Cell Signaling II

Question 1

Recognize the structure of the minor membrane phospholipid called phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]

Answer 1

• In order to understand the Gq pathway, we need to look at the phosphotidylinositols (also called phospotidic acid) in the plasma membrane which are in the inner layer or cytosolic side.
• PI kinase will phosphorylate on the position 4 hydroxyl to get the PI 4-phosphate (PI(4)P)
• This can then be phosphorylated again by PIP kinase at position 5 to get PI 4,5-bisphosphate [PI(4,5)P2]
• It turns out that this is subject to hydrolysis by an enzyme to create signal transduction molecules (PIP2 is)

Question 2

Describe the activation of phospholipase C-Beta by a Gq-coupled cell-surface receptor

Answer 2

A signal molecule will bind to the Gq-coupled protein receptor. This will then activate the GqPCR which can then associate with the G-protein and activate it by allowing it to lose the GDP and bind GTP. Then, the activated G-protein will activate the phospholipase C-beta. It is important to note that it is the same “initial steps” because it is still a G-protein!!! But, the specific GPCR is different and activates a different enzyme, not PKA!

Question 3

Indicate which bond in PI(4,5)P2 is hydrolyzed by phospholipase C-beta and name the resulting product

Answer 3

The phosphate on position 1 is cleaved to leave behind the DAG (diacylglycerol) and IP3 (inositol 1, 4, 5-triphosphate)

Question 4

Discuss the role of diacylglycerol in signal transduction

Answer 4

In the Gq pathway, it generates 2 signal molecules, the IP3 and the DAG. The DAG, in this case, can go on to phosphorylate the Protein Kinase C (PKC). The PKC will then go on and phosphorylate specific Ser/Thr on proteins. The PKC is another signal transduction pathway that doesn’t involve the IP3

Question 5

Compare the concentration of Ca2+ in the cytosol to that in the endoplasmic reticulum and outside the cell, and explain how this difference is maintained

Answer 5

The [Calcium] is very low in the cytosol in comparison to the ER and the outside of the cell. The concentration in the cytosol is kept very low (10^-7M) by mechanisms that move Ca2+ out of the cell or into the endoplasmic reticulum or mitochondria. This allows for rapid changes in cytosolic [Ca2+] by the opening Ca2+-channels in the plasma membrane or ER. There are Na+ -driven Ca2+ exchangers in which is an antiporter that pumps Na+ into the cytosol and the Ca2+ out. There is also a Ca2+-pump that uses ATP. There are also calcium binding molecules in the cytosol that will bind the calcium and finally there are H+ -driven Ca2+ exchangers that are also antiporters

Question 6

Describe the role of inositol triphosphate (IP3) in signal transduction

Answer 6

IP3 or inositol triphosphate is critical in the signal transduction pathway which stimulates the release of Ca2+ in the cytosol from the ER. The IP3 will go and bind to Ca2+ release channels and open them, allowing Ca2+ to go with its electrochemical gradient. This will increase the cytosolic concentration of Ca2+. Overall you can get a 10 to 20-fold increase in Ca2+ in the cytosol.

• Another thing that happens is that when the Ca is released, it can cause opening of plasma membrane calcium channels that allow calcium to come from the outside. This particular process can sustain the elevated calcium and also allows the calcium to replenish the ER stores of Ca2+
• Once the signal is finished (hormone or IP3 is degraded), the cytosolic Ca2+ will then be pumped back into the ER or out of the cell to turn off the calcium signal
• The increase in cytosolic calcium can have spatial and temporal affects
• When a starfish egg and a sperm comes in and brings with it a phospholipase C that produces a IP3 to release Ca2+.
• We are looking at a dye inside that binds to Ca2+ in the cytosol and makes it fluorese.
• Can see that where the sperm entered is a spike of Ca that then moves in like a wave-like fashion that moves through the egg.
• This is due to calcium-induced calcium release which means the release of calcium in one place will increase the calcium a bit further down and so this moves the wave across the surface. This actually changes the surface too so that another sperm cannot enter
• It is spatial in the sense that it starts at one place and temporal in because it takes time

Question 7

Using vasopressin-induced Ca2+ oscillations in a liver cell as an example, describe the effect of the hormone concentration on oscillatory frequency

Answer 7

• Vasopressin is a hormone that increases cytosolic calcium.
• Here it is acting on the liver cells.
• Looking at time in minutes and the calcium concentration determined by the dye.
• The calcium goes up and then it goes back down. You get these oscillations
• If you add a higher concentration of vasopressin you might expect the amplitude to go up but it doesn’t! The frequency changes!
• You get these spikes closer together (higher frequency) with more vasopressin
• The cell actually knows what to do with these changes in frequency

Overall, the more hormone present, the higher the frequency of the oscillations, not the amplitude!

Question 8

Describe the structure of Calmodulin, including the number and location of Ca2+ binding sites

Answer 8

• Calcium can do a lot of things and many of these effects are carried out by a protein called Calmodulin
• This is the secondary structure of calmodulin
• It sort of looks like a dumbbell
• There are 4 calcium binding sites, 2 in the N-terminal region and 2 in the C-terminal region and the affinity is higher in the C-terminal region
• At least 2-4 calcium bind to allow the calmodulin to bind to certain domains in other protein
• This binding of calcium calmodulin can modify this enzyme activity to turn it on or off depending on the protein
• This is a highly conserved protein  The amino acid sequence of human calmodulin is 90% identical to spinach so you cant mess around with the sequence much and have it still work right

Question 9

Discuss how the binding of Ca2+ to calmodulin affects its structure and function

Answer 9

The binding of at least 2 Ca2+ ions allow for a conformational change in the calmodulin that allows in to bind to many enzymes and membrane transport proteins and alter their activity. The binding of the calmodulin allows the protein that it is bind to to have a conformational change as well which can either activate it or inhibit it. CaM-Kinase II for example will be activated via Ca2+-calmodulin.

Question 10

Indicate how Ca2+ / calmodulin activates CaM-Kinase II

Answer 10

• This enzyme CaM-kinase II (Calcium Calmodulin dependent protein kinase II)
• We are just looking at one of the 12 subunits of the enzyme
• We are looking at the inactive state and there is a catalytic domain that actually does the phosphorylation
• There is also a C-terminal inhibitory domain that fits into the active site and prevents any substrate from getting in there
• This catalytic domain is inactive because of the inhibitory domain
• When there is a calcium signal, the calcium binds to the calmodulin, then the calcium calmodulin binds to the inhibitory domain and gets it out of the way so that the catalytic subunit is active.
• One of the first things the enzyme does is it phosphorylates itself! It undergoes auto-phosphorylation via ATP
• Now, the enzyme is fully active and can go around and phosphorylate other proteins.
• One thing the phosphate does is it increases the affinity for the calmodulin  It makes it harder for the calmodulin to get off
• But eventually the calcium spike will go down and calcium will get lower in the cytosol. Then the calcium will dissociate from the calmodulin, then the calmodulin will come off. But we are left with a subunit with the phosphate still on and as long as the phosphate is still on, it remains 50-80% active. So a protein phosphatase comes in and dephosphorylates the enzyme to deactivate it.
• This mechanism allows this enzyme to decode that calcium frequency that we saw

CaM-Kinase II phosphorylates other proteins on their Ser/Thr residues as well!!!

Question 11

Describe how autophosphorylation maintains CaM-Kinase II activity even when the cellular Ca2+ levels have decreased

Answer 11

The autophosphorylation allows the enzyme to remain 50-80% active because the phosphate group is still present, even when the calmodulin has dissociated from the protein. Therefore, the catalytic site can remain active and unblocked while the protein is still phosphorylated. When it encounters a phosphatase, however, the protein becomes dephosphorylated and this the inhibitory domain blocks the catalytic domain once again and renders the CaM-Kinase II inactive again.

Question 12

Indicate how CaM-Kinase II activity is turned off

Answer 12

When the calcium level decreases, the calmodulin loses its calcium and it dissociates from the CaM-Kinase II. Thus, the CaM-kinase II is left only phosphorylated. Now a protein kinase can come in and dephosphorylate the CaM-kinase II and render it inactive again

Question 13

Discuss how the frequency of Ca2+ oscillations can affect CaM-kinase II activity

Answer 13

If the oscillation frequencies are relatively low, then the CaM-kinase II will most likely be dephosphorylated by a phosphatase and no longer be able to phosphorylate the Ser/Thr residues of other proteins. However, with higher frequency of Oscillations, then the CaM-kinase II has a greater chance of remaining active because more calmodulin can bind to the CaM-kinase II if it is active and keep the CaM-kinase II active and continue to phosphorylate. You are giving the phosphatase less time to dephosphorylate and inactive the CaM-kinase II!!!

• The CaM-kinase II can decode the Calcium oscillations
• When there is a low frequency Ca oscillation, there is an increase in activity and then it goes down but not all the way down and then very slowly inactivates. It slowly inactivates because the phosphate is still on the CaM-kinase II keeping it 50-80% active. Can then get another spike and this continues to occur.
• If you have higher frequency of oscillations what happens is that you get another calcium spike before the last cycle is completed! So the phosphate wasn’t removed and then you get reactivation of it fully and activation of more enzyme. This enzyme allows the cell to respond in different ways to low and high Ca frequencies.
• It gives the cell a memory! The enzyme can remain a bit active and give it a memory.

Question 14

Discuss how activation of the inositol phospholipid pathway can have different effects in different cell types

Answer 14

The effect that a signaling molecule has on a cell depends in part on which set of proteins are available for phosphorylation. The response of a cell to a signaling molecule that activates the inositol phospholipid pathway depends in large part on which proteins are available for activation by Ca2+-calmodulin, as well as the proteins available for phosphorylation by Ca2+/calmodulin-dependent protein kinases and protein kinase C.

Question 15

Identify the major signal transduction mechanisms for the different adrenergic receptor subtypes

Answer 15

1) Alpha-1A: Gq
2) Alpha-1B: Gq
3) Alpha-1C: Gq
4) Alpha-2A: Gi, Go
5) Alpha-2B: Gi, Go
6) Alpha-2C: Gi, Go
7) Beta-1: Gs
8) Beta-2: Gs
9) Beta-3: Gs, Gi, Go

Question 16

Adrenergic Receptors

Answer 16

• Adrenergic receptors bind to epinephrine and norepinephrine
• They have 9 different G-protein coupled receptors and these multiple receptor proteins couple to different G-proteins and have different tissue locations and functions
• There are 9 different receptors for these hormones

Question 17

Discuss how the phenomenon of multiple receptors can be commandeered to create drugs that selectively mimic or block a fraction of the hormone’s effects

Answer 17

• This has been very difficult for biochemists to figure out what all of these receptors do but this turns out to be very good for medicine
• It is possible to activate or block a specific receptor without affecting the other receptors or the hormone which would in turn affect the other receptors and pathways –> this is good for drug development
• Drug companies have been very active in making specific ligands
• These represent a 7 transmembrane receptor
• Epinephrine would bind to all of these receptors and activate things so epinephrine isnt used much as a drug.
• Agonist: A drug that activates the receptor. It is something that binds to the receptor and activates it
• In this case, a drug would only bind to the alpha-1A receptor but not the others!
• Antagonist: A drug that binds to the receptor and prevents epinephrine from binding to it and thus not allowing it to be activated
• The epinephrine would perform its normal functions at the other receptors but not the one where there is a drug to inhibit it for
• These agonists and antagonists are an all or nothing thing –> They activate or they block. So there is a lot of work being done now to develop an allosteric drug that binds some place else and can change the conformation in a way to partially activate or inhibit it.

Question 18

Agonist

Answer 18

A drug that activates the receptor. It is something that binds to the receptor and activates it

Question 19

Antagonist

Answer 19

A drug that binds to the receptor and prevents epinephrine from binding to it and thus not allowing it to be activated

Question 20

Describe how bradykinin can stimulate the formation of NO in endothelial cells, and how NO can then cause relaxation of vascular smooth muscle cells and a decrease in blood pressure

Answer 20

• There is a hormone called bradykinin that can relax vascular smooth muscle and it has a 7 transmembrane receptor on the endothelial cell surface, it binds to it and this particular receptor is coupled to the Gq protein.
• When the Gq is activated, you have the alpha subunit with the GTP bound that goes and activates phosphlipase C-beta.
• That will cleave the PIP2 in the membrane and release IP3. The IP3 will go to the ER and bind to the ion channels and allow calcium to come out into the cytosol.
• Calcium in the cytosol also tends to open ion channels on the plasma membrane so there is calcium coming it too so you get an increase in calcium
• The calcium binds to the calmodulin and then the calcium-calmodulin complex binds to the endothelial nitric oxide synthase (NOS).
• The NOS then takes arginine and makes NO
• The NO is a gas so it can pass through membranes passively and then through the smooth muscle membrane
• Inside the smooth muscle there is a sGC protein or soluble guanylyl cyclase which will convert the GTP to a cGMP when the NO binds to its heme group and activates it.
• cGMP then activates the protein kinase G (PKG) which can then phosphorylate a set of proteins that leads to relaxation of the smooth muscle. This causes the vessel to dialate.
• There is a drug that mimics part of this pathway

Question 21

Nitric Oxide (NO)

Answer 21

-Now we are looking at Nitric oxide (NO) as a signaling molecule –> differs from nitrous oxide! (N2O)
-Nitric oxide is NO
-Nitric oxide is actually produced in the body
-Nitric oxide synthase (NOS) makes the NO in the body –> synthase does NOT require ATP
-This enzyme NOS uses arginine as a substrate and it also requires NADPH and molecular oxygen
-It is a 2 step process:
First the enzyme puts a hydroxyl group on the nitrogen
Then uses NADPH again and another molecular oxygen to replace the N-OH with just an =O, releasing a nitric oxide and you get citrulline left over

• Nitric oxide activates an enzyme called soluble guanylyl cyclase, binding to a heme group in this enzyme and when the NO binds to the heme it turns this enzyme on, increasing its activity and now it creates cGMP.
• cGMP then binds to protein kinase G and increases its activity
• PKG is a ser/thr kinase that can go phosphorylate other proteins

Question 22

Describe how nitrovasodialators work, and indicate whether or not endothelial cells are important in their mechanism of action

Answer 22

• There is a drug that mimics part of this pathway –> nitrovasodilators
• sodium nitroprusside, nitroglycerin, isosorbude dinitrate –> These are all NO donors!!!
• These drugs can either spontaneously or by the action of enzymes release NO from their structure
• The NO then undergoes the same pathway as previously but it skips the steps in the endothelium!!
• Bradykinin is an endothelium dependent signaling molecule because its receptor is on the endothelium and this is where it acts first!
• In the case of these drugs they are independent of the endothelium –> They are endothelium independent vasodilators
• These drugs are used to treat people with heart failure
• Large veins can dilate and store a lot more blood so when the heart goes to contract there is a lower pre-load volume and this makes it easier for the heart to pump because it is not pumping against a huge volume of blood.

Question 23

Describe how erectile dysfunction drugs like viagra work

Answer 23

These drugs are NO donors and allow it to skip the steps in the endothelium and have the NO go directly to the smooth muscle cells and cause vasodilation by increasing cGMP

• There is a drug that mimics part of this pathway  nitrovasodilators
• sodium nitroprusside, nitroglycerin, isosorbude dinitrate  These are all NO donors!!!
• These drugs can either spontaneously or by the action of enzymes release NO from their structure
• The NO then undergoes the same pathway as previously but it skips the steps in the endothelium!!
• Bradykinin is an endothelium dependent signaling molecule because its receptor is on the endothelium and this is where it acts first!
• In the case of these drugs they are independent of the endothelium  They are endothelium independent vasodilators
• These drugs are used to treat people with heart failure
• Large veins can dilate and store a lot more blood so when the heart goes to contract there is a lower pre-load volume and this makes it easier for the heart to pump because it is not pumping against a huge volume of blood.