Cell Biology Chapter 23 : Metabotropic Receptor Signal (Exam 3)

Name a couple of variants that have seven transmembrane domains and all interact with heterotrimeric G-proteins, that binds to different ligands.

1. Acetylcholine

2. Epinephrine

3. Norepinephrine

4. Dopamine

5. Serotonin

6. Parathyroid hormone

7. Angiotensin II

When activation a G-protein, the activated receptors which are GEFS has what kind of mechanism:

G-alpha subunit releases GDP then binds to GTP. The Gα subunit and Gβγ complex separate and interact with effectors

Explain the steps of when the alpha subunit of the heterotrimetric G-protein is bounded to GDP.

1. The receptor is not bounded to the ligand so the Gα subunit is bound to GDP and associated with the Gβγ complex.

2. When the ligand binds to the receptor, the receptor will then bind to a G protein. Gα will release GDP and acquires GTP.

3. Gα and Gβγ subunits separate.

4. G protein subunits activate or inhibit target proteins, initiating signal transduction events.

Different receptors may bind to different ________________

G-Proteins

G_as:

G_ai:

G_aq:

G_{as: activates adenylyl cyclase}

G_{ai: inhibits adenylyl cyclase}

G_{ai: activates phospholipase -}Cβ which breaks down phospholipids

Some Gβγ can regulate ____ ___________

ion channels

Adenylyl cyclase produces ________. This membrane bound enzyme converts ATP to _______.

cAMP

cAMP activates Protein kinase A. This is a highly ___________ ___________ ______________

conserved signaling pathway

Explain the process when cAMP activates protein kinase A.

A serine/threonine kinase that phosphorylates different proteins in different cell types to produce a variety of tissue-specific physiological outcomes.

Explain the mechanism when cAMP activates protein kinase A.

1. Protein kinase A is composed of two catalytic and two regulatory subunits. The regulatory subunits inhibit the catalytic subunits in the absence of cAMP.

2. Cyclic AMP activates protein kinase A by binding to the regulatory subunits, causing the regulatory subunits to induce a conformational change.

3. The catalytic subunits will then detach. They are now activated and can phosphorylate target proteins in the cell.

Glycogen degradation targets the ____ and ______. The hormone involved in this is epinephrine.

Muscle and liver

Fatty acid production targets ______ tissue. The hormone is epinephrine.

Adipose

Parathyroid hormone deals with what organ?

Bone

Natural ligands include epinephrine and norepinephrine. They have these things called adrenergic receptor. What’s the receptor for each?

Epinephrine: adrenaline

Norepinephrine: noradrenaline

Receptor subclasses include:

α and β

β1 is the subclass for what kind of tissue:

Cardiac muscle, skeletal muscle, and liver which activates the breakdown of glycogen

β2 is the subclass for what kind of tissue:

Airway epithelial smooth muscle

α1 is the subclass for what kind of tissue:

Vascular smooth muscle

(this controls the constricts of smooth tissue of arteries and blood pressure

What’s an affect on the skeletal muscle and liver?

Epinephrine or norepinephrine binds β1 receptors.

• both activate the canonical G(as) pathway to produce cAMP and increase PKA activity

• This is the same effect when glucagon binds glucagon receptors on liver

When does glucagon binds to glucagon receptors on the liver?

Glucagon binds glucagon receptors when the blood/sugar level gets too low

Explain the glycogen breakdown pathway.

1. cAMP binds to and activates protein kinase A

2. Protein kinase A phosphorylates phosphorylase kinase activating it

3. Active phosphorylase kinase phosphorylates phosphorylase b converting it to phosphorylase a, the active form of the enzyme.

4. Phosphorylase a catalyzes cleavage of a terminal glucose from glycogen as glucose-1-phosphate.

END GOAL: release glucose to the body

Signal amplification gives a cascade which implies that with each step we get a magnification. Explain the significance.

• Binding of epinephrine to a G-protein coupled receptor → 1 molecule

• Inactive G-protein to an Active G-protein → 10² molecules

• Inactive adenylyl cyclase to an active adenylyl cyclase → 10² molecules

Heart rate increase deals with what characteristics:

PKA phosphorylates [Ca 2+] channels → which increases Ca 2+ influx

G(as) directly interacts with [Ca 2+] channels → increases Ca 2+ influx

How does cAMP regulate transcription?

1. cAMP stimulates protein kinase A

2. Then this process heads over to a binding protein called CREB (cAMP -response element binding protein)

3. CREB is attached to CRE (cAMP response element) that’s a part of the promoter region of a gene (DNA)

4. It then gets phosphorylated with the CREB a being attached to an inorganic phosphate and a CBP protein known as the CREB binding protein which acts like the cofactor for CREB. CREB is attached to the CRE portion of the promoter region and transcription is then activated towards the end of the region of the gene

Explain the steps of receptor inactivation.

1. Ligand dissociates from receptor

2. Receptor then becomes phosphorylated by PKA or by a G-protein coupled receptor kinase

3. Sites that are phosphorylated by the G-protein couple receptor kinase become bounce by β-arrestin which is a beta blocker that blocks the G-protein from binding to the receptor

What does G-protein inactivation concludes?

1. GTP hydrolysis by the Gα subunit leads to the inactivation followed by the reassembly of the heterotrimeric complex.

2. Some Gα subtypes are more efficient GTPases than others.

3. Some will need a GAP to increase their GTPase activity to cause inactivation

4. Regulators of G-protein signaling (RGS proteins)

Explain the mechanism of G-protein inactivation

1. G-protein subunits activates or inhibits target proteins, initiating signal transduction events.

2. The Gα subunit hydrolysis it’s bound GTP to GDP, becoming inactive

3. Subunits recombine to form an inactive G-protein

cAMP hydrolysis includes cAMP-dependent phosphodiesterase which:

Breaks down cAMP in the cytosol (c-3 carbon)

What’s protein dephosphorylation?

This process removes phosphate groups. Protein phosphatases are the most constitutively active

Glycogen phosphorylase b can get activated by _______ ________ with the hydrolysis of ATP to become glycogen phosphorylase a.

Phosphorylase kinase

Glycogen phosphorylase a can get inactivated by _______ ________ into glycogen phosphorylase b.

Phosphorylase phosphatase

What’s the cholera toxin and the G(as) pathway?

Cholera toxin lives in marine environment, bacteria.

It’s an enterotoxin(attacks the digestive system) produced by virulent strains of Vibrio Cholerae

Can be ingested by eating uncleaned raw shellfish

This possess ADP ribosylase activity

In the Cholera toxin, what is ADP ribosylase activity?

It’s a normal enzymatic process that modifies a protein post-translationally. It starts cleaving nicotinamide from NAD then linking it to the rest of the molecules to Arginine residues in target proteins

Cholera toxin ADP ribosylates G(as) which interferes with its ______ _________. This prevents what?

GTPase activity

This prevent GTP hydrolysis so G(as) remains active

G(as) stimulation of adenylyl cyclase is prolonged when the Cholera toxin is ingested. What does this do?

This increases cAMP levels which becomes very high.

During when cholera is ingested, PKA activity becomes very high and begins to phosphorylate a ___ channel causing it to be more permeable to ___ ions.

Cl-

During the process by which cholera is ingested, once cells loose Cl-, what else happens?

Cells lose Cl-, followed by Na+, K+, H2O, HCO3-

( when Na+ goes, water also follows )

A few G protein couple receptors as known as (GPCRS) activate Gαi. Explain the process.

These are α2 adrenergic receptors

1. Gαi inhibits Adenylyl cyclase

2. Gai then is competing with G(as) for regulation of adenylyl cyclase

3. The level of adenylyl cyclase activity is depend on the relative signaling from receptors that activate G(as) such as β adrenergic receptors) vs the receptors that activate Gai such as the α2 adrenergic receptors.

Pertusis toxin and its Gai pathways includes what characteristics?

A toxin that can produce a respiratory infection leading to persistent coughing.

1. This Pertusis toxin ADP ribosylates Gai preventing it from binding adenylyl cyclase.

2. The loss of this inhibitory effect on adenylyl cyclase causes it to be more active

3. The direct connection between increase cAMP levels and the disease symptoms like coughing isn’t well understood

Some GPCRs activate Gαq which:

• α1 adrenergic receptors and muscarinic acetylcholine receptors

• G(aq) activates Phospholipase C-β(PLC-β)

PLC-β cleaves phosphatidylinositol 4,5 bisphosphate to produce:

• Inositol Trisphospate (IP3)

• Diacylglycerol (DAG)

Phosphatidylinositol 4,5 bisphosphate is:

A membrane lipid which cleaves the entire phosphate group on the C-1 leaving glycerol

IP3 which is known as the second messenger binds to a receptor on the _______ ______ which is a [Ca2+] channel.

Cystolic Ca2+ levels rise rapidly

Endoplasmic reticulum

Resting [Ca2+]= 1 × 10^-7 M (100nM)

Extracellular [Ca2+] = 1 × 10^-3 M (1mM)

Stimulated [Ca2+] = 1 × 10^-5 M (10uM)

When calcium binds to calmodulin:

The activates calmodulin binds and activates other proteins like phosphatases and kinases.

Phosphatases: PP2B (calcineurin) which is activated by calcium and firstly discovered in neurons

Kinases: calcium/ calmodulin kinase (CaM) kinase

Ca++ ATPases in the ER and the plasma membrane. When calcium is released from the ER→ Cytosol will have a high concentration so we need to decrease the [Ca2+]. How is this gonna happen?

Na+ is then pumped in, Ca2+ is pumped out. It’s a symport mechanism.

DAG and Ca2+ activate protein kinase __

C

When DAG and Ca2+ activate Protein Kinase C, what characteristics can be concluded?

• A ubiquitously expressed Ser/Thr kinase

• Many different isoforms

• Many cellular effects

What are some examples of cell functions that are regulated by inositol trisphosphate diacyglyercol?

Platelet activation

Muscle contraction

Insulin secretion

α1 receptor signaling is:

• present on vascular smooth muscle cells which surround blood vessels by binding by epinephrine or norepinephrine mobilizes Ca2+

• Ca2+ binds calmodulin and together they activate myosin light chain kinase

• Myosin light chain kinase phosphorylates myosin light chains which activates the motor proteins leading to contraction of the smooth muscle cells

• Contraction causes vasoconstriction

• Vasoconstriction is a major contributor to blood pressure

A baseline level signaling through this pathway maintains a steady - state level of blood pressure is the:

Vasomotor tone

The muscarinic receptor signaling includes:

1. Muscarinic receptors are present on vascular endothelial cells by binding epinephrine or norepinephrine mobilizes Ca 2+

2. Ca 2+ binds calmodulin and together they activate nitric oxide syntheses which produces the soluble intercellular signaling molecules nitric oxide (NO)

3. NO diffuses into adjacent smooth muscle cells where it binds and activates guanylyl cyclase

4. Guanylyl cyclase converts GTP into cGMP

5. cGMP activates protein kinase G

6. Protein kinase G phosphorylates multiple proteins resulting in vascular smooth muscle relaxation which causes vasodilation leading to decreased blood pressure