Cell Signaling III: Phosphatidyl Inositol, Calcium, and cyclic GMP

Cell Signaling III
G Proteins Cont’d; Ca2+ and InsP3; cGMP A second second messenger system was discovered in the late 1960s that used not cAMP but cGMP as the second messenger. The mechanism of synthesis of cGMP turned out in many cases to be similar, but not identical to that of cAMP. Namely, a hormone could bind a transmembrane receptor protein which activated an enzyme, guanylyl cyclase that converted GTP to cGMP. However, it turned out that no G protein was involved, and moreover the guanylyl cyclase activity is usually an integral part of the receptor protein (Fig. 20-3d in Lodish et al.), which is just the model originally proposed for receptors that activate adenylyl cyclase that had to be abandoned with the discovery of G proteins. An exception to this basic scheme of cell surface receptors arose when it was realized that one of the signaling molecules used in the body is not a compound dissolved in the bodily fluids, such as the ones we’ve been discussing, but a gas, Nitric Oxide, NO. NO is produced from arginine by the enzyme NO synthase, and once produced can diffuse through the membrane of the cell that makes it and into nearby cells, because it crosses membranes readily (like O2 or CO2 does). It is very labile and thus is quite rapidly inactivated by reactions with water or O2, so it has a lifetime of at most a few seconds. That means that it can only affect cells in its immediate vicinity. It has recently been shown that the way that NO works on its target cells is to activate a soluble (not membrane-associated) the enzyme guanylyl cyclase to make cGMP. So cGMP is a second messenger for NO. In this case, the NO acts directly on the synthetic enzyme guanylyl cyclase, bypassing a receptor or G-protein intermediary.
In blood vessels cGMP causes relaxation of the smooth muscle cells. Blood vessels are made up primarily of two cell types--smooth muscle and endothelial cells. It's known that the nervous system can control blood pressure; the adrenergic system causes blood vessel contraction and increased blood pressure. The opposite system, which uses acetylcholine, causes relaxation of the blood vessels and lowering of the blood pressure. (The acetylcholine response is basically the opposite of the "flight or fight" syndrome, and is sometimes called the "rest and digest" affect. It lowers heart rate, causes pupillary contraction, speeds peristalsis, lowers blood pressure, etc.). For a long time people thought that the neurons that released acetylcholine (ACh) acted directly on the smooth muscle cells in blood vessels to cause them to relax. However, careful studies showed that the neurons make synapses on the endothelial cells, not on the smooth muscle, and thus must indirectly affect muscle. Studies in the late '80s showed that ACh binds endothelial cells and induces them to make NO. Thus NO is a second messenger for ACh (actually it's a third messenger because the second messenger is calcium, see below). Then NO diffuses out of the endothelial cells into the nearby smooth muscle cells, where it activates guanylyl cyclase and causes an increase in cGMP. This in turn causes relaxation of the muscle cells (see Fig. 20-42b. Another gas, CO, is thought to act similarly as a signaling molecule. The three people who first demonstrated that gases, like NO, act as intercellular signaling molecules won Nobel Prizes two years ago.
For a long time people believed that cAMP and cGMP would be enough second messenger molecules. The prevalent idea was that they were a kind of yin and yang of messenger molecules--where cAMP would stimulate cells to do something, and cGMP would inhibit them. Thus the activity of the cell would be determined by the relative amounts of cAMP and cGMP that were present. However, this view lost ground for several reasons. First, the discovery of Gi proteins meant that cAMP activity could be regulated directly by altering the activity of adenylyl cyclase. More tellingly, some signaling molecules didn’t cause a change in either cAMP or cGMP in the cells that respond to them.
One clue came many years ago from studies that showed that when some cells were stimulated by signaling molecules, they broke down some minor lipids in their membrane--phosphatidylinositol. (Fig. 20-38). Originally this was interpreted as a secondary effect of the signaling molecule--that somehow the breakdown of this lipid altered membrane fluidity to enhance the movement of the necessary proteins. However, it eventually became clear that this wasn’t the case and that the lipid itself was somehow involved in the signal transduction. After much effort by many labs, the general outlines of this signaling pathway became clear in the early 1980s and it has been recognized increasingly as one of the main signaling pathways in cells in the last decade and a half.
How does this system work? Just as in the other systems, there are three crucial proteins--a receptor, a G protein and an enzyme. (The book doesn't have a good figure for this, but Fig. 20-39 is the best they do; read the caption because not all the steps are illustrated.) The receptor binds a signaling molecule such as acetylcholine, angiotensin, glucose (in beta cells of the pancreas--i.e. the insulin secretion response), vasopressin, thyrotropin releasing hormone, etc. The now-activated receptor binds to a G protein, usually a class of G protein called the Gq class, to distinguish if from Gs or Gi, catalyzing the usual GTP/GDP swap and dissociation of the alpha subunit. The Ga subunit then binds to and activates an enzyme. The enzyme activated by this G protein is called phospholipase C (don’t ask me why--I think because they first found phospholipases A and B, and so were up to C.). As the name of the enzyme implies, its substrate is phospholipids, particularly the phospholipid phosphatidyl inositol 4-5 bisphosphate (PIP₂). (See Fig. 20-38a).
The enzyme cuts the PIP2, which releases inositol 1,4,5-trishosphate (InsP3 or IP₃) into the cytosol--it’s soluble because its highly charged, but the diacylglycerol (DAG) (i.e. glycerol with two fatty acid esters attached) remains stuck in the lipid bilayer.
Now the story gets complicated. The InsP3 can diffuse throughout the cytosol, and bind to an InsP3 receptor protein located in the membrane of the endoplasmic reticulum. (Fig. 20-39). This InsP3 receptor is a kind of Ca²⁺ selective transmembrane protein--i.e. it is a calcium channel. Normally it is impermeable to Ca²⁺, but when it is bound by InsP3, it opens up, allowing Ca²⁺ to pass through the ER membrane. Now inside the ER the Ca²⁺ concentration is around 1 mM, while in the cytosol it is more like 100 nM. That is, the concentration in the cytosol is about 10,000 X less than in the ER, and if Ca²⁺ can diffuse out of the ER, it does. So IP₃binding to its receptor causes a rapid increase in the [Ca^2+]] in the cytosol of 10-50 fold. And guess what? Ca²⁺ can act as a second messenger molecule for many different cellular processes.
One of the enzymes in the cell that is Ca²⁺ dependent is protein kinase C (C means Ca²⁺ dependent). Normally inactive and in the cytosol, this enzyme is activated when it is bound by two different signaling substances. One is Ca²⁺, which appears to cause it to move from the cytosol to associate with the inner side of the plasma membrane. The other is diacylglycerol, which PKC binds to if it finds DAG available in the membrane. When will DG be available? When PLC has produced it in response to a first messenger molecule. Thus, the effects of PLC on PIP2 are to raise the concentration of both Ca²⁺ and DAG, both of which are required to activate PKC. PKC, like PKA, is a serine/threonine kinase that phosphorylates these amino acids on protein side chains. It’s especially active in phosphorylating membrane proteins, which typically, changes the membrane’s permeability to certain ions. Other proteins phosphorylated by PKC appear to be gene regulatory proteins that can move into the nucleus when they are phosphorylated and turn specific genes on or off.
A second effect of the rise in Ca²⁺ in the cytoplasm as a result of the IP₃ is the activation of a calcium binding protein called calmodulin. Calmodulin binds 4 molecules of Ca²⁺ (Fig. 20-41) and once that happens, it can interact with a bunch of other proteins. ( A group of these are also protein kinases, called, ingeniously enough, the Ca²⁺/Calmodulin dependent protein kinases (CaM Kinases).
Normally inactive, the CaM kinases are activated by calmodulin (which is activated by elevated Ca²⁺) and can phosphorylate a bunch of other proteins, again on serine or threonine residues. CaM kinase appears to be important in release of transmitter by neurons in response to an action potential and at least some synaptic proteins are phosphorylated both by CaM kinase and PKA, though at different places. It also phosphorylates and activates glycogen phosphorylase, an enzyme that is also turned on by PKA. Again the effects of CaM kinase are widespread but not well characterized, as is the case for the other kinases we have discussed.
In all these cases however, I hope you see a common theme. Namely that activation of a kinase can have two kinds of effects. One is to change the activity of a group of enzymes that get phosphorylated--sometimes turning them on, sometimes turning them off--and this changes the reactions that the cell can conduct. The other is to activate transcription regulating factors. These alterations in turn change the properties of the cell. In the example that we started with, the breakdown of glycogen, adrenaline both speeds up breakdown by activating glycogen phosphorylase and slows down the synthesis of glycogen by inhibiting glycogen synthase, so that glucose production is greatly favored. This happens in the liver and muscles of animals that are getting ready to fight or flee. If the animal actually does begin to use its muscles, Ca²⁺ is released into the muscle cell cytosol (from the muscle ER, which is called sarcoplasmic reticulum). This not only promotes movement of the actomyosin filaments and muscle contraction, but it activates CaM kinase, which in turn activates glycogen phosphorylase even more. This in turn speeds up glycogen breakdown. So cAMP helps muscles get ready for action by increasing [glucose] for breakdown by glycolysis and the CA cycle to make ATP for work. And if the muscle actually begins to work, the increase in Ca²⁺ accelerates the breakdown to glycogen to glucose and increases the rate at which ATP is synthesized. So these two systems act in concert--or synergistically as cell biologists would say, and they actually converge on the same enzyme, glycogen phosphorylase which is a substrate for both PKA and CaM kinase.
As if this weren’t complex enough, it’s becoming increasingly clear that these various second messenger systems can interact with each other. Not only do cAMP and CaM converge on common pathways like glycogen phosphorylase, under some conditions, they can each act to increase or decrease the rate of production of the other. I already mentioned how the Ca²⁺ level can affect the activity of PKC. It also can activate or inhibit adenylyl cyclase--the enzyme that makes cAMP--and phosphodiesterase, the enzyme that breaks down cAMP. PKA can phosphorylate Ca²⁺ channels, such as the IP₃ receptor in the ER, and change their properties. cGMP seems to regulate a second form of Ca²⁺ channel in sarcoplasmic reticulum and other ER membranes, called the ryanodine receptor. (Ryanodine is a drug used to regulate blood pressure; one of the so-called Ca²⁺ channel blockers, and it was used to identify the first Ca channel known). The cGMP activates an enzyme called ADP ribosyl cyclase which acts on NAD⁺ to form a cyclic molecule called cyclic ADP ribose which in turn can bind to and open the ryanodine Ca channel. This also allows Ca to flow into the cytoplasm and raises the concentration intracellularly. So, an increase in cGMP can in some cells increase Ca²⁺ which in turn can act to do all the things we’ve discussed. (Note that Ca²⁺ can have a positive, or sometimes negative, feedback on its own concentration in the cells. The Ca²⁺channels are more likely to open in the presence of high levels of Ca²⁺ in the cytosol than in the presence of low levels).
The point here is that cell signaling is a complex process and scientists have only just begun to scratch the surface of this complexity. However, it’s already clear that the various second messenger systems that exist (I haven’t mentioned them all) don’t operate independently but interact in ways that can either augment a response when two pathways are activated and augment each other, or cancel a response when the two pathways inhibit each other. (In some cells Ca activates phosphodiesterase and short circuits the response to cAMP, for example). Can you think of reasons why nature might want to design these complex interactive pathways?
Finally, the systems I’ve been describing must be turned off as well as turned on. How does that happen? Well, you already know from our previous discussions that there are Ca² "pumps" in the cell that can use ATP to move Ca²⁺ across membranes or that can exchange Na⁺ and Ca²⁺. Such pumps exist in the ER membrane, the plasma membrane, and the mitochondria. Usually the fastest transport is accomplished through the plasma membrane and almost as fast through the ER. The mitochondria seem to be an emergency pathway that only kicks in when the other mechanisms are overwhelmed by high Ca²⁺. The mitochondria take up Ca²⁺temporarily, at the expense of the ATP they’ve made, but they are not healthy with high internal Ca²⁺, so they must eventually excrete the Ca²⁺ back into the cytosol so it can be moved into the ER or out of the cell or they will self-destruct. As the Ca²⁺ levels in the cytosol return to the resting level of 100 nM, Ca²⁺ unbinds from the various proteins that it bound to, such as calmodulin, PKC, etc. And these proteins therefore become inactive again. Eventually phosphatases remove the phosphate groups from the various proteins that got phosphorylated by these kinases, returning those proteins to their resting state.
There are phosphatases that degrade cGMP and IP₃ as well, and so these inactivated, usually pretty quickly. Thus the concentration of these compounds only remains high as long as they are being synthesized, which in turn depends on the continued presence of the extracellular signal. If its concentration falls, the concentration of the intracellular signal also falls fairly quickly as well, which turns off the complex cascade of events that is set in motion by the rise of the intracellular signal.
What should you remember about all of this? Well you should understand the function of receptors, G proteins and the proteins activated by G proteins. You should understand in general terms the role of kinases in cell signaling and of cascade systems. (Do you know of any other biological cascade systems?) And finally you should understand that cells have systems that actively reverse the effects of the extracellular signaling molecule once it disappears from the extracellular fluid and unbinds from its membrane receptor.
What do all the following substances have in common?:
phorbol esters
calcium ionophores (A23187, ionomycin)
pertussis toxin
cholera toxin
forskolin
caffeine, theophylline
They’re all substances that affect second messenger pathways in cells. The first two affect the PIP2 system and the others affect the cAMP system. One way to determine what the second messenger is for a system is to find out whether one or more of these substances can interfere with or simulate the response.
Phorbol esters are called tumor promoters because they increase the likelihood of an animal’s getting cancer from some other cause, though they don’t cause cancer directly. They bind to and activate protein kinase C, in the absence of diacylglycerol, thus bypassing one arm of the second messenger response. If phorbol esters can mimic a response, then it’s a good bet that PKC is part of the response pathway. Similarly the Ca²⁺ ionophores can allow Ca²⁺ out of the ER and thus mimic the effect of raising IP3 levels in the cell.
All the other substances increase the levels of cAMP in cells, as I've mentioned previously. The use of such activators and inhibitors has proved invaluable in establishing which second messengers are part of which responses; similar strategies are used in all signaling systems that have been studied.