Cyclic AMP
Many of these receptors share common features, which are these. First, the receptor protein has two conformational states, and the shift of the protein between the states is regulated by whether the ligand is present. In the absence of ligand the receptor is inactive. When a receptor binds the ligand, it changes shape so that it can bind to other proteins that are also associated with the plasma membrane. These other proteins bind to guanine nucleotides--GTP and GDP--and for that reason they are called G proteins. G proteins themselves are normally inactive, but when an active receptor binds to them, they are induced in turn to become active themselves. In their active form they are able to bind to a third protein, an enzyme or other protein. Usually, the enzyme is an integral membrane protein, but sometimes not. As you would guess, the enzyme is normally inactive, but binding to the G protein can activate the enzyme to catalyze some reaction. Many different signal transduction systems make use of this basic model; what differs among them are the details of the which receptors are involved, which G proteins are involved, and which enzymes are involved.
Enough of this generality. Let’s take a look at the first situation in which a second messenger was shown to be involved in a signaling process, which was also the first instance where G proteins were discovered. In the late 1950s a man named Earl Sutherland and his coworkers were trying to understand how adrenaline or noradrenaline (properly called epinephrine and norepinephrine, respectively, in the U.S.) induces breakdown of glycogen in liver cells. As part of the sympathetic nervous system-mediated fight or flight response, the adrenal gland dumps lots of adrenaline into the blood stream of animals that are aroused or under stress. (There are lots of other aspects to this response as well). Somehow the liver cells of the animal respond by ceasing to make glycogen and instead begin breaking down the glycogen to glucose -1 -phosphate, which can feed directly into glycolysis and the citric acid cycle to produce ATP or be released into the blood so that other cells in the body can take it up and use it to make ATP. That is, when an animal is frightened or aroused, it prepares to make lots of ATP to sustain lots of movement--running or fighting.
Sutherland wondered how the liver cells did this trick--how did they detect and respond to the adrenaline in the blood? He applied adrenaline to liver cells, quickly ground them up, then took the cytoplasm from these treated cells and mixed it with the cytoplasm of untreated cells. The mixed cytoplasm broke down glycogen well. This implied to him that there was something inside the treated cells that mediated the effects of adrenaline or noradrenaline. Using this assay, he took different parts or fractions from the cytoplasm from untreated cells to see if these fractions could induce the glycogen breakdown in untreated cytoplasm. He showed that the active stuff was a small, soluble molecule (it escaped dialysis tubing, for example), and eventually he purified it and characterized it. It turned out to be a relative of ATP that had only one phosphate--i.e., the molecule was AMP, but a slightly different form of AMP than any that had ever been seen before. Usually the phosphate group is attached to the 5’ carbon of the ribose ring, but in Sutherland’s molecule, the phosphate formed a bridge between oxygens on the 5’ and the 3’ carbons. It was not only a phosphate ester, but a diester, and it’s called cyclic 3’,5’ adenosine monophosphate, or cyclic AMP for short (Fig. 20-4).
He further showed that cAMP was made from ATP in a one step reaction by an enzyme called adenylyl or adenylate cyclase---it splits off two phosphate groups from ATP and twists the rest of the molecule up on itself. Sutherland showed that cAMP levels in the muscle cell rose rapidly after they were treated with adrenaline and he surmised that this was because the adenylyl cyclase was activated. Sure enough when he measured the enzyme’s activity in quiescent cells, it was low, but it rose dramatically within seconds after cells were exposed to adrenaline. So Sutherland proposed that cAMP was a "second messenger" for adrenaline and that binding of adrenaline to its receptor somehow activated adenylyl cyclase.
For a long time it was believed that the receptor and adenylyl cyclase were part of the same molecule. In the 1970s a scientist named Martin Rodbell showed that there were more receptors per cell than molecules of adenylyl cyclase, so he proposed that they couldn’t be directly coupled--i.e., an adenylyl cyclase molecule could bind to more than one kind of receptor. He also showed that GTP was required for formation of cAMP. He concluded that there was some sort of GTP requiring coupling mechanism between the two proteins. Other scientists, Albert Gilman and Elliott Ross, who were interested in the mechanism of this process tried to generate mutant cells that did not make cAMP in response to adrenaline. They expected to get what kind of mutants?--in the receptor or in the adenylyl cyclase. They did find such mutants, but they also found some mutant cells that had normal receptors and normal adenylyl cyclase, but that still couldn’t produce cAMP in response to adrenaline. Why not? When they looked closely they discovered that these mutations affected a different protein. They showed that the protein could bind GTP and they set out to purify it, which they did, in 1980. This was the first so-called G protein ever discovered. What was learned from studies of the cAMP system set the framework for studies of other second messenger systems, which have been shown to have a number of features in common.
So how does noradrenaline control cAMP levels? The current model is this (Fig.. 20-16). First noradrenaline binds to its receptor protein (called the b-adrenergic receptor). As a result of the binding, the receptor can interact with the quiescent form of the G protein, by diffusing in the plane of the lipid bilayer. This G protein consists of three subunits--called a, b, g--that bind GDP in their inactive state. When this inactive G protein trimer interacts with the activated beta-adrenergic receptor, this catalyzes an exchange reaction in which GDP is replaced by GTP. This exchange in turn promotes the dissociation of the G protein trimer into the asubunit and the b,g dimer. The a subunit, to which the GTP is bound can now diffuse in the plane of the membrane until it contacts inactive adenylyl cyclase. This interaction, between the adenylyl cyclase and the a subunit of the G protein, activates the ad cyclase, so that it can now convert ATP to cAMP at the rate of several thousand reactions per second. (Show overhead on G protein on/off cycle). This continues until the GTP is hydrolyzed by the a subunit back to GDP; the a subunit is a slow GTPase, and so it essentially turns itself off after a time. This breakdown of GTP to GDP causes dissociation of the a subunit and the adenylyl cyclase, which turns off the adenylyl cyclase and allows the other subunits to rebind to form the original trimer (check out the quicktime movie on extracellular signaling).
As we’ll see later, this basic scheme--receptor activates G protein activates enzyme--is very common. A variant on the scheme happens in some cells. A different kind of receptor for adrenaline is called the a-adrenergic receptor; binding of adrenaline to the alpha receptor actually lowers the cAMP levels in cells (Fig. 20-18). Thus binding of adrenaline to the alpha receptor has the opposite effect of binding to the beta receptor or of any signaling molecule that elevates cAMP. Thisantagonism is mediated by a different kind of G protein, called Gi for inhibitory G protein. (What do you think the G protein that’s associated with the b -adrenergic receptor is called? Gs for stimulatory). Gi is also a trimer of 3 subunits, and the beta and gamma subunits are the same as in Gs. The a subunit is different however, though both a i and a s bind GTP/GDP. The difference is that when Gi is activated by the receptor, and the alpha subunit dissociates, just as does the alpha subunit of Gs, this alpha subunit cannot bind to and activate the adenylyl cyclase. In fact it appears that both ai and the bg dimers can inhibit adenylyl cyclase, and thereby prohibit synthesis of cAMP.
The specificity of the system, of course, lies in the nature of the receptor proteins, part of which binds specifically to a signaling molecule, like norepinephrine, and part of which interacts with a specific G protein. All of the receptors that bind to G proteins have a common structure--they all have an extracellular N-terminal portion, they pass through the membrane 7 times, and their C terminus is intracellular (Fig. 20-10). They share some sequence homology, but their main common feature is their overall structure and arrangement in the plasma membrane. Experiments in which chimeric adrenergic receptors were made from pieces of the a andb adrenergic receptors (which are, respectively, inhibitory and excitatory) showed that the part of the receptor that interacts with the ligand (norepinephrine) is near the C-terminus, probably between transmembrane segments 6 and 7, while the part that interacts with a G protein is the intracellular loop between transmembrane segments 5 and 6 (Fig. 20-14). All such proteins are called G-protein-coupled receptors and they are among the most common kinds of proteins known. For example, all of the genes of the roundworm Caenorhabditis elegans have been completely sequenced. C. elegans has about 18,000 genes total, of which about 6% or roughly 1100 are thought to be G-protein coupled receptors. Another hundred or so are G proteins themselves. In other words, cell signaling must be pretty important to organisms if they devote such a large fraction of their genetic material to this purpose. (The reason that there are so many G-protein coupled receptors is that all the proteins involved in detecting odors are G-protein-coupled receptors. That is, in your olfactory sense organs are lots of nerve cells, each one of which has a particular kind of receptor protein that binds certain airborne molecules (called odorants). Binding of an odorant to its receptor activates the nerve cell and generates an action potential that travels to your brain and gets interpreted as a smell. Humans supposedly can detect thousands of different odors; worms can detect hundreds. Rhodopsin, the light absorbing pigment in the eye is also a G-protein-coupled receptor, so two of our special senses depend on these proteins).
To summarize, in the cAMP system a binding of a signaling molecule to its receptor in the plasma membrane activates the exchange of GTP for GDP in a G protein, the dissociation of G protein into subunits, and the interaction of the G protein subunits with adenylyl cyclase, which either activates it to produce cAMP or inhibits it from doing so. There are dozens of signaling molecules that have been shown to raise the level of cAMP in cells--some are hormones like adenaline, some are neurotransmitters like dopamine, some are protein hormones like glucagon, but all act through a fundamentally similar mechanism. (For their work on the discovery of the G protein mechanism, Gilman and Rodbell shared the Nobel Prize in Medicine or Physiology in 1993, and Sutherland had earlier won a Nobel Prize for his discovery of cAMP).
Well, once cAMP concentration in the cell increases in response to the external signal, is that all there is? Not quite. As I indicated earlier, the cell will respond to an extracellular signal either by changing the activity of preexisting enzyme molecules or by changing the rate of synthesis of enzymes. cAMP can do both. Sutherland was the one who worked out the basic details of how this happens. The cAMP works by binding to the inactive form of an enzyme, called protein kinase A (PKA, or according to Lodish et al., APK, but that's not a term in general use). This inactive form is a tetramer consisting of two identical catalytic subunits and two identical regulatory subunits (Fig. 3-27a). When the cAMP level in the cell rises, it can bind to the regulatory subunit of PKA, and this causes dissociation of the tetramer into its component monomers--two regulatory subunits with cAMP attached, and two active catalytic subunits. (I.e., the regulatory subunits inactivate the catalytic subunits).
Now a protein kinase is a special class of enzyme; its substrate is other proteins (hence the "protein" part of the name), and the reaction it catalyzes is the transfer of a phosphate group from ATP to a serine, threonine or tyrosine residue in a protein. Enzymes that transfer phosphate from ATP to some other molecule are called kinases. That accounts for the P and K. The A in PKA is short for cyclic AMP. That is, PKA is activated by cAMP to transfer phosphates from ATP to protein substrates (PKA is a serine/threonine kinase).
These active PKA subunits now can bind to and phosphorylate a lot of different enzymes, and different kinds of cells have different substrates for PKA. One of the reasons that cAMP has different effects on different kinds of cells is that different cells contain different proteins that are substrates for PKA. In liver cells, the ones studied by Sutherland, two of the enzymes phosphorylated by PKA are important in glycogen metabolism. One is called glycogen phosphorylase kinase, which is normally inactive. If it is phosphorylated by PKA, it becomes an active enzyme, and what it does is phosphorylate glycogen phosphorylase. (overhead) The phosphorylated glycogen phosphorylase is now activated and can catalyze the splitting off of glucose-1-phosphate molecules from glycogen. Thus one effect of cAMP is to speed up the rate at which glycogen is converted to glucose (Fig. 20-34 and -35)..
Another substrate of PKA is glycogen synthase. This enzyme is normally active in the cell, but when it is phosphorylated, by active PKA, it becomes inactive. This is the opposite effect of the other enzymes that we’ve been discussing, which are activated by phosphorylation. This one is inactivated by phosphorylation and when this happens it inhibits the formation of glycogen. Thus two of the effects of the raising cAMP levels in liver and muscle is to slow down the synthesis of glycogen and speed up its conversion to glucose, both of which act to increase the level of glucose in the cell, and in the blood stream (the liver secretes the glucose), so that the animal is ready for running or combat. (Fig. 34b and 35).
This basic scheme works in lots of different cells although the outcome on the cell’s metabolism is different. (Table 20-3). Thus although a particular hormone activates the same internal mechanism, increased cAMP, the consequence is quite different. That’s because why? Because different proteins in the different cells are activated by PKA; that is, not just glycogen synthase or glycogen phosphorylase, but many other proteins are targets of PKA.
What happens when the threat to the organism passes? First, the adrenal gland stops secreting adrenaline. Thus adrenaline levels in the blood fall, the concentration of adrenaline surrounding the cell decreases and thus adrenaline dissociates from its receptor. The receptor can now glom onto the G protein and prevent it from interacting with adenylyl cyclase, so adenylyl cyclase becomes inactive. Thus, cAMP is no longer being made. In fact cAMP is constantly degraded into AMP by an enzyme called phosphodiesterase, so the cAMP levels begin to fall when cAMP is no longer being made. Once this happens, cAMP dissociates from the regulatory subunit of PKA, so that the PKA can reassemble--catalytic and regulatory subunits together. This of course inactivates the catalytic subunit so that it no longer can phosphorylate proteins. Finally there is a group of enzymes, called phosphatases, that cut phosphate groups off the serine and threonine residues that PKA phosphorylated. These phosphatases thus return glycogen synthase and glycogen phosphorylase kinase and glycogen phosphorylase back to their original unphosphorylated forms (Fig. 20-35a) which shifts the balance in the cell away from glycogen breakdown to glycogen synthesis. Thus, using this elaborate cascade of events, cells can rapidly respond to, and amplify, signals that impinge upon their surface, and they can rapidly reverse the response when the signal is no longer present. (This is called a "cascade system", and its advantage is rapid amplification of a signal-Fig. 20-37).
Interestingly, a number of drugs or toxins affect the cAMP system. For example, the inactivation of Gs by hydrolysis of GTP is prevented by cholera toxin, which "locks" the G protein into the on position, causes continuous production of cAMP in the absence of an extracellular signal. In intestinal cells, high levels of cAMP promote water secretion, and the consequences of cholera are excess excretion of water into the lumen of the intestine (and thus diarrhea), dehydration, and ultimately death. Pertussis toxin, which is made by the bacterium that causes whooping cough, prevents the GDP/GTP exchange for Gi proteins, thus inhibiting the inhibitory protein. This in turn causes excess production of cAMP in some cells. Caffeine (from coffee, tea, and cola drinks) inhibits phosphodiesterase, as does theophylline (from black tea), and both thus slow the breakdown of cAMP and promote increases in the intracellular levels of the compound. All these drugs and toxins thus act to elevate cAMP and thus mimic the effects of signaling molecules that themselves activate adenylyl cyclase.
I indicated earlier that cAMP not only affects the activity of proteins but also the rate at which proteins are synthesized: that is, it affects gene expression. Much less is known about the mechanism by which this happens in eukaryotes (it’s been well studied in bacteria, but it’s clear that the mechanisms are quite different in the two systems). In eukaryotes it appears that there is a regulatory protein that can activate transcription, that is normally inactive (called the CREB protein). However, if the cell is activated by signaling molecule to produce increased levels of cAMP, one of the consequences is that PKA phosphorylates CREB protein. This enables the CREB protein to enter the nucleus and bind to a short stretch of bases in the DNA called the cyclic AMP response element (CRE) (see Fig. 20-48a). This sequence is next to all the genes activated by cAMP. Now phosphorylated CREB protein (which means cyclic AMP response element binding protein) binds CRE on the DNA and accelerates the rate of transcription of the adjacent genes (e.g. the gene for somatostatin, a peptide hormone.), presumably by a mechanism similar to that by which the steroid hormone receptor complex works, by providing a binding site for RNA polymerase that increases the likelihood that it will transcribe the gene.
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