Signal transduction II

What is the structure of PKA?

2 regulatory subunits and 2 catalytic subunits 4 cAMP (total) bind to the regulatory subunits (2 each)

Ga family: II

Gi: inhibits adenylyl cyclase

Ga family: III

Gq: activates phospholipase C-B

_____ and _____ are antagonistic pathways that both have opposing effects on adenylyl cyclase.

Gs & Gi

Ga family: I

Gs: activates adenylyl cyclase; activates Ca2+ channels Golf: activates adenylyl cyclase in olfactory sensory neurons

Regulation of Rho family GTPase signaling

1. GEF activates Rho-->Rho-GTP 2. activated Rho binds to effector and induces down-stream signaling 3. GAP inactivates Rho-->Rho-GDP 4. a GDI (rho guanine nucleotide dissociation inhibitor) binds Rho-GDP and sequesters it in the cytosol, regulating Rho protein targeting to the plasma membrane

What are the signals that are associated with G12/13

1. Lysophosphatidic acid 2. Thrombin 3. Thromboxane

Explain the process of Rho inducing contraction in smooth muscle, clot retraction, and endothelial permeability.

1. Signal binds GPCR, which acts as a GEF to activate a13 2. a13-GTP activates RGS (Which is bound in the PM) 3. RGS then recruits DH-PH, the GEF of Rho, from the cytoplasm to the PM 4. Rho's GEF activates RhoA, which activates its effectors (ROCK--involved in actin turnover; mDia involved in actin polymerization) 5. Rho-GTP then phosphorylates MLC (+) and MLCP (-)

What are the stimulatory ligands for adenylyl cyclase activation and what are the inhibitory oens?

1. epinephrine, Glucagon, ATCH 2. PGE, adenosine

What is the process of a ligand binding to GPCR that leads to rise in intracellular cAMP?

1. ligand binds to receptor 2. Gs-GTP binds to adenylyl cyclase & activates it, allowing ATP to be converted to cAMP 3. Signal is amplified 4. Gsa uses intrinsic GTPase activity to hydrolyze GTP-->Gs-GDP(inactivated), which dissociates from adenylyl cyclase, inactivating it

What are the cytoplasmic targets of PKA?

1. metabolic pathways 2. calcium regulation 3. cytoskeletal pathways/contractility

What are the consequences of adrenaline acting on the beta receptors (mediated by cAMP) in the fight or flight response?

1. triglyceride breakdown in adipose cells 2. increase in heart rate and force of contraction of heart 3. Glycogen breakdown in muscle and liver cells (first cAMP regulated process to be discovered)

What is the MOA of cholera toxin?

Activates Gs to drive cAMP production Catalyzes the ADP-ribosylation of Gs, which prevents the GTP from being hydrolyzed

How is the fight or flight response mediated? What accounts for the different types of responses seen throughout the body?

By adrenaline acting on G-protein coupled receptors The alpha and beta adrenergic receptors are coupled to different heterotrimeric G proteins, which elicit different responses

RhoA lowers the amount of __________________ to induce contraction

Ca2+

What is the mechanism that allows for endothelial permeability using RhoA?

Contraction in the endothelial cells produces paracellular pores between them, which increases micro vessel permeability

cAMP is synthesized from ATP through a ____________ reaction.

Cyclization

PKA targets can be __________ or __________.

Cytoplasmic Nuclear

How is the GCPR signal turned off?

Down regulation; GRK phosphorylates GPCR-->BARR binds to P-P-GPCR and recruits AP-2 and clathrin--> Dynamin & endocytosis--> receptor can be recycled or destroyed

In GCPR signaling, what is the first messenger and what is the second messenger?

First messenger is the hormone itself, second messenger is cAMP

Half of all drugs work through

G-protein coupled receptors

Ga subfamily: IV

G12/13: activates Rho family monomeric GTPases (via Rho-GEF) to regulate the actin cytoskeleton

What is the GEF for alpha subunit and what is the GAP?

GEF is the GCPR once a ligand binds to it GAP is the RGS (regulators of G protein signaling)

What is the largest family of cell-surface receptors?

GPCRs

What is the MOA of pertussis toxin?

Inhibits Gi, which promotes cAMP production. It does this by catalyzing the ADP-ribosylation of Gi, preventing the G protein from interacting with receptors

How does Rho regulate the contraction of actin?

It phosphorylates MLC, inducing contraction while also inhibiting (phosphorylating) MLCP, which promotes contraction.

What is the only means of degrading cAMP?

Phosphodiesterase (PDEs)

What is the nuclear target of PKA? What's the process? What does this regulate?

Phosphorylation of CREB 1. PKA catalytic subunits move into the nucleus and phosphorylate CREB (activating it) 2. P-CREB binds to DNA & recruits histone acetyl transferases, and then transcription is initiated This regulates fuel metabolism, spermatogenesis, memory, and steroidogenesis

What is signaling in the G(12/13) family involved in?

Rho activation (small GTPase), which causes changes in the actin distribution/contractibility in cells

Structure of GCPR

The receptors is a single polypeptide with 7 transmembrane segments G proteins is a heterotrimeric protein Effector is adenylyl cyclase, Phospholipases C & A2, & cGMP phosphodiesterase

What are A kinase anchoring proteins (AKAPs)?

They target PKA to specific sites within the cell They will have PKA bound and a PDE bound, which high levels of cAMP will cause catalytic units of PKA to dissociate from the AKAP/PKA unit and phosphorylate the PDE, which will in turn inactivate PKA

What are the non-selective inhibitors of PDEs?

Xanthines (caffeine & theophylline)

Which subunit of the G protein is activated by GTP binding and has intrinsic GTPase activity?

alpha subunit

What is the main way that cAMP exerts its effects?

by activating cAMP dependent protein kinase (PKA), which is a serine/threonine kinase