L10-11: Second Messengers & Hormonal Regulation of Metabolism

Question 1

What is the substrate for NO synthase? What affect does NO have on the vasculature? What enzyme is affected by NO? How is NO signaling turned off?

Answer 1

• NO produced from arginine by the enzyme nitric oxide synthase. It is a gas and diffuses readily in cell, acting locally due to short half-life and conversion into nitrates, nitrites when reacted with o2 and water. - Typical function of NO is to cause relaxation of smooth muscle. - Typical mechanims: ACh is released from nerve terminal, causes activation of NO synthase in endothelial cells, triggering conversion of arginine into NO. NO diffuses to neighbouring smooth muscle cells, binds guanylate cyclase in the cytoplasm, stimulating production of cGMP, which activates cGMP-dependent protein kinase, leading to smooth muscle relaxation. - Inactivation: NO has a short half-life as it is highly reactive with oxygen and water, produced nitrates and nitrites. In addition, cGMP is degraded by phosphodiesterasterases yielding GMP, which are not able to activate cGMP-dependent protein kinases that lead to smooth muscle relaxation.

Question 2

What is the significance of the esterification of DAG at carbon 2?

Answer 2

• This is often arachidonic acid, with are precusors of eicosanoids, which are lipid-derived signaling molecules such as prostaglandins and leukotrienes.

Question 2

What is cholera? What is it caused by? How does cholera toxin work? What are the important symptoms of cholera?

Answer 2

• Cholera is bacterial disease that causes massive diarrhea, can lead to death as the result of dehydration. - It is caused by Vibrio cholerae that produces the cholera toxin. - Cholera toxin catalyzes ADP-ribosylation of alpha-subunit Gs proteins at an arginine residue that causes the subunit to be in the GTP-bound active state. As a result, signaling by this molecule cannot be turned off, cAMP levels remain high, PKA remains active. CFTR is phosphorylated. Secretion of Cl, Na and water into the intestinal lumen, ie. massive diarrhea.

Question 3

Define hormone. Distinguish between endocrine, paracrine and autocrine signaling.

Answer 3

• Hormones are chemical substances secreted into the blood by specialized cells that have regulatory effects on specific organs or tissue - - a.) endocrine signaling: when hormone has regulatory effect on site distant from site of hormone secretion - b.) paracrine signaling: when hormone has regulatory effect on site adjacent/surrounding the site of hormone synthesis and release - c.) autocrine signaling: when hormone is released from a cell and has regulatory effect on that same cell

Question 4

Why is the formation of cAMP and irreversible reaction?

Answer 4

• Generation of cAMP from ATP via adenylate cyclase produces pyrophosphate (PPi). PPi is hydrolyzed into 2xPi via pyrophosphatase, which drives the formation of cAMP.

Question 4

In broad terms, how does the insulin receptor function?

Answer 4

• The insulin receptor is a catalytic receptor. Upon binding insulin, the receptor autophosphorylates at the tyrosine residue. This causes docking by other molecules (incl. IRS1), which become phosphorylated by the receptor. These phosphorylated docked molecules serve as binding sites for other molecules (build signaling complexes) that mediate the action of insulin inside the cell.

Question 4

What is pertussis? What is it caused by? How does pertussis toxin work?

Answer 4

• Pertussis is a respiratory bacterial infection. - It is caused by Bordetella pertussis, which produces the pertussis toxin. - Pertussis toxin catalyzes ADP-ribosylation of alpha-subunit Gi protein as a specific cysteine residue, which lowers the affinity of the G-protein for GTP. As a result, the G-protein is trapped in the inactive conformation and cannot inhibit adenylate cyclase resulting in increased cAMP levels. Unclear the exact mechanism that leads to respiratory issues; however.

Question 5

What are G-proteins? Distinguish between Gs, Gi and Gq. Explain how G-proteins function.

Answer 5

• G-proteins are heterotrimeric proteins (with alpha, beta and gamma subunits) that are associated to the plasma membrane. When bound to GDP, they are in their inactive form. When bound to GTP, they are in their active form. They are GTPases and can self hydrolyze GTP to GDP and inactivate themselves. - There are many different types of G-proteins, including: a.) Gs, b.) Gi and c.) Gq. a.) Gs typically stimulate the activity of downstream enzymes b.) Gi typically inhibit the activity of downstream enzymes c.) Gq typically stimulate the activity of downstream enzymes - When receptors become activated, GDP-bound G-proteins bind them. This binding triggers and exchange of GDP for GTP, which activates the G-protein and causes dissociation of the alpha subunit from the beta-gamma dimer. Activated G-protein (alpha with GTP bound) associates with effector enzymes and alters their activity (inhibition or activation, depending on what type of G-protein it is). Intrinsic GTPase activity of the alpha subunit hydrolyses GTP to GDP and the G-protein is deactivated, re-associates with the beta-gamma dimer, able to participate in further signal transduction.

Question 5

What type of hormone is insulin?

Answer 5

• peptide

Question 7

Differentiate between hydrophilic and hydrophobic hormones. Where are their receptors mainly located? What are their principal modes of action?

Answer 7

• Hydrophilic hormones are water soluble hormones that cannot cross the plasma membrane of a cell. They bind to plasma membrane receptors at the surface of target cells. Binding results in two things: generation of intracellular second messenger or activation of receptor enzymatic activity for the purposes of causing rapid changes to activity of metabolic enzymes. - Hydrophobic (lipophilic) hormones are lipid soluble hormones that are mainly steroid derived (ie. from cholesterol) and are able to pass through the plasma membrane and bind receptors in the nucleus or cytoplasm of target cells. Binding results in altered transcription for the purposes of altering enzyme synthesis (slower).

Question 7

What type of hormone is vasopressin/AVP/ADH?

Answer 7

• peptide

Question 8

How is adenylate cyclase activated? Deactivated? Inactivated?

Answer 8

• Adenylate cyclase is activated by Gs proteins. Deactivated when Gs proteins are inactivated by their GTPase or when Gi proteins bind adenylate cyclase.

Question 9

Explain the different types of adrenergic receptors.

Answer 9

• 1.) Alpha receptors a.) alpha-1 subtype: Gq protein coupled = activation of PLC b.) alpha-2 subtype: Gi protein coupled = inhibition of adenylate cyclase - 2.) Beta receptors three subtypes (beta-1,2,3) are Gs protein coupled = activation of adenylate cyclase

Question 11

What are examples of hormones that function through the cAMP signal transduction pathway? Function of hormone?

Answer 11

• Epinephrine (function: glycogenolysis in muscle, fat hydrolysis in adipose, increased HR and FOC) - Glucagon (function: glycogenolysis in liver, fat hydrolysis in adipose) - AVP/ADH/vasopressin (function: water re-absorption in kidney)

Question 12

What type of hormone is glucagon?

Answer 12

• peptide

Question 14

What reaction does cholera toxin catalyze? What reaction does pertussis toxin catalyze? What g-protein subtypes are implicated? What is the result?

Answer 14

• Both toxins catalyze ADP-ribosylation, which is the attachment of ADP-ribose to specific residues. - Cholera toxin catalyzes this reaction in Gs alpha subunit proteins, causing subunit to remain in active state, leading to high adenylate cyclase and cAMP levels. - Pertussis toxin catalyzes this reaction in Gi alpha subunit proteins, causing subunit to remain in inactive state, leading to high adenylate cyclase and cAMP levels.

Question 16

Describe how the IP3-DAG signal transduction system works. Include all the necessary players from activation to deactivation.

Answer 16

• Phospholipase C is activated by G-proteins (upon hormone/receptor binding) and hydrolyzes PIP2 (phosphatidylinositol 4,5 bisphosphate), a membrane lipid, generating two second messengers: IP3 (inositol 1,4,5 triphosphate) and DAG (diacylglycerol). - IP3 is water soluble and diffuses into cytosol where it binds to receptors on ER, triggering release of Ca2+ down its concentration gradient and into the cytosol. Ca2+ activates cytosolic protein kinase C (PKC) who have downstream effects. - DAG is liphophilic and remains membrane-associated, but easilty diffuses in the plane of the membrane. With Ca2+, it activates plasma membrane associated PKC. - Ca2+ released by the mechanism above can also bind calmodulin, causing it to interact with other protein kinases, phosphatases and ion channels. - Inactivation: 1.) receptor down-regulation/internalization/degration; 2.) protein phosphatases reverse action of PKC; 3.) Ca2+ pumped out of cytosol (into ER or to ECF); 4.) IP3 is dephosphorylated by phosphatase converting it into derivative that isn’t active; 5.) DAG is phosphorylated yielding phosphatidic acid or hydrolyzed to glycerol and FAs.

Question 16

What type of protein is YopH? What is it made by? What does it do?

Answer 16

• YopH is a protein tyrosine phosphatase. - It is encoded by a plasmid in Yersinia pestis, the bacterium responsible for the bubonic plague. - It dephosphorylates certain key proteins and prevents host immune system from mounting an effective defense against the bacterium.

Question 18

What are catalytic receptors and why do we say that they are catalytic? Name some catalytic activities of catalytic receptors? What is the most common catalytic activity of receptors?

Answer 18

• Catalytic receptors refer to receptors that have intrinsic enzyme activity. They have ligand-binding domains and intracellular domains with enzymatic activity. Most are protein tyrosine kinases. - They are catalytic in that they can transfer gamma-phosphate from ATP to their own tyrosine residues (autophosphorylation) and/or to other proteins.

Question 19

What are examples of hormones that function through IP3-DAG signal transduction pathway? Function of hormone?

Answer 19

• PDGF (function: cell growth and proliferation) - Oxytocin (function: vascular smooth muscle contraction, lactation, parturition) - Epinephrine (function: glycogenolysis)

Question 20

Explain how nitroglycerin can reduce blood pressure.

Answer 20

• Nitroglycerin is converted to NO in the body. When binding to guanylate cyclase in smooth muscle lining blood vessels, it causes them to relax and volume within the vasculature to expand. This causes a reduction in blood pressure.

Question 21

Describe how the cAMP signal transduction system works. Include all the necessary players from activation to deactivation.

Answer 21

• cAMP, or cyclic AMP, is a secondary messenger. It is generated through the enzyme adenylate cyclase by converting ATP. - Gs proteins activate adenylate cyclase and cause rapid rises in cAMP levels. - cAMP acts as a secondary messenger by targeting protein kinase A (PKA, also known as cAMP-dependent protein kinase). It transfers gamma-phosphate groups from ATP to the hydroxyl groups of serine / threonine residues on the regulatory subunits, which cause conformational change and dissociation from the catalytic subunits, which then perform their specific downstream functions. - Deactivation of cAMP signaling: 1.) cAMP is hydrolyzed by cAMP phosphodiesterase to AMP and is no longer capable of activating PKA. 2.) intrinsic GTPase activity of G-protein returns protein to inactive states and adenylate cyclase is no longer activated. 3.) Hormone-receptor are no longer in active state (either internalized/degraded or inactivated by phosphorylation. 4.) Downstream enzymes phosphorylated by PKA are dephosphorylated by protein phosphatases and therefore are deactivated. - Gi proteins typically inhibit adenylate cyclase and cause rapid decreases in cAMP levels.

Question 22

What are the typical results of downstream signaling as a result of binding hydrophilic vs hydrophobic hormones?

Answer 22

• Hydrophilic hormones generally cause rapid action changes to activity of metabolic enzymes - Hydrophobic (lipophilic) hormones generally cause alterations to enzyme synthesis (by altering transcription), which is a slower process. - Hydrophilic result in more rapid changes, while lipophilic result in slower changes.

Question 23

Explain the process of signal amplification

Answer 23

• Signal amplification refers to the process which contributes to rapid cellular responses as the result of hormone/ligand binding. If one ligand/hormone molecule binds, it can cause production of 20 second messengers, which can bind 20 downstream kinases, which can each trigger another 20 etc. etc. The result of one ligand/hormone binding, could result in the activation of 1000s of enzymes.

Question 24

What type of hormone is epinephrine?

Answer 24

• catecholamine

Question 25

Explain how Viagra works.

Answer 25

• Viagra is a phosphodiesterase inhibitor and prevents deactivation of cGMP, allowing their levels to remain elevated for longer. cGMP is what binds to cGMP-dependent protein kinases that cause smooth muscle to relaxed. As a result, increase in blood flow to the penis is prolonged, treating erectile dysfunction.

Question 27

What is meant by the term second messenger? Give examples of second messengers.

Answer 27

• These are low mol. Weight, typically diffusible molecules that relay a signal within a cell. - Examples: cAMP, cGMP, Ca2+, IP3, DAG, NO

Question 28

What signal transduction mechanism is most commonly utilized by the receptor for glucagon?

Answer 28

• cAMP signaling pathway, which leads to glycogenolysis.

Question 29

Distinguish between Type 1 and Type 2 diabetes mellitus

Answer 29

• Type 1 diabetes is an autoimmune disease that leads to the absence of insulin as the result of the destruction of beta-cells. - Type 2 diabetes is a disease where tissues become less sensitive to insulin as the result of defects in signal transduction between the insulin receptor and downstream components. Possible link to obesity and transient/extended periods of hyperglycemia.

Question 30

Through what two cell-signaling mechanisms does epinephrine function? What are the receptor types it binds to? Where are these receptors found? What G proteins are implicated in each?

Answer 30

• Epinephrine can signal through cAMP and DAG-IP3 pathways. - It binds beta-adrenergic receptors in skeletal muscle through the cAMP (and adenylate cyclase via Gs proteins) signaling pathway to trigger glycogenolysis. - It binds alpha-1-adrenergic receptors in the liver through the DAG-IP3 (and PLC via Gq proteins) signaling pathway to trigger glycogenolysis.

Question 31

What are some examples of hydrophilic hormones?

Answer 31

Insulin, epinephrine, oxytocin

Question 32

What are some examples of hydrophobic/lipophilic hormones?

Answer 32

Retinoic acid, glucocorticoids (cortisol), thyroid hormones

Question 33

What signaling cascades to Gs, Gi and Gq affect?

Answer 33

• Gs – stimulation of cAMP via adenylate cyclase
• Gi – inhibition of cAMP via adenylate cyclase
• Gq – stimulation of IP3/DAG via PLC