Signal Transduction IV

Question 1

What is the general pathway for the adenylate cyclase signaling pathway?

Answer 1

Stimulatory
1. Gsalpha binds to AC, which increases the conversion of ATP to cAMP.
2. Increased cAMP concentraion activates Protein Kinase A (PKA) to phosphorylate various proteins to transduce the signal
3. Signal Reduction: Phosphodiesterases and phosphatases

Inhibitory
1. Gialpha binding to AC to inhibit AC

Examples!

Gsalpha: Beta-adrenergic receptors
Gialpha: alpha2-adrenergic receptor
Ligands: epinephrine (adrenaline)/norephinephrine (noradrenaline)

Drug Targeting:
alpha 2 adrenergic receptor- antihypertension
Beta adrenergic receptors- beta blockers, heart conditions

Question 2

How does Gq trigger Ca2+ signaling? Why is this important?

Answer 2

Maintaining low cytoplasmic [Ca2+]
* Huge difference in free calcium levels inside (10^-7 M) and outside (10^-3 M) the cell: ratio= 10^4.
* Small increase (to 10^-6 M) rapidly activates certain cellular functions.
* Ca2+ is released from the ER as a result of Galphaq activation.
* Active transport maintains low levels of free calcium in the cytosol
* NCX–> Sodium-calcium exchanger
* CA2+: ATPase
* SERCA: Sarco/endoplasmic reticulum Ca2+ ATPase

Calmodulin
* Calcodulin binds Ca2+ at two terminal domains (EF hands). Binding induces conformational changes in CaM that expose hydrophobic surfaces.

This alternative CaM conformation binds many cellular signaling proteins:
* e.g. Calmodulin-dependent kinases (CaM kinases)
* Alters CaM kinase activity, including authophosphorylation activity

Question 3

Describe the process of phospholipid derived signaling.

Answer 3

• Receptors GPCRs and RTKs lead to activation of phospholipases and phospholipid kinases that hydrolyze or phosphorylate various classes of phospholipids (like phosphatidylinositol)
• One example is phospholipase C is a membrane associated enzume that hydrolyzes PIP2. (ex. a step in Ca2+ mediated signaling)
• RTK example= Phosphatidylnositol 3 Kinase (PI3K)

Question 4

Why are signaling pathways integrated rather than exclusive or independent?

Answer 4

The signaling pathways are interconnected because the GPCRs and RTKs work together to produce gene regulatory proteins and cytosolic or membrane proteins. Although they are different processes, both pathways work together to regulate how much of each thing is needed in the cell and maintain equilibrium/favorable conditions.

Question 5

What is the general mechanism for phosphoinositide signaling?

Answer 5

1. Ligand binding mediates effector activation through GPCR and trimeric G protein as before
2. Generates two second messengers, diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3)
3. IP3 gates calcium release through Ca2+ channel from the ER
4. Calcium binding to cytosolic proteins alters their functions, e.g calcmodulin and protein kinase C (PKC).
5. Ca2+ binding promotes PKC binding membranes where another activator, DAG, is located.

Gq- muscarinic acetylcholine receptors, Angiotensin II receptors

Question 6

What are some examples of phospholipases?

Answer 6

• Phospholipase C (PLC-Beta, PLC-gamma, PLC-delta)
• Phospholipase D
• Phospholipase A2

Note: When you cleave different positions of phospholipid; different substrate specificity.

Question 7

How do phospholipases generate 2nd messengers?

Answer 7

When a signaling molecule binds to a receptor like GPCR, it activates intracellular signaling pathways. The activated receptor stimulates phospholipases, particularly phospholipases C or phospholipase A2.

PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which are two key messengers. IP3 stimulates the release of calcium ions in the ER. DAG activates protein kinase C, which phosphorylates various target proteins to propagate the signal.

PLCBeta is activated by interactions with trimeric G proteins, such as GPCR. PLCgamma is acitvated by tyrosine phosphorylation (RTKs)

PLD hydrolyzes phosphatidylcholine to phasphatidic acid and choline. PA is enzymatically converted to diacylglyercal (DAG).

Question 8

What are the important components of phosphatidylinositol phosphorylation?

Answer 8

P13-kinase: Phosphorylation of 3’-OH
* PIP2–> PIP3

PIP3
* 2nd messenger of numerous signaling pathways
* Recognized by PH domains
* PH domains: recruit enzymes to the plasma membrane and stimulate its activity.
* Key pathways: Akt signaling promotes cell survual by repressing cell-death signaling pathways; also regulates glucose transport and glycogen metabolism.

Phospholipid kinases: activated by RTKs–> signaling happens when binding is there

Question 9

What can protein kinase C capable of doing in phospholipid derived signaling?

Answer 9

PKC phosphorylates transcription factors, ion channels, other kinases gor growth, apoptosis, and differentiation