7T: Second Messengers; Signaling Networks

Question 1

Adenylate Cyclase function

Answer 1

To produce CAMP by hydrolyzing ATP to produce PPi. and then cyclizes the remaining portion of the ATP to create cAMP. This is a slow reaction without the activator GalphaS

Question 2

regulation of adenylate cyclase

Answer 2

GalphaI: inhibitory. So inhibits production of cAMP
GalphaS: stimulatory. So stimulates the production of cAMP
some Beta/Gamma units can also regulate AC
Ca2+ can also regulate AC

Question 3

Targets of cAMP

Answer 3

PKA: protein kinase A. Phosphorylates substrates. Is activated by cAMP. Composed of 4 subunits: 2 catalytic and 2 regulatory. When cAMP binds the two regulatory subunits of PKA, this causes a conformational change and a release of the two catalytic subunits.
EPAC:

Question 4

Major targets of active PKA

Answer 4

• can phosphorylate the GCPR, causing heterologous desensitization
• can phosphorylate metabolic enzymes
• can phosphorylate CREB, a transcription factor, that upregulates transcription of cAMP response elements
• can phosphorylate CFTR, a downstream target of cholera toxin and involved in cystic fibrosis

Question 5

How does Cholera toxin effect AC?

Answer 5

Cholera toxin ADPribosylates GalphaS subunits that inhibits their ability to hydrolyze GTP. So, since GalphaS stimulates cAMP production people with cholera cant hydrolyze GTP and they produce a lot of cAMP. Normally cAMP activates PKA, which activates CFTR, which allows Cl- to move out of the cell. Na+ follow along with water. Since this process can’t turn off, a lot of Na+, Cl-, and water flow out and cause the diarrhea.

Question 6

activating mutations of the LH receptor

Answer 6

• activating mutations of the LH receptor will lead to ligand-independent GalphaS activation.
• this leads to premature testosterone production
• precocious puberty (ex. spermatogenesis by age 3)

Question 7

inhibiting mutations of the LH receptor

Answer 7

• the mutation doesn’t allow GalphaS activation

- antagonizes puberty and leads to pseudohermaphroditism

Question 8

DAG, IP3, and Ca2+

Answer 8

• PLC (phospholipase C) cleaves PIP2 into DAG and IP3
• DAG stays in the membrane b/c it is hydrophillic
• IP3 is soluble so it is released from the membrane
• PLC activity is increased by GalphaQ subunits
• DAG partially activates PKC
• IP3 then binds to into receptors on the ER which releases Ca2+ into the cytoplasm
• The Ca2+ then fully activates PKC the rest of the way

Question 9

What does Ca2+ regulate?

Answer 9

• PKC activation
• AC activation or inhibition depending on the type of AC
• PLA2 regulation
• Calmodulin (soaks up the Ca2+), which can regulate kinases and phosphatases
• ryanodine receptors on the sarcomere in muscle contraction
• NOS (which leads to increased cGMP by GC, which increases vasodilation)

Question 10

How are the second messengers cAMP, DAG, IP3, and NO regulated?

Answer 10

cAMP: PDE (phosphodiesterase) cleaves the cAMP to AMP. This causes the regulatory unit of protein kinase A to re-associate with the catalytic subunit and inactivate it.
- same thing for cGMP
DAG and IP3: There are enzymes that will degrade DAG and IP3. As these levels drop, IP3 receptors close, DAG degrades and PKC becomes partially inactivated (Ca2+ still bound). Ca2+ is decreased by Ca2+ binding molecules, or actively transported by SERCA back into the sarcoplasmic reticulum, which leads to complete deactivation of PKC.
NO: Cells have detoxifying enzymes to convert NO into an inert form. Can also be regulated by degradation of the iNOS mRNA, which leads to less NO.

Question 11

iNOS vs. eNOS

Answer 11

iNOS: is an isoform of NOS that is expressed in immune cells such as macrophages. iNOS is not regulated by Ca2+ levels
eNOS: is expressed in endothelial cells. Produced NO, which activates Guanylate Cyclase, which produces cGMP, which promotes vasodilation. Regulated by Ca2+

Question 12

Cushing’s syndrome

Answer 12

• caused by excess cortisol production
• corticotropin effects cAMP levels, which then activates PKA, which will then produce cortisol
  2 forms:
  a. corticotropin-dependent: aberrant corticotropin secretion
  b. corticotropin-independent: mutation in catalytic subunit of protein-kinase A, that can’t bind back to the regulatory subunit and is always “on”.

Question 13

function of nitroglycerin

Answer 13

Bypasses eNOS function. Cells convert nitroglycerin to NO, which activates GC leading to increased cGMP, leading to vasodilation

Question 14

What is signal “crosstalk”

Answer 14

means that signaling is not isolated or independent from each other. This leads to multiple signals utilizing shared components generating specific and divergent outcomes

Question 15

MOA of Viagra

Answer 15

• Viagra mimics cGMP
• competitively interferes with cGMP binding to PDE, so this leads to excess cGMP, which means prolonged vasodilation and smooth muscle relaxation

Question 16

MOA of nifedipine and verapamil

Answer 16

• competitively inhibits the Ca2+ channels in the sarcoplasmic reticulum to release into the cell
• so Ca2+ is not released, causing muscle relaxation (and arterial dilation)