP-ribosylation of Gs protein, disrupting GTPase activity and locking the protein in its active state.

Activation of IP3-Gated Ca2+ Channel

Increases in IP3 following Gq activation stimulate Ca2+ release from the endoplasmic reticulum, elevating intracellular calcium levels.

Gq Signaling Pathway and Second Messenger Production

Gq, upon activation, stimulates phospholipase C to produce IP3 and DAG, which coordinate to elevate intracellular calcium and activate PKC pathways.

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Description

Learn about the role of cyclic AMP (cAMP) as a second messenger for various regulatory molecules and the functions of stimulatory G protein (Gs) and inhibitory G protein (Gi). Discover how cAMP is hydrolyzed to 5′ AMP and its significance in cellular signaling pathways.